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LIST OF CONTENTS
Subjects
Page
LIST OF ABBREVIATION
INTRODUCTION
AIM OF THE WORK
12
LITERATURE REVIEW
15
1- Human burns
15
2- Types of infection in burned patients
21
3- Pathogenic bacteria isolated from burned patients
24
4- The antibiotics activity against pathogenic bacteria
35
5- Resistant of bacteria to antimicrobial agent
41
6- Antimicrobial resistant in P. Aeruginosa
59
7- Antimicrobial resistant in Enterobacteriacae
60
8- Bactericidal action of different antimicrobial agent
61
9- Essential oils as antibacterial agents
67
10- Molecular studies
71
80
MATERIAL AND METHODS

1- Patients
80
2- Materials and equipment for samples collection and processing
80
3- Isolation and purification of pathogenic bacteria isolated from
80
medical specimens
4- Identification for bacterial isolates
84
5- Antibiotic susceptibility test
94
6- Determination of MICs and MBCs of different antibiotic against
95
selected isolates
7- Bactericidal activity of different antimicrobial agent
96
8- Antimicrobial activity of some essential oils against multi resistant
97
bacterial isolates
9- Screening for the virulence factors and degrading enzymes
produced by tested S. aureus (12, 30, 50 and 95) using agar well
99
diffusion assay
10-Effect of certain bactericidal concentration on the enzyme activities 100
(virulence factors)
11-Effect of tea tree oil on the enzyme activities (virulence factors)
101
12- Molecular identification and characterization assays of van A & 101

mec A genes
107
RESULTS
1- History of burned patients
107
2- Factors affecting mortality
111
3- Distribution of collected isolates
118
4. Morphological and biochemical tests for identification of all 125

bacterial isolates
5. Prevalence of microorganisms in burned patients
131
6. Antibiotic susceptibility test
136
7. Determination of minimum inhibitory concentration (MIC) and 145

minimum bacterocidal concentration (MBC)
8. Bactericidal action of different disinfectants against the four 151
multiresistant
9. Susceptibility of multiresistant S. aureus (12, 30, 50 and 95) to 153
certain essential oils
10- Screening for the virulence factors and degrading enzymes 159
produced by multiresistant S. aureus
11. Molecular identification and characterisation assays of van A & 162
mec A gene
DISCUSSION
179
SUMMARY
203
REFERENCES
210
LIST OF ABBREVIATIONS
AR
Antimicrobial resistance
ARDS
Adult respiratory distress syndrome
AST
Antibiotic sensitivity testing
BSI
Bloodstream infection
CA-MRSA
Community associated- MRSA
CoNS
Coagulase-negative staphylococci
DHFR
Dihydrofolate reductase
EF-G
Elongation factor G
FIC
Fractional Inhibitory Concentration
IRS
Isoleucyl-tRNA synthetase
MRSA
Methicillin resistant S. aureus
NNIS
National Nosocomial Infections Surveillance
PBPs
penicillin-binding proteins
PVL
Panton-Valentine leukocidin
PCR
Polymerase chain reaction
Resistance
SSIs
Surgical site infections
TSS
Toxic shock syndrome
UTI
Urinary tract infection
VFs
Virulence factors
VISA
vancomycin-intermediate S. aureus
VRE
Vancomycin resistant Enterococcus.
VRSA
Vancomycin-resistant S. aureus
Microgram
DNA
Deoxy ribonucleic acid
EPS
Exopolysaccharide
Erm
Erythromycin ribosome methylation ( macrolide),
Female
Gram
GISA
Glycopeptide intermediate Staphylococcus aureus
Hours
Kbp
Kilo base pairs

3
Kda
Kilo Dalton
Liter
Male
MBC
Minimal bactericidal concentration
MIC
Minimum inhibitory concentration
Min.
Minute
MR
Methyl red
MRSA
Methicillin-Resistant Staphylococcus aureus
OF
Oxidation fermentation
S. aureus
Staphylococcus aureus
Unit
Introduction
Introduction
Infection in the burned patient is a leading cause of morbidity and
mortality and remains one of the most challenging concerns for the burn
unit (Cochran et al, 2010).
Burn is one of the most common and devastating forms of trauma.
Patients with serious thermal injury require immediate specialized care in
order to minimize morbidity and mortality. Significant thermal injuries
induce a state of Immuno-suppression, which predisposes infectious
complications in burned patients (Church et al, 2011).
Burn patients are ideal hosts for opportunistic infections, the burn
site remains relatively sterile during the first 24 hour; thereafter,
colonization of the wound by Gram negative bacteria is common (Pruitt et
al, 2008)
Sources of organisms are found in the patients own endogenous
(normal) flora, from exogenous sources in the environment, and from
healthcare personnel. Exogenous organisms from the hospital environment
are generally more resistant to antimicrobial agents than endogenous
organisms. Organisms associated with infection in burn patients include
Gram-positive, Gram-negative, and yeast. The distribution of organisms
changes over time in the individual patient and such changes can be
ameliorated with appropriate management of the burn wound and patient.
The typical burn wound is initially colonized predominantly with Grampositive organisms, which are fairly quickly replaced by antibioticsusceptible Gram-negative organisms, usually within a week of the burn
injury. If wound closure is delayed and the patient becomes infected,
requiring treatment with broad-spectrum antibiotics, these flora may be
replaced by yeasts and antibiotic-resistant bacteria (Church et al, 2011).
Introduction
Antimicrobial resistance can increase complications and costs

associated with procedures and burn treatment. An infected wound
complicates the postoperative course and results in prolonged stay in the
hospital and delayed recovery (Li, 2002).
The pathogens that infect the burns are primarily Gram-positive
bacteria such as methicillin-resistant Staphylococcus aureus (MRSA),
enterococci, group A beta-hemolytic Streptococcus, coagulase negative
Staphylococcus, and Gram-negative bacteria such as Pseudomonas
aeruginosa, and Klebsiella species. These latter pathogens are notable for
their increasing resistance to a broad array of different antimicrobial agents.
In addition, burn wounds are commonly infected with fungal pathogens
(Gladwin, 2007).
The patient has three principal defenses against infection: physical
defenses, nonspecific immune responses, and specific immune responses.
Changes in these defenses determine the patients susceptibility to infection
(Cloete, 2003).
Wound infections have been a problem in the field of surgery for a
long time. Advances in control of infections have not completely eradicated
the problem because of development of resistance. Burn wound infection
can be subdivided into local or non-invasive infection and invasive
infection (Gladwin, 2007).
Urinary tract infection (UTI) has received little attention in burned
patients. Risk factors specific to burned patients includes the presence of
burns in certain patients and the increased length of time patients require
catheterization in the treatment of extensive injuries. In pediatric burns,
nosocomial UTI occurs almost exclusively in patients with indwelling
urinary catheters. Signs and symptoms of UTI may be present or obscured
in burn patients relative to other conditions accompanying the injury
(Schmitz et al., 2000).
Introduction
Respiratory complications rank as the major cause of death in burn

patients. Respiratory complications include inhalation injuries, aspiration of
fluids by unconscious patients, bacterial pneumonia, pulmonary edema, and
post injury respiratory failure. Direct-inhalation injuries, which can lead to
other respiratory complications, are especially common. The three basic
categories of direct-inhalation injuries are inhalation of dry heat and soot,
carbon monoxide poisoning, and smoke inhalation (Angioni et al., 2006).
The emergence of antibiotics resistance is one of the most dangerous
phenomena of the last twenty years and knowledge of resistance
mechanisms means fully understanding their spread into all environments.
Antibiotic resistance in bacteria is a serious problem facing society today.
There are many reasons for this problem, one of which is an overuse of
antibiotics. Through evolution, tougher bacteria that cannot be killed by
antibiotics are surviving and reproducing. This is not necessarily a problem
in people with strong immune systems, but in hospitals where many people
have compromised immune systems, harboring such bacteria can kill a
person (Stefani and Agodi, 2008).
The primary cause of antibiotic resistance is genetic mutation in
bacteria. The prevalence of antibiotic resistant bacteria is a result of
antibiotic use both within medicine and veterinary medicine. The greater
the duration of exposure the greater the risk of the development of
resistance irrespective of the severity of the need for antibiotics. As
resistance becomes more common there becomes a greater need for
alternative treatments. However despite a push for new antibiotic therapies
there has been a continued decline in the number of newly approved drugs.
Antibiotic resistance therefore poses a significant problem (Todar, 2008).
MRSA become an endemic organism in many burn units. Gramnegative organisms have long been known to cause serious infection in
burn patients. Gram negative bacteremia has been associated with a 50%
increase in predicted mortality for patients with bacteremia compared to
8
Introduction
those without bacteremia. This is in contrast to Gram-positive bacteremia,

which was associated with no increase in predicted mortality (Stefani and
Agodi, 2008).
Vancomycin resistance has been reported in clinical isolates of both
coagulase-negative
staphylococci
and
Staphylococcus aureus.
The
emerging threat of widespread vancomycin resistance poses a serious

public health concern given the fact that vancomycin has long been the
preferred treatment of antibiotic-resistant Gram-positive organisms
(Dorman and Deans, 2000).
The detection and identification of microorganisms based on their
16S rRNA have many advantages. First, each bacterial cell contains
multiple copies of the 16S rRNA in its ribosomes. Hence, the technique is
sensitive enough to detect single bacterial cells. Second, 16S rRNA genes
are highly conserved throughout bacterial evolution. (Maniatis et al.,
2000).
One of the key factors enabling S. aureus to survive, colonize,
proliferate and cause human infections is the expression of virulence
factors (VFs). Furthermore, S. aureus produces an array of VFs, which can
be broadly grouped into those involved in 1) bacterial attachment, 2)
evasion of host defenses and 3) tissue invasion. It has also been shown that
not every S. aureus strain produces every VF, or does not produce each to
the same degree and it is tempting to then assume that those strains
expressing more or higher levels of VFs are the more pathogenic (Todar,
2008).
A large number of essential oils and their constituents have been
investigated for their antimicrobial properties against series of bacteria
(Miura et al., 2002 and Dapkevicius et al., 2002). Thyme and tea tree oil
are the best antiseptic substance available for external wounds, it is
valuable in the treatment of infections, also it has been traditionally used
9
Introduction
for a number of applications including skin problems such as burns,

eczema and cuts bruises, treatment of circulation problems, joint
inflammation, respiratory infections, tonsillitis, pharyngitis, bronchitis,
cough, excellent in treating cold (Aqil et al., 2005). Thyme oil is a strong
antioxidant, it has antibacterial activity against 25 different genera of
bacteria (Shan et al., 2007).
The essential oil of thyme and tea tree showed a wide antibacterial
activity against microorganisms that had developed resistance to antibiotics
as MRSA and vancomycin resistant S. aureus (Yarwood et al., 2004).
Tea tree oil have the ability to reduce or stop the production of
virulence factors, this may be another mechanism by which tea tree oil
works to prevent or clear S. aureus infections (Nostro et al., 2004).
A combination of plant extracts with antibiotics help to minimize
concentrations
and
reduce
sensory
impact.
Furthermore,
these
combinations may also control some bacteria that are known to show
consistently high resistance to antimicrobials (Aqil et al., 2005).
10
11
Aim of the Work
Aim of the Work

The present study is undertaken to study the antibiotic resistant
bacteria contaminating burn wounds, blood, urine and sputum of the
burned patients. This study will help to assess the burden of infections and
antimicrobial susceptibility testing will help to formulate antibiotic policy
for better management of these patients. Antibiotic resistant bacteria from
these sources are important and cause serious problem in clinical field. The
present study is undertaken with the following aims and objectives:
1.
Isolation and purification of pathogenic bacteria from burned patients.
2.
Identification of isolated bacteria according to morphological

characteristics and biochemical tests.
3.
Studying the susceptibility and resistance of isolated bacteria to

different antibiotics and determination of MICs and MBCs of some
antibiotics against multiresistant isolates
4.
Determination of bactericidal action of ethanol and H2O2 as

antimicrobial agent.
5.
Studying the susceptibility of multiresistant isolates to certain essential

oils.
6.
Determination of the minimum inhibitory concentration and minimum

bactericidal concentration of thyme and tea tree oil for multiresistant
isolates.
7.
Studying the influences of combination between thyme or tea tree oil

and vancomycin antibiotic on the growth of multiresistant isolates.
8.
Screening for the virulence factors and degrading enzymes produced

by tested isolates.
9.
Studying the effect of bactericidal action of different antimicrobial

agent on virulence factors of tested isolates.
10. Investigation the effects of tea tree oil on the production of virulence
factors by multiresistant isolates.
11. Molecular studies on multiresistant isolates:
12
Aim of the Work
a. Confirmed identification of multiresistant isolates by 16S rDNA and

gene sequencing
b. Detection of the resistant gene by using multiplex PCR technique
and specific primers pairs.
13
14
Review of Literature
1. Human burns
Burn infection is problematic because it delays healing, encourages
scarring and may result in bacteremia, sepsis or multiple-organ dysfunction
syndrome (a.k.a. organ failure) whereby organs from several systems are
unable to maintain homeostasis on their own, requiring immediate medical
attention. Bacteria are the most common pathogens of burn wounds. These
microbes form multi-species biofilms on burn wounds within 48 72 hours
of injury (Agnihotri et al., 2004). In patients with severe burns over more
than 40% of the total body surface area (TBSA), 75% of all deaths are
currently related to sepsis from burn wound infection or other infection
complications and/or inhalation injury (Atiyeh et al., 2005).
1.1. HUMAN skin - A major host innate defense:
An intact human skin surface is vital to the preservation of body

fluid homeostasis, thermoregulation, and the host's protection against
infection. The skin also has immunological, neurosensory, and metabolic
functions such as vitamin D metabolism. Thermal injury creates a breach in
the surface of the skin. A basic knowledge of skin anatomy and physiology
is required to understand emergency burn assessment and approaches to
burn care (DeBoer and O'Connor, 2004).
Basic skin anatomy, showing the depth of injury for first, second, and third-degree burns(Roth
and Hughes, 2004).
15
The skin is derived from ectoderm and mesoderm and has two
anatomic layers: the epidermis or outermost nonvascular layer consists of
several layers of epidermal cells that vary in thickness over various body
surfaces, and the dermis or corium is largely made of collagen and contains
the microcirculation, a complex vascular plexus of arterioles and venules.
The two skin layers are bound together by a complex mechanism that is
essential for normal function. Burn injury is a very painful form of trauma
because of the multitude of pain receptors and nerves that traverse the skin
layers. Beneath the skin lie the subcutaneous tissues, muscle, and bone
(Dyer, 2008).
1.2. Causes and classification of burned injury :
1.2.1-Causes of burned injury:
a. Thermal injury
Direct contact with flame, a hot surface or hot liquid (scald), or a
source of heat conduction, convection, or radiation causes a degree of
cellular damage to the skin that varies with the temperature and duration of
exposure (Baker et al., 2009). As the temperature rises further, protein
denaturation occurs, oxygen radicals are liberated, and eventually cells die
with the formation of the burn eschar (Moritz and Heuriquez, 2005).
b. Chemical injury
Chemical interaction may also damage protein structures. A
classification system that was described and remains in use groups
chemicals according to their mode of action. (Amshel et al., 2000).
c. Electrical injury
Electrical burns are caused by either an electric shock or an
uncontrolled short circuit (a burn from a hot, electrified heating element is
not considered an electrical burn). Common occurrences of electrical burns
include workplace injuries, or being defibrillated or cardioverted without a
16
conductive gel. Lightning is also a rare cause of electrical burns (Moritz

and Heuriquez, 2005).
d. Radiation
Radiation burns are caused by protracted exposure to UV light (as
from the sun), tanning booths, radiation therapy (in people undergoing
cancer therapy), sunlamps, radioactive fallout, and X-rays (Baker et al.,
2009). By far the most common burn associated with radiation is sun
exposure, specifically two wavelengths of light UVA, and UVB, the latter
being more dangerous. More severe cases of sun burn result in what is
known as sun poisoning or "heatstroke". Microwave burns are caused by
the thermal effects of microwave radiation (Hartford, 2009).
1.2.2. Classification of burned injury:
Three degrees of burns: Burns can be classified according to
mechanism of injury, depth, extent & associated injuries, according to the
surface area of injury and according to presence of inhalation injury.
I-According to the depth of injury :
Currently, burns are described according to the depth of injury to the
dermis and are loosely classified into first, second, third, and fourth
degrees.
The degrees of burn injury are listed as the following (American
Burn Association, 2000).
Nomencla
ture
First
degree
Layer
involved
Epidermis
Appearan
ce
Redness
(erythema)
Textur
e
Dry
Sensati
on
Painful
Time to
healing
1week or
less
Complication
None
Second
degree
(superficial
partial
thickness)
Extends
into
superficial
(papillary)
dermis
Red with
clear
blister.
Blanches
with
pressure
Moist
Painful
2-3wks
Local
infection/
cellulit
17
Example
Second
degree
(deep
partial
thickness)
Extends
into deep
(reticular)
dermis
Red-andwhite with
bloody
blisters.
Less
blanching.
Moist
Painful
Weeks may
progress
to
third
degree
Scarring,
contractures
(may
require
excision
and
skin
grafting)
Third
degree
(full
thickness)
Extends
through
entire
dermis
Stiff and
white
/brown
Dry,
leather
y
Painles
Requires
excision
Scarring,
contractures
,
amputation
Fourth
degree
Extends
through
skin, and
muscle and
bone
Black;
charred
with eschar
Dry
Painles
Requires
excision
Amputation
, functional
impairment,
gangrene,
and death.
II-According to the severity of injury:

In order to determine the need for referral to a specialized burn unit,
the American Burn Association devised a classification system to aid in the
decision-making process. Under this system, burns can be classified as
major, moderate and minor. This is assessed based on a number of factors,
including total body surface area (TBSA) burnt, the involvement of specific
anatomical zones, age of the person and associated injuries (American
Burn Association, 2000)
a. Major burns are defined as:
Age 10-50yrs: partial thickness burns >25% of total body surface area
Age <10 or >50: partial thickness burns >20% of total body surface area
Burns involving the hands, face, or feet
Electrical burns
Burns associated with fractures or other trauma
Burns in infants and the elderly
Burns in persons at high-risk of developing complications
These burns typically require referral to a specialized burn treatment center.
18
b. Moderate burns are defined as:
Age 10-50yrs: partial thickness burns involving 15-25% of total body

surface area.
Age <10 or >50: partial thickness burns involving 10-20% of TBSA.
Full thickness burns involving 2-10% of total body surface area.
Persons suffering these burns often need to be hospitalised for burn care.
c. Minor burns are:
Age 10-50yrs: partial thickness burns <15% of total body surface area
Age <10 or >50: partial thickness burns involving <10% of total body
surface area
Full thickness burns <2% of total body surface area, without associated
injuries.
These burns usually do not require hospitalization.

III-According to the surface area of injury.
Burns can also be assessed in terms of total body surface area
(TBSA), which is the percentage affected by partial thickness or full
thickness burns (Lionelli et al., 2005). The rule of nines is used as a quick
and useful way to estimate the affected TBSA (David, 2010).
19
Body diagram for estimation of total burned surface area (%TBSA) in adults, using the rule of
nines (numbers are for anterior only and posterior only) (Roth and Hughes. 2004)
Body diagram for estimation of total burned surface area (%TBSA) in children, using the rule of
nines (numbers include anterior and posterior) (Roth, and Hughes. 2004).
IV-According to presence of Inhalation Injury

Inhalation injury occurs in anywhere from 3 to 21% of burn patients
and is a major cause of mortality; 80% of fire-related deaths occur from
hypoxia due to oxygen deprivation or from inhalation of the toxins found in
smoke. Inhalation injury of the lung in adults is usually proportional to the
depth and extent of body surface area burn. Hypoxia occurs when the
carbon monoxide generated by combustion is inhaled and binds to
20
hemoglobin. The pathogenesis of pulmonary injury from smoke inhalation

has been well described (Atiyeh et al., 2005).
1.3. Impact of Patient Demographics and Burn Severity:
Very young children and the elderly have an increased risk of being
burned and worse clinical outcomes than patients in other age groups.
Individuals with deliberate self-inflected burn injuries and the disabled
have been shown to have more severe injuries and longer hospital stays
than those with accidental injuries (Backstein et al., 1993). Obese adults
and those who have an underlying medical condition such as diabetes have
also been shown to have higher morbidity and mortality. AIDS patients
appear to have more complications due to infection, delayed wound
healing, and increased mortality, although reported outcome data for
human immunodeficiency virus-infected and AIDS patients are limited. It
is
expected
that
burn
patients
with
other
types
of
severe
immunosuppression would have similar problems, particularly increased

problems with wound infection and sepsis and a higher mortality, although
this group has not been studied (Backstein et al., 1993).
Burns in the elderly constitute more severe injuries than in the
general population and result in a higher number of fatalities. A recent
review of adult patients admitted to a burn center over a 7-year period
showed that 221 of 1,557 (11%) were >59 years of age and a higher
proportion were women (McGill et al., 2000).
2. Types of bacterial infection in burned patients
2.1. Burn Wound Infection
Overall, the incidence of burn wound infection has declined in recent
years with the change to early excision and wound closure. As the size of
the wound increases, so does the risk of infection. Causes of burn wound
infection relate to the loss of the protective barrier of the skin and
thrombosis of the subcutaneous blood vessels. The resulting a vascular
21
wound bed makes an excellent medium, which can support the growth of
microorganisms as well as prevent the penetration of systemically,
administered antimicrobial drugs. Burn wound infection can be subdivided
into local or non-invasive infection and invasive infection. Local wound
infection is characterized by erythema or cellulitis, purulent, drainage, graft
loss, fever >38.5C and leukocytosis. Invasive wound infection is
characterized by conversion of partial-thickness to full-thickness injury,
necrosis of small blood vessels, edema, erythema, and tenderness at the
wound edges. Systemically, the patient may be hypothermic or
hyperthermic, hypotensive, have a decreased urine output and illeus.
Laboratory
results
will
reveal
leukocytosis
or
leukopenia,
thrombocytopenia, positive blood cultures, hyperglycemia and invasion of

organisms into viable tissue on histopathologic examination of the wound
(McVay et al., 2007).
All surgical wounds are contaminated by microbes, but in most
cases, infection does not develop because innate host defenses are quite
efficient in the elimination of contaminants. A complex interplay between
host, microbial, and surgical factors ultimately determines the prevention or
establishment of a wound infection (Baxter, 2000). The most common
group of bacteria responsible for wound infections were S. aureus,
coagulase negative staphylococci, enterococci, E. coli, Proteus mirabilis,
Enterobacter species, K. pneumoniae, other P. aeruginosa (NNIS, 1996).
2.2. Bloodstream and Intravascular Catheter Infection
Bloodstream infection occurs more often in burn patients than in any
other patient population (Zaragoza et al., 2003). Intravascular catheterassociated bloodstream infection rates are higher in t3he burn population
than in any other. These two facts are related to the hematogenous seeding
of catheters that often occurs related to the colonized or infected burn
wound and to the often-necessary placement of catheters near or through
22
the wound in patients with extensive injuries (Murray and Hospenthal,

2008).
S.aureus remains a common cause of community onset bloodstream
infections (Collignon et al., 2005). Staphylococcal bacteraemia mortality
rate was approximately 20% to 50% between 1992 and 1998 in Belgium
(Blot et al., 2002). The increased risk in staphylococcal bacteraemia is
mostly attributed to catherisation and patients with a high nasal carriage
(85%) of S. aureus in hospital settings. It is estimated that more than 50%
of S. aureus associated bacteraemia are acquired in the hospital after
surgical operation or resulting from constant use of contaminated
intravascularcatheters (Mylotte and Tayara, 2000).
2.3. Urinary Tract Infections (UTI) in burned patients.
Urinary tract infection (UTI) has received little attention in burn
patients (Greenfield and Manus, 1997). Thought to be a benign infection
by many, it is associated with a 2% to 4% risk of bacteremia and a case
fatality rate in non-burn patients which is 3 times as high as patients
without UTI. Risk factors specific to burn patients includes the presence of
perineal burns in certain patients and the increased length of time patients
require catheterization in the treatment of extensive injuries. In pediatric
burns, nosocomial UTI occurs almost (Ramakrishnan, 2005).
Burn patients may develop a urinary tract infection in association
with prolonged bladder catheterization. Patients will develop significant
bacteriuria after 72 h. of urinary catheter insertion, so these devices should
be removed after the initial period of fluid resuscitation and output
monitoring (Gastmeier et al., 2002).
2.4. Respiratory tract infections in burned patients
The burn patient is very prone to infection, particularly after a
smoke-inhalation injury. The hyper metabolic state produces a marked
increase in oxygen needs and carbon dioxide production.
Adult
respiratory distress syndrome (ARDS) is a severe complication of the

sepsis process, which is very difficult to reverse in the burn patient.
23
Burn patients with a combination of inhalation injury and a major body

burn have the greatest risk of pneumonia, with a rate exceeding 50%.
The high incidence is due to the presence of virulent organisms in the
intensive care environment and immune suppressed state of the burn
patient. The events occurring in the nosocomial lung infections are:
Colonization or bacterial overgrowth of the oro- and nasopharynx with
potential pathogens occurs in about 50% burned patients. Nearly 100% of
burn patients with respiratory problem have colonized their oropharynx
with pathogens (Garner and Heppell, 2005).
3. Pathogenic bacteria isolated from burned patients.
Bacteria rapidly colonize open skin wounds after burn injury.
Microorganisms colonizing the burn wound originate from the patient's
endogenous skin and gastrointestinal and respiratory flora (Barret and
Herndon, 2003). Immediately following injury, Gram-positive bacteria
from the patient's endogenous skin flora or the external environment
predominantly colonize the burn wound. Endogenous gram-negative
bacteria from the patient's gastrointestinal flora also rapidly colonize the
burn wound surface in the first few days after injury (Fuchs et al., 2002).
Different pathogens involved Gram positive and Gram negative
bacteria are isolated from burned patients. The following table of
(Agnihotri et al., 2004) indicates bacterial species causing burn wound
infection.
-Microorganisms causing invasive burn wound infection are listed as the
following (Agnihotri et al., 2004)
Group
Gram-positive
organisms
Gram-negative
organisms
Species
Staphylococcus aureus
Vancomycin-resistant Staphylococcus aureus
Methicillin-resistant S. aureus
Coagulase-negative staphylococci
Enterococcus spp.
Vancomycin-resistant enterococci
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
24
Serratia marcescens
Enterobacter spp.
Proteus spp.
Acinetobacter spp.
3.1. Staphylococcus aureus

S. aureus is a catalase positive, facultative anaerobic, Gram
positive cocci. Within the genus Staphylococcus, S. aureus is the most
significant of the pathogenic species and is easily distinguished from the
other species because it is coagulase positive; the remainder are coagulase
negative (Noble, 1997). The normal microbial habitat of S. aureus is warmblooded mammals, with approximately 20% of humans permanently
colonized with this organism, and as much as 50% of the population
transiently colonized (Tenover and Gaynes, 2000).
3.1.1. Infections caused by S. aureus
In addition to its role as a commensal organism, S. aureus can cause
many different infections, ranging from those that are relatively mild and
superficial to those that are life threatening or fatal. The particular strain of
S. aureus causing the infection may be derived from a patients own flora,
or may be community or hospital acquired. In fact, S. aureus is one of the
most common causes of both community and hospital-acquired infections
(Boyce, Projan and Novick, 1997).
S. aureus causing diseases ranging from superficial skin andwound
infections to severe illnesses such as septicaemia, endocarditis and toxic
shock syndrome. S.aureus is particularly a problem in hospitals because it
spreadseasilyintheseenvironmentsandcausespotentiallyfatalinfectionsin
immuno-compromised hospitalpatients(Liu and Chambers, 2003).
25
S. aureus infections, which can be divided into three types: (i)

superficial lesions such as wound infection, (ii) toxinoses such as food
poisoning, scalded skin syndrome and toxic shock syndrome, and (iii)
systemic
and
life-threatening
conditions
such
as
endocarditis,
osteomyelitis, pneumonia, brain abscesses, meningitis, and bacteremia

(Roth and Hughes, 2004).
a. Skin and superficial infections
S. aureus is very quick to opportunistically infect broken or
compromised skin and as such causes a wide range of superficial skin
infections. S. aureus can cause folliculitis, cellulitis, mastitis, furuncles
(boils), carbuncles and pyoderma. The organism also causes staphylococcal
scalded skin syndrome and may cause impetigo or school sores. In
several of these infections, research has shown that the bacterial strain
causing the infection is indistinguishable from that colonising, suggesting
that a patients own flora often serves as the reservoir for infection. In
addition to the conditions named above, S. aureus is also frequently found
as a colonising or infecting organism in the skin lesions associated with
ecxema or atopic dermatitis. Skin ulcers in elderly and/or diabetic patients
may also be colonised and/or infected with S. aureus, as may surgical
wounds. The treatment of S. aureus skin infections is very much dependant
on the type of infection and can vary from surgical drainage only to topical
antimicrobial agents to oral antibiotics (Noble, 1997).
b. Other infections
S. aureus is a very versatile pathogen and is able to infect virtually
every human organ system and body site (Archer, 1998). It has caused
severe life-threatening infections of the central nervous system, joints and
bone,
circulatory
system,
respiratory
tract,
urinary
tract,
and
gastrointestinal tract. In addition, S. aureus can cause endocarditis,

meningitis, brain abscesses and infections of the eyes (Ing et al., 1997).
26
c. S. aureus the principal etiological agent of burn infections.

S. aureus became the principal etiological agent of burn wound
infections shortly after the introduction of penicillin G in the early 1950s.
Although Staphylococcus aureus remains a common cause of early burn
wound infection, (Altoparlak et al., 2004).
S. aureus named also golden staph and Oro staphira. It is the most
common cause of staph infections. It is a spherical bacterium, frequently
part of the skin flora found in the nose and on skin. About 20% of the
populations
are
long-term
carriers
of
S.
aureus.http://en.wikipedia.org/wiki/Staphylococcus_aureus - cite_note-pmid92278640#cite_note-pmid9227864-0 Also S. aureus can cause a range of illnesses from
minor skin infections, such as pimples, impetigo (may also be caused by

Streptococcus pyogenes), boils (furuncles), cellulitis folliculitis, carbuncles,
scalded skin syndrome and abscesses, to life-threatening diseases such as
pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome
(TSS), bacteremia and septicemia. Its incidence is from skin, soft tissue,
respiratory, bone, joint, endovascular to wound infections. It is still one of
the five most common causes of nosocomial infections, often causing
postsurgical wound infections (Kluytmans et al., 1997).
S. aureus was well documented as a human opportunistic pathogen
and was one of the most frequently identified pathogens in clinical
laboratories. It remained among the most important nosocomial pathogens
because of the diversity and the severity of infection caused by these
organisms (Lowy, 1998).
S. aureus infections are often acute, pyogenic, and if untreated may
spread to surrounding tissues or via bacteremia to metastatic sites involve
other organs (Kloos and Banmerman, 1999).
Gram-positive bacteria that survive the thermal insult, such as
staphylococci located deep within sweat glands and hair follicles, heavily
27
colonize the wound surface within the first 48 h. unless topical

antimicrobial agents are used (Kloos, 1998).
Staphylococci have a record of developing resistance quickly and
successfully to antimicrobials. This defense response is a consequence of
the acquisition and transfer of antibiotic resistance plasmids and the
possession of intrinsic resistance mechanisms, some of which may have
evolved prior to the formation of the genus Staphylococcus. The acquired
defense systems by staphylococci may have originated from antibiotic
producing organisms, where they may have been developed and then
passed on to other genera. Presumably, the genus Staphylococcus has been
one of the recipient genera as a consequence of coming in contact with
antibiotic producing bacteria and fungi in their natural habitat (Livermore,
2000).
Amongst the staphylococcal species, coagulase-positive S. aureus
andcoagulase negative (CNS) (S.epidermidis) are of clinical importance
(S. aureus is a coloniser of the nasal passages, causing skin infections,
which range from boils, furuncles, impetigo and sties to more serious
complications such as endocarditis, scalded skin syndrome, surgicalwoundinfectionsand toxic shock syndrome (Prescott,2002).
Although
methicillin-resistant
S.aureus
(MRSA)
have
been
entrenched in hospital settings for several decades, MRSA strains have

recently emerged outside the hospital becoming known as community
associated- MRSA (CA-MRSA) or superbug strains of the organism, which
now account for the majority of staphylococcal infections seen in the clinic
(Todar, 2008).
28
3.1.2. S. aureus virulence factors

Virulence factors are the mechanismsandstrategies used by bacteria
to initiate andestablishinfections (Roth and Hughes, 2004).
One of the key factors enabling S. aureus to survive, colonize,
proliferate and cause human infections is the expression of virulence
factors (VFs). Furthermore, S. aureus produces an array of VFs, which can
be broadly grouped into those involved in bacterial attachment evasion of
host defenses. Work to date suggests that each single VF alone is not
sufficient to cause infection and that it is much more likely that several VFs
work together in concert to cause infection and disease (Projan and
Novick, 1997). It has also been shown that not every S. aureus strain
produces every VF, or does not produce each to the same degree, and it is
tempting to then assume that those strains expressing more or higher levels
of VFs are the more pathogenic (Karlsson and Arvidson, 2002).
The risk of invasive burn wound infection is influenced by the extent
and depth of the burn injury, various host factors, and the quantity and
virulence of the microbial flora colonizing the wound. Common burn
wound pathogens such as S. aureus produce a number of virulence factors
that are important in the pathogenesis of invasive infection. Virulence
factors mediate a number of processes, including adhesion, nutrient
acquisition, immune system evasion, leukocyte killing, tissue destruction,
and bloodstream invasion.
Also carries many intrinsic and acquired
antimicrobial resistance traits that make infected burn wounds difficult to

treat (Laupland et al., 2005).
S. aureus also has a diverse array of virulence factors that facilitate
adherence to host tissues, immune system evasion, and destruction of host
cells and tissues, including coagulase, protein A, leukocidins, hemolysins,
and superantigens. Resistance to methicillin in Staphylococcus aureus, and
more
recently
emergence
of
resistance
29
to
glycopeptides
and
oxazolidinones, also complicate the treatment of burn wound infections and

sepsis caused by this highly virulent organism (Meka et al., 2004).
Some examples of virulence factors produced by certain bacteria,
and the role they have in pathogenicity, 1- Fimbriae, or pili the hair-like
structures on the surface of the bacterial body. These hairs are able to
attach to certain sites of our body, and in this way the bacteria cannot be
washed away (removal of bacteria from sensitive sites is one of our
defenses). For instance, uropathogenic E. coli produces fimbria, 2-Flagella,
these are long tails with which the bacteria can swim, (Kloos and
Banmerman, 1999). 3-Toxins, some bacteria produce toxic compounds
that cause harm to their host, 4-Invasion, some bacteria have learned to
invade our cells. This is particularly important for pathogenic bacteria that
colonize the gut (Trudy, 2008).
It has been documented that there is probably no other bacterium that
produces as many cellular components, enzymes, extracellular toxins and
haemolysins as S. aureus (Todar, 2008).
Bacterial cell wall as the virulance factor
The cellwal of S. aureus is composed of a thick peptidoglycan layer,
which contributes to the virulence of the bacterium. The peptidoglycan
stimulates the production of cytokines by macrophages resulting in
complement system activation and platelet aggregation (Barret and
Herndon. 2003).
Biofilms
Biofilms are complex communities of surface-attached aggregates of
microorganisms embedded in a self-secreted extracellular polysaccharide
matrix, or slime. They are found in a wide range of natural and artificial
environments and provide their constituent microbial cells with a plethora
of protected dynamic microenvironments (Stoodley et al., 2002). Once
mature, biofilms act as efficient barriers against antimicrobial agents and
30
the host immune system, resulting in persistent colonization and/or

infection at the site of biofilm formation (Erol et al., 2004).
Although biofilms are best known for their role in foreign devicerelated infections, recent studies have confirmed the importance of biofilms
in the pathogenesis of burn wound infections. Bacteria within a biofilm
typically undergo a phenotypic change whereby microbial virulence factor
production is altered and metabolic rate and motility are reduced (Stoodley
et al., 2002).
Pigment
Some
strains
of
''S.
aureus''
are
capable
of
producing
''staphyloxanthin' a carotenoid pigment that acts as a virulence factor. It has

an antioxidant action that helps the microbe to evade killing with reactive
oxygen used by the host immune system. It is thought that staphyloxanthin
is responsible for S. aureus' characteristic golden colour. This pigment may
be key to the ability of ''S. aureus'' to survive immune system attacks.
Drugs designed to inhibit the bacterium's production of the staphyloxanthin
may weaken it and renew its susceptibility to antibiotics. In fact, because of
similarities in the pathways for biosynthesis of staphyloxanthin and human
cholesterol, a drug developed in the context of cholesterol-lowering therapy
was shown to block S. aureus pigmentation and disease progression in a
mouse infection model (Projan and Novick, 1997).
Toxin and protein
Upon entry into the host tissue, immune cells phagocytose S. aureus
cells, which promotes the production of proteolytic enzymes and toxins that
facilitate the spread to adjoining tissues and the release of the staphylococci
into the bloodstream resulting in bacteraemia (Timbury et al., 2002). The
infected endothelial cells produce tissue necrosis factoras part of the
immune response to infection, which results in necrosis and abscess
formation.
31
The pathogenicity of S. aureus is determined by the production of

toxins, such as the 33-kdprotein-alpha toxin, exfoliatin A, exfoliatin B and
Panton. These toxins can be harmful to the host and cause skin diseases
(carbuncles, boils, folliculitis and impetigo) and othercomplications, such
as endocarditis, meningitis as well as toxic shock syndrome (TSS) (File,
2008).
Infections caused by S. aureus can occur in two stages: (i) S. aureus
cells enter the body through damaged endovascular points of the host where
platelet thrombin complex have formed and attach via microbial surface
components and (ii) the bacterial cells may attach to endothelial cells via
adhesion receptor interactions or by bridging ligands, including serum
components such as fibrinogen (Gemmell and Ford, 2002).
Summary of toxins and toxic components produced by S. aureus were
listed by Timbury et al. (2002) as the following;
Toxin
Haemolysins,an
Coagulase
Fibrinolysin
Leukocidin
Hyaluronidase
DNAse
ProteinA
EpidermolytictoxinsA
andB
Enterotoxin(s)
Toxicshocksyndrome
toxin
Proteases
Activity
Cytolytic;lyse erythrocytesofvariousanimal
species
Clotsplasma, alsousedinclinicalmicrobiology
laboratoriestodifferentiatebetweenS.aureus
and CNS
Digetsfibrin
Killsleukocytes
Breaksdownhyaluronicacid
HydrolysesDNA
Lypolytic(producesopacityinegg-yolk
medium
Epidermalsplittingandexfoliation
Foodpoisoningtoxinsthatcausevomitingand
Diarrhea
Shock,rashanddesquamation
S. aureus produces several extracellular proteases, including metalloserine and cysteine proteases (Dubin, 2002). These proteases are thought to
32
be involved in evading host defenses and invading tissue (Archer, 1998).

The general function of all proteases is to cleave proteins and in doing so,
this may inactivate key proteins and antimicrobial peptides involved in host
defenses. An example of this is the serine protease of S. aureus that has
been shown to cleave IgG antibodies (Projan and Novick, 1997). Finally,
the proteases may destroy host tissue proteins, leading to generalised tissue
destruction (Projan and Novick, 1997) and at the same time the creation
of valuable nutrients for microbial growth. In addition to the functions
described above, the expression of proteases may contribute to the severity
of superficial S. aureus infections; given that S. aureus strains isolated from
patients with atopic dermatitis have been shown to produce much higher
levels of proteases than strains from healthy volunteers (Miedzobrodzki et
al., 2002).
Coagulase
Coagulase is produced by S. aureus as an extracellular protein and is
thought to have a role in cellular attachment. This is important given that
attachment is one of the first steps in the pathogenesis of infection (Archer,
1998). However, the role of coagulase as a virulence factor is still a matter
of some debate (Projan and Novick, 1997). This is based on studies with
animal models showing that coagulase-deficient mutants do not show
reduced virulence compared to coagulase competent strains. Nonetheless,
given that most S. aureus strains express coagulase, and that this protein
binds to and activates prothrombin and causes the coagulation of serum, it
would seem likely that coagulase plays an important role in disease
(Projan and Novick, Carter et al., 2003).
3.2. Pseudomonas aeruginosa
P. aeruginosa are the most common source of burn infections. They
are Gram-negative bacilli that possess a single supercoiled circular
chromosome. P. aeruginosa are facultative aerobes capable of fermentation
33
that live in the human gut. These bacteria are known for their tendency to
cause disease in immuno-compromised patients, such as those with AIDS
or cystic fibrosis, but rarely in healthy individuals P. aeruginosa use
quorum sensing to induce the production of virulence factors such as
proteases, hemolysins, exotoxin A and pyocyanin (Lyczak et al., 2002).
3.2.1. The virulence factors of P. aeruginosa
These bacteria posses' two quorum-sensing systems: one that
regulates proteases and another that regulates hemolysins. Though separate
systems, the two interact through products of the protease system
controlling
the
hemoysin
system
at
both
transcriptional
and
posttranslational levels. While it has been widely held that quorum sensing
is required for biofilm formation, new research has been put forth that
challenges that position. A team of scientists out of Auburn University
investigated the development of P. aeruginosa biofilms using a mouse burn
model (Lyczak et al., 2002). Third-degree thermal injury was induced and
mice were then infected with two strains of P. aeruginosa, one wild type
(WT) and the other quorum-sensing deficient (QS). Eschar samples were
examined using flouresence and scanning electron microscopy at 8, 24 and
48 h after infection. Both strains developed biofilms at the same pace,
although WT biofilms were somewhat denser than the QS strain. It had
been previously demonstrated that the WT strain was more virulent than
the QS. This study suggests that the difference in virulence is not due to
impaired biofilms formation; rather it is likely due to differences in the
expression of virulence factors. It seems that quorum-sensing knockout
strains lost some other genes in the process of mutation. Proteases are
virulence factors that degrade the integrity of the hosts physical barriers by
splitting proteins and amino acids, allowing for deeper infiltration of
infection (Schaber et al., 2007). Exotoxin A halts the synthesis of proteins,
causing local tissue damage, immune-suppression and cell death.
Hemolysins act like detergents to break down lipids in epithelial cells
34
allowing, like the proteases, the bacilli to penetrate the host resulting in the
spread of infection. In addition, genes encoding proteins for pili and
flagella are also important to P. aeruginosa since without these structures,
bacteria are unable to navigate their environment and form biofilms. P.
aeruginosa infections are difficult to treat because these microbes possess
multiple strategies for eluding predators, including multidrug efflux pumps,
antibiotic-modifying enzymes, and tough outer membranes with low
permeability (Vidaillac et al., 2009)
4. The antibiotics activity against pathogenic bacteria.
4.1. Definition of antibiotics:
Antibiotics are low-molecular weight microbial metabolites that at
low concentrations inhibit the growth of microorganisms. The term low
molecular weight substance refers to molecule with a defined chemical
structure having a relative mass of at most a few thousand Daltons
(Franklin and Snow, 1990).
Antibiotics may be informally defined as the sub-group of anti-infective
that are derived from bacterial sources and are used to treat bacterial
infections (Mackie and McCartney, 1980). Also, defined as a drug used
to treat infections caused by bacteria and other microorganisms (Davies,
1990). Originally, an antibiotic was a substance produced by one
microorganism that selectively inhibits the growth of another. Synthetic
antibiotics, usually chemically related to natural antibiotics, have since
been produced that accomplish comparable tasks (Bush et al., 1997).
4.2. Mode of action of antibiotics on bacterial cell:The development of antibiotics for the bacterial infections represents
one of the most remarkable achievements of last century. The most
contributing factors for developing resistance were the excessive use and/or
abuse of antibiotics (WHO, 1980).
35
The antibacterial (antibiotics) can be divided into two categories: 1those that inhibit the growth of Gram positive bacteria and are inactive
against Gram negative bacteria, and 2- those that inhibit both Gram
positive and Gram negative bacteria. These latter are often called broadspectrum antibiotics. It is very seldom that an antibiotic is found that is
active only against Gram negative bacteria (Walker, 1998).
Antibiotics are characterized by their chemical composition and
mode of action in the target organism. The main targets for action in
bacteria include the prokaryotic cell wall, the cell membrane, protein
synthesis, enzymatic pathways, and DNA replication (Brooks et al., 2001).
The microbiological and clinical factors considered in the selection
of appropriate antimicrobial therapy of anaerobic infections include: (1) the
in-vitro activity of the drug against the pathogens; (2) the ability of the
compounds to penetrate the infected site and resist inactivation; (3) the
proven efficacy of the antimicrobial agents in well controlled, randomized
prospective studies as compared to established therapy and (4) comparison
of the side effects of the drugs. The resistance of anaerobic bacteria to a
number of antimicrobial agents has an impact on the first three factors
(Giacommetti et al., 2000).
Diagram indicated the common mechanism of killing by bactericidal antibiotics with diverse targets
(Dwyer et al., 2007).
36
Mechanisms of action of antibiotics had been previously described by

Ghazal (1984). Antibiotics classified according to their mode of action into:
antibiotics inhibiting the synthesis of cell wall as pencillins and
cephalosporins; antibiotics upsetting the function of the cell membrane as
albomycins, gramicidins and candicidins; antibiotics that inhibited synthesis
of nucleic acids selectively as actinomycin, neomycin and kanamycin
inhibited synthesis of RNA and aclidione, novobiocin which inhibited
synthesis of DNA; antibiotics inhibited synthesis of purines and pyrimidines
as azaserine and decoinine; antibiotics inhibited synthesis of protein as
tetracycline, chloramphenicol, kanamycin, neomycin and streptomycin;
antibiotics inhibited respiration as actinomycins, oligocins and usnic acid;
antibiotics inhibited oxidative phosphorylation as valinomycin, gramicidins
and oligomycin and antibiotics with antimetabolic proportieas. The character
of biological action of antibiotics depended mainly on their chemical
nature, concentration, and the particular species of organism, the
microstructure of its cells, conditions under which the microorganism
functions and on other factor. This classification did not however mean that
antibiotics inhibited only the mentioned vital processes of the cell, at the
same time, each particular antibiotic classified in the above system by the
mechanism of its concentration or other conditions. There were four major
sites in the bacterial cell that were sufficiently different from the human
cell that they serve as the basis for the action of clinically effective drugs:
cell wall, ribosome, nucleic acids and cell membrane (Levinson and
Jawetz, 2000).
37
The activity and mode of action of some antibiotics were listed by CLS I.
(2008) as the following.
Antibiotic
Penicillin
Cephalosporin
Activity
Gram-positive bacteria
Broad spectrum
Site or mode of action

Wall synthesis
Wall synthesis
Polymyxin B
Gram-negative bacteria
Cell membrane
Erythromycin
Gram-positive bacteria
Protein synthesis
Neomycin
Broad spectrum
Protein synthesis
Streptomycin
Gram-negative bacteria
Protein synthesis
Tetracycline
Broad spectrum
Protein synthesis
Gentamicin
Broad spectrum
Protein synthesis
Rifamycin
Tuberculosis
Protein synthesis
An antibiotic inhibits the bacterial growth if it: penetrate bacterial

cell, interact with a structure involved in an essential function, and
substationally inhibits this function (Chopra et al., 1978).
4.3. Mode of action of vancomycin on S. aureus as inhibitor cell wall
synthesis.
Vancomycin is an antibiotic drug which is structurally classified as a
glycopeptide. It mainly acts by prevention of cell-wall biosynthesis of
bacteria. It is especially effective against gram-positive bacteria and has
been known to be effective against resistant strains of MRSA. Vancomycin
is known as the drug of last resort as it generally used when all other
treatments have failed. However, bacteria have started developing
resistance to vancomycin as well- leading to use of other antibiotics
(Reynolds, 1990).
Vancomycin acts by inhibiting cell wall synthesis of bacteria.
Peptidoglycan layer of the cell wall is rigid due to its highly cross-linked
structure (Johannes et al., 2005). During the synthesis of the peptidoglycan
layer of bacteria, new building blocks of peptidoglycan get inserted (i.e.
38
monomers of N-acetylmuramic acid and N- acetylglucosamine) into the

membrane. Reformation of the peptide cross links occurs by the enzyme
transpeptidase. Vancomycin binds to the building blocks (i.e. NAG and
NAM) of the peptidoglycan and prevents the transpeptidase from acting on
these new formed blocks and thus prevents cross-linking of the
peptidoglycan layer. By doing so, vancomycin makes the peptidoglycan
layer less rigid and more permeable. This causes cellular contents of the
bacteria to leak out and eventually death of the bacteria. Mutations in the
transpeptidase enzyme can lead to increased resistance to vancomycin
(Bugg et al., 1991)
Vancomycin binds with high affinity to the D-Ala-D-Ala C-terminus
of the pentapeptide, thus blocking the addition of late precursors by
transglycosylation to the nascent peptidoglycan chain and preventing
subsequent cross-linking by transpeptidation. Vancomycin does not
penetrate into the cytoplasm; therefore, interaction with its target can take
place only after translocation of the precursors to the outer surface of the
membrane (Melzer et al., Johannes et al., 2005).
4.4. Response of bacteria to antibiotic:
The major difference between growth and agar dilution MIC,
Probably reflect differences between end points that are often arbitrarily
defined. In broth dilution test, a single surviving colony forming unit can
grow to produce visible turbidity. Consequently, agar MICs tend to be
lower than broth dilution MICs. The growth of bacteria sensitive to some
antibiotics was sometimes not stopped at concentration exceeding
significantly the minimum bacteriostatic ones. Part of cells continues
growing, multiplying, hence number of cells and the bulk of the biomass
increase, this phenomenon is described as "residual growth" (Low et al.,
2002).
39
Response of microorganisms to antibiotic action can be measured by

using agar dilution or broth dilution susceptibility test. In agar dilution
method test, the lowest concentration of drug, which completely inhibits
the growth, disregarding a single colony or faint haze of growth, was
defined as the minimum inhibitory concentration of the drug (MIC). While
in broth dilution test, MIC was denoted as the lowest concentration of drug,
which prevented visible growth after the incubation for 18 to 24 hours
(WHO, 1978).
Depending on the nature of the antibiotic, its concentration, time of
action, the microstructure of the organism's cell and its inoculum
concentration, the environmental conditions such as temperature, pH and
other factors, the antibiotic substances may be bacteriostatic which arrest the
growth of cells (eg. erythromycin, tetracycline), bactericidal which killed cells
(eg. Penicillins cephalosporins and aminoglycosides) or certain strains of
bacteria may be tolerant (Conte and Barriere, 1981).
The range of MIC for 66 S. aureus isolates for ciprofloxacin,
ceftazidime, ofloxacin, amikacin, cefotaxime were 0.5-128, 0.5-128, 0.12564, 0.25-256 and 0.5-256 g/ ml respectively (Prashanth and Badrinath,
2004).
The MICs of cefotaxime were 0.125 mg/liter for S. aureus, 0.01 to
0.06 mg/liter for E. coli, 0.03 mg/liter for K. oxytoca, and 0.06 mg/liter for
K. pneumoniae. The MIC of cefotaxime for the E. coli strains (i.e., 0.125
mg/liter) was achieved in all cases. The lowest concentration achieved in
the cord blood (0.51 mg/liter) exceeded three times the MIC (Robert et al.,
1989). The cefotaxime cord concentration/mother concentration ratios
ranged from 5 to 172% (mean, 77%). The cefotaxime concentration in cord
blood was at least seven times higher than its MIC for E. coli (i.e., 0.4
mg/liter), and the cord concentration/mother concentration ratios ranged
from 31 to 187% (mean, 100%)(Raymond et al., 2008).
40
S. aureus was tested for antimicrobial susceptibility by disk

diffusion; a vancomycin-agar screen plate (brain heart infusion agar
containing 6 g/mL vancomycin) also was inoculated. Growth on the
vancomycin screen plate and a 12 mm zone of inhibition around the
vancomycin disk suggested that the isolate had decreased susceptibility to
vancomycin. Further testing by Etest confirmed that the isolate was
resistant to vancomycin (MIC=64 g/mL). Following notification of the
Pennsylvania Department of Health (PDH), the isolate was forwarded to
CDC, where it was confirmed to be VRSA (vancomycin MIC=32 g/mL
by broth microdilution testing) (Assadullah et al., 2003).
The minimal inhibitory concentrations of seven antimicrobial agents
were determined via agar dilution: the MICs were 64 g/ml for ampicillin,
8 g/ml for chloramphenicol, 64 g/ml for ciprofloxacin, 16 g/ml for
enrofloxacin, 128 g/ml for erythromycin, 128 g/ml for gentamicin,
and 128 g/ml for tetracycline. The proportion of S. aureus isolates
resistant to each antimicrobial agent was as follows: 28.9% for ampicillin,
2.6% for chloramphenicol, 84.2% for ciprofloxacin, 83.3% for
enrofloxacin, 46.5% for erythromycin, 20.2% for gentamicin, and 56.1%
for tetracycline (Shin and Lee, 2007).
The activity of several antibiotics against 225 clinical isolates of S.
aureus and 252 isolates of Streptococcus agalactiae. Only glycopeptides
and linezolid were active against all the isolates of S. aureus, whereas the
-lactams were also active against S. agalactia (Mataseje et al., 2009).
5. Resistance of bacteria to antimicrobial agents.
Resistance of bacteria to antibiotics was defined by Falcow (1985) as:Resistance: A bacterial strain derived from a species that is susceptible to
an antibiotic is said to be resistant when it is not inhibited the growth of
that species.
Multi resistance: When a bacterium is resistant to two or more structurally
unrelated antibiotics.
41
Cross-resistance: When a bacterial strain is resistant to an antibiotic and

also resistant to other antibiotic of the same family.
Resistance was defined by Cloete (2003) as the temporary or
permanent ability of an organism and its progeny to remain viable and/or
multiply under conditions that would destroy or inhibit other members of
the strain. Bacteria may be defined as resistant when they are not
susceptible to a concentration of antibacterial agent used in practice.
However, different bacteria react differently to bactericides, either due to
inherent differences such as unique cell envelope composition and nonsusceptible proteins, or to the development of resistance, either by
adaptation or by genetic exchange.
Organisms resistant to one antibiotic might remain sensitive to other
preparations. This holds true for antibiotics with different mechanisms of
their action (Low et al., 2002). Penicillin resistant bacteria remain sensitive
to streptomycin resistant forms were inhibited by neomycin; forms resistant
to penicillin and streptomycin were susceptible to the action of novobiocin,
etc. Also, Neu (1999) denoted that, as antibiotics act on microorganisms
sensitive to them, the latter often developed forms that were resistant to
those antibiotics. Bacteria always seen to be able to find ways of avoiding
being killed by antibiotics.
*Type of resistance and mechanisms for more common antibiotics
(Falcow, 1985).
Antibiotic
-lactam antibiotics
(penicillins,
cephalosporins)
Chloramphinicol
Erythromycin
Rifamycin
Tetracyclin
Type of
mechanism
Location of genetic
determinate
Description of
mechanism
Inactivation
Generally Extra
chromosomal
-lactamases that open

the -lactam ring
Inactivation,
Modification of
target
Modification of
target
Modification of
target
Cellular
permeability
Extrachromosomal,
Chromosomal
Acetylation by inducible
enzyme Modification of
ribosomal receptors
Alteration of protein of
ribosomal 50S subunit.
Alteration of -subunit
of RNA polymerase
Decreased effeciancy of
transport. The resistance
is partially induced
Chromosomal,
Extrachromosomal
Chromosomal
Extrachromosomal
42
5.1. Biologic Clinical Resistance

Biologic resistance refers to changes on the structure or physiology
of the organism making it less susceptible to a particular antibiotic. When
the drug is no longer effective for clinical use, the organism has achieved
clinical
resistance
(Schaaff
et
al.,
2002).
Resistance
may
be
environmentally or micro organism- mediated.

5.2. Sources of microbial resistance
Development and spread of bacterial resistance is usually attributed
to the abuse of antibiotics. The use of these agents for any infection in any
dose and over any time period forces microbes to adapt or die (selective
pressure). The microbes which survive are those that carry genes for
resistance to antimicrobial agents. However, antibiotic resistance is not a
single phesnomenon, and many resistance mechanisms have been identified
and analyzed for most clinical pathogens and almost all antibiotics for
clinical use (Satter, 1997).
5.3. Bacterial resistance in the environment
Antibiotic resistance probably has different origins in nature and
must be as ancient as antibiotic synthesis. Resistance genes pre-existed in
nature, in soil and water, and their presence was probably related to the
production of antibacterial agents, synthesized naturally in the environment
by saprophytic organisms (Schaeffer, and Stern, 2003)
5.4. Common pathways for antimicrobial resistance
Whether resistance is intrinsic or acquired bacteria share pathways to
induce resistance to antimicrobial agents. These pathways include
enzymatic degradation or modification of the antimicrobial agent,
decreased uptake or accumulation of the antimicrobial agent, altered
antimicrobial target, circumvention of the consequences of antimicrobial
43
action, uncoupling of antimicrobial agent-target interactions and

subsequent effects on bacterial metabolism or any combination of the
above mechanisms (Neu, 1999).
There are many different mechanisms by which microorganisms can
express resistance to antibiotics, and some are very well documented: 1)
they may produce enzymes that destroy the active drug, 2) they can change
membrane permeability to block entry or express transport systems that
actively pump the drug out into the extracellular environment, 3) they
express altered structural targets, 4) they express metabolic pathways that
bypass the reaction inhibited by the drug, or 5) they express altered
enzymes that are less affected by the drug (Salyers and Whitt, 2001).
5.5. The biochemical bases of resistance mechanism
Transformation of the antibiotic into an inactive form, modification
of the cell s target site for the antibiotics, modification of the permeability
of the bacterium to the antibiotic, and increased the production of the
structure inhibited by antibiotic (Falcow, 1985).
There are many different mechanisms by which microorganisms can
express resistance to antibiotics, and some are very well documented: 1)
they may produce enzymes that destroy the active drug, 2) they can change
membrane permeability to block entry or express transport systems that
actively pump the drug out into the extracellular environment, 3) they
express altered structural targets, 4) they express metabolic pathways that
bypass the reaction inhibited by the drug, or 5) they express altered
enzymes that are less affected by the drug (Salyers and Whitt, 2001).
The origin of drug resistance may be genetic or non-genetic.
Microorganisms that are metabolically inactive (non-multiplying or
sporulated) may be phenotypically resistant to drugs. Also, microorganisms
may lose the specific structural drug target for several generations, and thus
become temporarily resistant. These non-genetic mechanisms of drug
44
resistance, though important in many circumstances, are temporary

reversible phenotypical states. However, drug resistance that emerges as a
result of genetic change and subsequent selection by antimicrobial drugs,
and thus may become permanent in the bacterium and be passed onto its
next generations, comprises one of the greatest problems in human and
veterinary medical care (Brooks et al., 2001).
A-Resistant by exopolysacchrides production:The growth of bacteria was often accompanied by the production of
polysaccharides which was found outside the cell wall. These
exopolysaccharides might be found as a capsule attached to the bacteria or
they might be released to the environment as slime or both (Cloete, 2003).
In natural environment, bacteria can often be found as sessile
communities, usually referred to as biofilms. Most biofilms produced an
extracellular matrix (extracellular polymeric structure or EPS), composed of
both polysaccharides and proteins, which could constitute a relevant part of
their total dry weight. This extensive EPS production takes place even if
biofilms were growing in oligotrophic environments, despite the high energy
consumption required by EPS biosynthesis, suggesting that growth of the EPS
matrix confers important advantages on the microorganisms, EPS production
can also protect bacteria against predation by bacteriophages which can be
present up to 10:1 ratio to bacteria in natural environments such as seawater.
Finally, the EPS was likely to contribute, together with specific gene
expression and lower growth rates, to biofilm resistance to a number of
environmental stresses, such as treatment with biocides (Davies, 1990).
Exopolysaccharides play an important role in microbial resistance to
antimicrobials. Exopolysaccharide matrix produced by antimicrobial
biofilm (Biofilms are dense bacterial communities attached to a solid
surface and surrounded by an exopolysaccharide matrix) acts as an
effective barrier that restricts penetration of chemically reactive biocides,
45
cationic antibiotics and antimicrobial peptides. The reduced uptake of

antimicrobial
agents
and
biocides
caused
by
the
biofilm
exopolysaccharides may lead to development of resistant against such

agents. It should be noted that some antimicrobial compounds at sub-lethal
concentrations actually promote exopolysaccharides synthesis. Bacterial
growth was inhibited and considerably more exopolysaccharide was
formed than in the absence of the agents (Mylotte andTayara,2000)
In general, Gram-negative bacteria were less sensitive than Grampositive bacteria to antibiotics and synthetic antibacterial compounds
Resistance of Gram-negative bacteria and sensitivity of Gram-positive
bacteria to antibiotics were studied by several investigators and they
referred the difference in bacterial response to antibiotics due to difference
in the structure of the outer envelope of Gram-positive and Gram-negative
bacteria (Murray and Hospenthal, 2008).
Biofilms gain resistance to various antimicrobial agents, and the
presence of antibiotic resistance genes is thought to contribute to a biofilmmediated antibiotic resistance. The interplay showed between the
tetracycline resistance efflux pump TetA(C) and the ampicillin resistance
gene (blaTEM-1) in biofilms of Escherichia coli harboring pBR322 in the
presence of the mixture of ampicillin and tetracycline (May et al., 2009).
B- Resistant by outer membrane.
Mutations that result in decreased outer membrane permeability have
been observed in K. Pneumoniae, E. aerogenes, E. cloacae and S. marscens
(Cloete, 2003).
Bacterial resistance to the antibiotics may be attributed to decreased
permeability of the antibiotic or disruption of bacterial envelope or the
outer layer in Gram-negative cells or to increased lipid biosynthesis in the
bacterial cells. The resistant strain of S. aureus contained higher amount of
lipid and more DPG than the sensitive strain (Meka et al., 2004).
46
C- Resistant by drug efflux systems.

In Gram- negative bacteria, resistance is usually due to active efflux
systems of the drug, or protection of the ribosome encoded by acquired
plasmid. Sometimes chromosomal mutations affect the outer membrane
permeability (Murray and Hospenthal, 2008).
Drug efflux is a major mechanism of multiple drug resistance in
bacteria. Generally, this is accomplished by efflux systems, responsible for
unidirectional pumping of cytotoxic drugs to the extracellular environment.
Although these efflux systems are usually chromosomally encoded, some
may be present on plasmids. In addition to antibiotics, these pumps may
export dyes, detergents, disinfectants, inhibitors, organic solvents, and
lactones (Fange et al., 2009).
Diagram illustrating drug flows over the cell envelope, drug inactivation and target binding.
Drugs enter the cell by passive transport (rate constant k passive) and exit by passive transport
(rate constant k passive), active pumping (rate constant k active), drug degradation in the
cytoplasm (rate constant k deg.) (Fange et al., 2009).
D- Genetic resistance:
Resistance to antimicrobial agents may be mediated by agents that
are encoded on the host cell chromosomes, plasmids or transpospons,
which are capable of integrating into the plasmid and/or into the
chromosome. Bacteria may acquire exogenous genetic material that leads to
47
antimicrobial resistance (Davies, 1997). Plasmid typing or plasmid finger

printing is a relatively new addition to the typing systems used in clinical
microbiology. Plasmid DNA content is a unique and relatively stable
characteristic of most pathogenic bacteria, and as such, it has gained
importance as an epidemiologic tool (Finks et al., 2009).
E- Resistant by enzyme production
Beta-lactamases,
penicillinases
and
cephalosporinases
were
chromosomal or plasmid mediated present in both Gram-negative and

Gram-positive bacteria. The resistance of the population depended on the
production of beta-lactamase by the mass of bacteria. In Gram-negative
beta-lactamase were produced in very much smaller amount than in Grampositive and they were cell bound existing in the periplasmic space and
they are not released into the surrounding media (Ambler, 1999).
5.6. Resistant to different antibiotics.
Antibiotic resistance is a problem that continues to challenge the
healthcare sector. In particular, multidrug resistance is now common in
familiar pathogens such as S. aureus and Mycobacterium tuberculosis, as
well as emerging pathogens such as Acinetobacter baumannii. New
antibiotics and new therapeutic strategies are needed to address this
challenge. Advances in identifying new sources of antibiotic natural
products and expanding antibiotic chemical diversity are providing
chemical leads for new drugs. Inhibitors of resistance mechanisms and
microbial virulence are orthogonal strategies that are also generating new
chemicals that can extend the life of existing antibiotics. This new
chemistry, coupled with a growing understanding of the mechanisms,
origins and distribution of antibiotic resistance, position us to tackle the
challenges of antibiotic resistance in the 21st century (Murphy et al.,
2003).
48
In the early 1940s, the introduction of benzylpenicillin (penicillin G)

temporarily solved the problem of staphylococcal infections, but the
continued use of this agent caused the selection of resistant strains, which
produced
penicillinase
(beta-lactamase),
similarly,
the
successive
introduction of streptomycin, tetracycline, chloramphenicol, and the

macrolides, was faced by the emergence of resistant organisms with a wide
spectrum of resistance. The introduction of semisynthetic -lactamase
resistant penicillins such as methicillin and oxacillin brought about a
general decline in the pgeyalence of multiresistant S.aureus during the
early 1960s (Boyce et al., 1993).
The pathogens that infect the wound are primarily Gram-positive
bacteria such as (MRSA), (VRSA) and Gram-negative bacteria such as
Acinetobacter
baumannii-calcoaceticus
complex,
Pseudomonas
aeruginosa, and Klebsiella species. These latter pathogens are notable for
their increasing resistance to a broad array of different antimicrobial agents.
Emerging antimicrobial resistance trends in burn wound bacterial
pathogens represent a serious therapeutic challenge for clinicians caring for
burned patients (Gerard and Arlene, 2007).
5.7. Historical aspects of antibiotic resistant staphylococci.
The appearance of antibiotic resistant staphylococci over the past
years has been regarded as an inevitable genetic response to the selective
pressure imposed by antimicrobial therapy; it also illustrates the ability of
microbial population to readily adapt to changes in their environment
(Saunder et al., 1984).
5.7.1. Resistance to -lactam and glycopeptides antibiotics
Resistance to -lactam antibiotics in staphylococci can be mediated
by three different mechanisms; -lactamase mediated resistance, intrinsic
49
resistance, and tolerance to the killing action of -lactam antibiotics

(Ambler, 1999).
Glycopeptides and -lactams are the major antibiotics available for the
treatment of infections due to Gram-positive bacteria. Emergence of crossresistance to these drugs by a single mechanism has been considered as
unlikely because they inhibit peptidoglycan polymerization by different
mechanisms (Aqil et al., 2005).
5.8. Methicillin and vancomycin resistance mechanisms in S. aureus.
Methicillin-resistant coagulase-negative staphylococci, vancomycinresistant enterococci, and multiply resistant Gram-negative bacteria that
possess several types of beta-lactamases, including extended-spectrum
beta-lactamases, ampC beta-lactamases, and metallo-beta-lactamases, have
been emerging as serious pathogens in hospitalized patients (Boyce, et al.,
1993).
Antibiotic-resistant
organisms
such
as
MRSA,
(VRSA),
vancomycin-resistant enterococci, and multiply-resistant Gram-negative

rods, including P. aeruginosa and Acinetobacter spp., have been associated
with infections of the burn wound and other anatomic sites in patients with
major thermal injury, occasionally in the form of nosocomial outbreaks.
Risk factors for acquisition of an antibiotic-resistant organism include
receipt of antibiotics prior to the development of infection, extended
duration of hospitalization, previous hospitalization, invasive procedures,
comatose state, and advancing age (Todar, 2008).
S. aureus (colloquially known as "Staph. aureus" or a Staph
infection) is one of the major resistant pathogens. Found on the mucous
membranes and the human skin of around a third of the population, it is
extremely adaptable to antibiotic pressure. It was one of the earlier bacteria
in which penicillin resistance was foundin 1947, just four years after the
drug started being mass-produced. Methicillin was then the antibiotic of
50
choice, but has since been replaced by oxicillin due to significant kidney
toxicity. MRSA (methicillin-resistant S. aureus) was first detected in
Britain in 1961 and is now "quite common" in hospitals. MRSA was
responsible for 37% of fatal cases of sepsis in the UK in 1999, up from 4%
in 1991. Half of all S. aureus infections in the US are resistant to penicillin,
methicillin, tetracycline and erythromycin (Todar, 2008).
Community-acquired MRSA (CA-MRSA) has now emerged as an
epidemic that is responsible for rapidly progressive, fatal diseases including
necrotizing pneumonia, severe sepsis and necrotizing fasciitis. MRSA is
the most frequently identified antimicrobial drug-resistant pathogen in US
hospitals. The epidemiology of infections caused by MRSA is rapidly
changing. In the past 10 years, infections caused by this organism have
emerged in the community. The 2 MRSA clones in the United States most
closely associated with community outbreaks, USA400 (MW2 strain, ST1
lineage) and USA300, often contain Panton-Valentine leukocidin (PVL)
genes and, more frequently, have been associated with skin and soft tissue
infections. Outbreaks of CA-MRSA infections have been reported in
correctional facilities, among athletic teams, among military recruits, in
newborn nurseries, and among men who have sex with men. CA-MRSA
infections now appear to be endemic in many urban regions and cause most
CA-S. aureus infections. (Clark et al., 2005).
A-Mechanism of resistance methicillin in S. aureus
Methicillin resistance is defined as the strains of S. aureus that are
resistant to the isoxazoyl penicillins such as methicillin, oxacillin and
flucloxacillin. MRSA are cross-resistant to all currently licensed betalactam antibiotics. The expression of methicillin resistance by S. aureus
strains is by virtue of acquired penicillin binding proteinPBP2a, encoded by
mec A gene.
Structurally, PBP2a possesses both transglycosylase and
transpeptidase. PBP2a confer resistance to all beta-lactam antibiotics. The

51
origin of mec A gene is unknown. Expression of methicillin resistance in S.

aureus is commonly under regulatory control by mec I or by Bla I gene.
The mec I and bla I repressors are controlled by the mec RI and bla RI
transducers. Expression of methicillin resistance in S. aureus is also
influenced by the expression of other genetic loci called fem ("factors
essential for methicillin resistance") or aux ("auxiliary") genes. Many fem
and aux factors have now been identified, which are involved in formation
of the staphylococcal cell wall(Teruyo et al., 2001).
The mec A gene is located within a larger region of chromosome
known as the staphylococcal cassette chromosome mec (SCCmec) region
(21-67 kb). SCCmec is a mobile element, with mobility conferred by the
presence of the ccrA and ccrB genes. The basic elements of SCCmec are
the mecRI-mecI-pbp2a region and ccrA. Nosocomial isolates have larger
SCCmec, owing to the accumulation over time of integrated plasmids or
transposons that contribute to the multi-drug resistance(CLSI, 2008).
The mecAgene present in MRSA strains encodes the altered protein
(PBP2a),whichisnot inactivatedbymethicillin(Gazeetal.,2008).The
mecA gene resides on the staphylococcal cassette chromosome mec
(SCCmec)andisexpressedbytheregulatorgenes mecR1andmecI(Lowy,
1998).TheregulatorgenemecR1 isactivatedby beta - lactamantibiotics
and serves as a signal transducer that inactivates the mecI repressor gene
product. SomeSCCmectypescontain geneticelementsforotherantibiotic
resistance, such as Tn554, a transposon responsible for resistance to
macrolides, clindamycin and streptogramin B, while theT181 plasmid
accounts for resistance to tetracyclines .There are five different types of
SCCmecwithvaryingsizes,includingSCCmectypeI,II,III,IVandVwith
sizes34,53,67,2124and28kbrespectively.These five types(I-V) have
been used to classify and distinguish between HA-MRSA and CA-MRSA
strains.The mecgenecomplexesarestructuredasfollows:classA,IS431-
52
mecA-mecR1-mecI; classB, IS431-mecA-mecR1-IS1271; class C, IS431mecA-mecR1 IS431andclassD,IS431-mecA-mecR1 (Itoetal.,2004)

MRSA strains that have acquired the vanA operon from
glycopeptide-resistant enterococci are designated VRSA. The first two
VRSA isolates were recovered in the United States in 2002(Zhu, 2008).
Since then, an additional seven MRSA isolates carrying the vanA gene
cluster have been detected in the United States (one in New York and six in
Michigan), and two tentative identifications of VRSA have been reported
in India and Iran (Kennedy, 2010).
In last decade MRSA with high level of VRSA have been reported
and generally the patients with VRSA infection were also infected with a
vancomycin resistant Enterococcus (VRE). Considering that the high level
of vancomycin-resistance in VRSA isolates seems to involve the horizontal
transfer of Tn1546 transposon containing vanA gene from coinfecting VRE
strains, the authors have studied the in vitro conjugative transfer of this
resistance from vanA enterococci to S. aureus. Staphylococcus aureus, a
major cause of community and hospital-acquired infections, has developed
resistance to many antimicrobial agents and often glycopeptides such as
vancomycin remained one of the most effective drugs to treat MRSA
infections. Unfortunately, in the last decade, MRSA strains with reduced
susceptibility to vancomycin have been reported with increasing frequency
and the appearance of this resistance can make treatment of S. aureus
infections increasingly difficult (Zhu, 2008).
B- Mechanism of resistance to Vancomycin
S. aureus is one of the most common causes of hospital- and
community-acquired infections, and treatment of staphylococcal infections
is complicated by the ability of this bacterial species to become resistant to
antibiotics. Vancomycin is the drug of choice for therapy of infections due
to methicillin (meticillin)-resistant S. aureus (MRSA), but increase in
53
vancomycin use has led to the emergence of two types of glycopeptideresistant S. aureus. The first one, designated vancomycin intermediateresistant S. aureus, is associated with a thickened and poorly cross-linked
cell wall, resulting in accumulation of acyl-D-alanyl-D-alanine (X-D-Ala-DAla) targets in the periphery that sequester glycopeptides. The second type,
vancomycin-resistant S. aureus (VRSA), is due to acquisition from
Enterococcus spp. of the vanA operon, carried by transposon Tn1546,
resulting in high-level resistance (Murphy et al., 2003).
The resistance of Staphyococi to vancomcin has been found to be
reversible under laboratory conditions. (Rybak and Akins, 2001). One
study indicated that vancomycin resistance in S. aureus may occur under
the selective pressure of prolonged vancomycin use and that the resistance
to vancomycin in S. aureus is reversible on removal of the drug .
Moreover, mechanism for vancomycin resistance in these bacteria (Walsh
and Howe, 2002).
Actually, thickening of the cell wall of vancomycin- resistant
staphylococci has been found to be associated with complex reorganization of cell wall metabolism with extra cell wall material showing
reduced peptidoglycan cross - linking of D-Ala-D-Ala termini of side
chains (Zaragoza et al., 2003).
Vancomycin act as the only effective agent available at the time.
However, strains with intermediate (4-8 g/ml) levels of resistance, termed
GISA (glycopeptide intermediate S. aureus) or VISA, began appearing in
the late 1990s. The first identified case was in Japan in 1996, and strains
have since been found in hospitals in England, France and the US. The first
documented strain with complete (>16 g/ml) resistance to vancomycin,
termed VRSA appeared in the United States in 2002 (Cui et al., 2009).
The transfer of van resistance genes from Enterococcus species to S.
aureus, which results in high levels of resistance to vancomycin, was
54
obtained in vitro and in an animal model. Most important, this transfer also
occurs in vivo. Three MRSA) isolates with high or moderate levels of
resistance to vancomycin and teicoplanin have been isolated from patients
in Michigan (MI-VRSA), Pennsylvania (PA-VRSA), and New York after
acquisition of the van A gene cluster (Kacica and McDonald 2004).
Analysis of the nucleotide sequences flanking Tn1546 indicated that
the transposon was flanked by 5-bp duplications of target DNA typical of
the Tn3 family of elements to which Tn1546 belongs. This observation
confirms that Tn1546 has transposed into the plasmid of MI-MRSA.
Comparative analysis of peptidoglycan precursors and of D,D-dipeptidase
(vanX) and D,D-carboxypeptidase (vanY) activities indicated high similar
levels of expression of the vanA gene clusters in the MI-VRSA and PAVRSA strains. Thus, the difference in glycopeptide resistance between the
2 isolates is not due to a difference in van gene expression (Liu and
Chambers, 2003).
Mechanism of vancomycin resistance in VISA strains due to
vancomycin binds with the D-alanyl-D-alamine C terminus of the bacterial
cell precursors, thereby preventing cross-linking by transpeptidation
resulting in inhibition of cell wall production by attacking sites responsible
for cell wall production. VISA and hetero-VISA strains have been found to
have thickened cell wall with reduced glycoprotein. This could be due to
changes in peptidoglycan synthesis resulting in increased residues of D
alanyl-D-alanine, which bind vancomycin molecules and prevent them
from reaching the target sites(Zhu, 2008).
Mechanism of vancomycin resistance in VRSA strains also have
been found to have thicker cell walls than the sensitive strains. As with
VISA strains, there is also increased peptidoglycan synthesis. It has been
shown that vancomycin is only trapped in the outer layers and sequestered
by the bacteria and not deactivated. Exchange of genetic material is yet
55
another mechanism postulated for VRSA. It has been suggested that

patients at risk for VRSA are co-infected or co-colonized with VRE and
MRSA, which enables transfer of vanA gene from VRE to MRSA in a
biofilm environment leading to a VRSA strain. In a case, it was reported
that the patient had resistant E. fecalis in the wound, which caused the
conjugative transfer of vanA geneFinks et al., 2009)
Vancomycin acts by binding to the C-terminal acyl-D-alanyl-Dalanine (acyl-D-Ala-D-Ala) of pentapeptide peptidoglycan precursors and
inhibits transglycosylation and transpeptidation reactions, thus preventing
cell wall formation and with the acyl-D-Ala-D-Ala residues being
incorporated into peptidoglycan precursors as dipeptides synthesized by the
host D-Ala:D-Ala ligase (Ddl) (Kennedy, 2010). VanA-type resistance is
characterized by high-level inducible resistance to vancomycin and
teicoplanin due to synthesis of peptidoglycan pentadepsipeptide precursors
ending in D-Ala-D-lactate (D-Ala-D-Lac). This alteration is responsible for
the diminished binding affinity of glycopeptides for their target. VanA-type
resistance is mediated by a gene cluster often carried by Tn1546 which
composed of nine proteins five of which are necessary for the expression of
resistance (vanA, vanR, vanS, vanH and vanX) (Cui et al., 2009). Because
vancomycin does not interact with cell wall biosynthetic enzymes but
forms complexes with peptidoglycan precursors, its activity is not
determined by the affinity for a target enzyme but by the substrate
specificity of the enzymes that determine the structure of peptidoglycan
precursors. Resistance to vancomycin is due to the presence of operons that
encode enzymes (1) for synthesis of low-affinity precursors, in which the
C-terminal D-Ala residue is replaced by D-lactate (D-Lac) or D-serine (DSer), thus modifying the vancomyin-binding target; and (2) for elimination
of the high-affinity precursors that are normally produced by the host, thus
removing the vancomycin-binding target (Zhu, 2008).
56
SchematicrepresentationofthemechanismsofS.aureusintermediateresistanceto vancomycin.The
vancomycin-intermediate S. aureus strains synthesise additional quantities of peptidoglycan with
increased numbers of D-Ala-D-Ala residues thatbind vancomycin, thus preventing the molecule to
bindtoitsbacterialtarget(cellwall) (Zhu, 2008).
Van A-type resistance, which is characterized by inducible high

levels of resistance to vancomycin, was the first type of resistance
described and is mediated by transposon Tn1546 and elements closely
related to it. The transposon encodes a dehydrogenase (VanH), which
reduces pyruvate to D-Lac, and the van A ligase, which catalyzes the
formation of an ester bond between D-Ala and D-Lac. The resulting D-AlaD-Lac depsipeptide replaces the D-Ala-D-Ala dipeptide in peptidoglycan
synthesis, a substitution that decreases the affinity of the molecule for
glycopeptides considerably (CDC, 2002).
Six types of vancomycin resistance have been characterized on both
a phenotypic and a genotypic basis in enterococci and Staphylococci. Five
of these types (van A, B, D, E, and G) correspond to acquired resistance.
Classification of glycopeptide resistance is currently based on the primary
sequence of the structural genes for the resistance ligases rather than on the
levels of resistance to glycopeptides, because the MIC ranges of
vancomycin and teicoplanin against the various types overlap. vanA-type
strains display high levels of inducible resistance to both vancomycin and
57
teicoplanin, whereas van B-type strains have variable levels of inducible

resistance to vancomycin only. Van D-type strains are characterized by
constitutive resistance to moderate levels of the 2 glycopeptides. Van C,
van E, and van G type strains are resistant to low levels of vancomycin but
remain susceptible to teicoplanin (Reynolds et al., 2005).
VanA. van A is the most frequently encountered type of glycopeptide
resistance in enterococci and, to date is the only one detected in S. aureus.
The prototype Tn1546 vanA-type resistance element, which was originally
detected on a plasmid in E. faecium clinical isolate, is an 11-kb transposon.
It encodes 9 polypeptides that can be assigned to various functional groups:
transposition (ORF1 and ORF2), regulation of resistance gene expression
(vanR and vanS), synthesis of the D-Ala-D-Lac dipeptide (vanH and
vanA), and hydrolysis of peptidoglycan precursors (vanX and vanY); the
function of vanZ remains unknown. The van R and vanS proteins are part
of a 2-component regulatory system that modulates transcription of the
resistance gene cluster (Reynolds et al., 2005). This system is composed of
a cytoplasmic van R response regulator, which acts as a transcriptional
activator, and a membrane-bound van S histidine kinase (Depardieu et al.,
2006).
VanB. As in VanA-type staphylococci, acquired van B-type resistance is
due to synthesis of peptidoglycan precursors ending in the dipeptide D-AlaD-Lac instead of the dipeptide D-Ala-D-Ala (Reynolds et al., 2005).
The organization and functionality of the vanB cluster is similar to that of
vanA but differs in its regulation, because vancomycin, but not teicoplanin,
is an inducer of the vanB cluster. There is no correlation between the vanB
subtype and the level of resistance to vancomycin (Depardieu et al., 2006).
VanD. Acquired vanD-type resistance is due to constitutive production of
peptidoglycan precursors ending in D-Ala-D-Lac. The organization of the
van D operon, which is located exclusively in the chromosome in strains
58
that have been studied, is similar to that of van A and van B. Van D-type
strains share other characteristics that distinguish them from van A- and
van B-type enterococci (Reynolds et al., 2005).
VanC. The van C phenotype is expressed constitutively or inducibly as a
result of the production of peptidoglycan precursors ending in D-Ser. Three
vanC genes encoding D-Ala:D-Ser ligases have been described: vanC-1 in
E. gallinarum, vanC-2 in E. casseliflavus, and vanC-3in E. flavescens
(Abadia et al., 2002).
VanE. The vanE phenotype corresponds to low-level resistance to
vancomycin and susceptibility to teicoplanin due to synthesis of
peptidoglycan precursors terminating in D-Ala-D-Ser as in intrinsically
resistant Enterococcus species. The vanE cluster has an organization
identical to that of the vanC operon (Abadia et al., 2002).
VanG. Acquired vanG type is characterized by resistance to low levels of
vancomycin (MIC, 16 g/mL) but susceptibility to teicoplanin (MIC, 0.5
g/mL) and by inducible synthesis of peptidoglycan precursors ending in
D-Ala-D-Ser. The chromosomal vanG cluster is composed of 7 genes
recruited from various van operons (Reynolds et al., 2005).
6. Antimicrobial resistance in P. aeruginosa.
P. aeruginosa remains a serious cause of infection and septic
mortality in burn patients, particularly when nosocomially acquired. A
prototypic burn patient who developed serious nosocomially acquired
Pseudomonas infection is described as an index case which initiated
investigations and measures taken to identify the source of the infection.
The effect of changes in wound care to avoid further nosocomial infections
was measured to provide data on outcome and cost of care. The
bacteriology of Pseudomonas is reviewed to increase the burn care
providers understanding of the behaviour of this very common and serious
pathogen in the burn care setting, before reviewing the approach to
59
detection of the organism and treatment both medically and surgically.

After controlling the nosocomial spread of Pseudomonas in our burn unit,
we investigated the morbidity and mortality associated with nosocomial
infection with an aminoglycoside resistant Pseudomonas and the associated
costs compared to a group of case-matched control patients with similar
severity of burn injury that did not acquire resistant Pseudomonas during
hospitalization (Kohler et al., 1999).
7. Antibiotic resistance in Enterobacteriacae
Extensive antibiotic resistance has developed in Gram-negative
bacteria due to both innate resistance in some species and the fact that they
are highly adept at acquiring antibiotic-resistant determinants from each
other (Waterer and Wunderink, 2001).
A survey in the united states revealed 30-50% resistance of clinical
isolates of E. coli to older -lactams. Most of this resistance is due to
plasmid or transpose-encoded -lactamases which are typically active
against ampicillin and other penicillins, cephalosporins, and to a lesser
extent cetamandole. Third generation cephalosporins, cephamyclins and
irnipeneni are not hydrolyzed by these enzymes which are found in a
variety of Enterobacteriacae (McGrown et al., 1989).
It was noted that some strains of E. coil and K. pneumoniae acquired
plasmid-mediated resistance to third generation cephalosporin that can
spread from species to species and are usually blocked by lactamase
inhibitors.
Following
the
over
use
of
the
expanded
spectrum
cephalosporins, sever outbreaks caused by expanded spectrum Lactamases (ESBL) producing E. coli and K. pneumoniae have been
reported (Kohler et al., 1999).
The most disturbing feature of P. aeruginosa is its broad resistance
to antibiotics. This resistance may also be plasmid mediated. There are at
least thirteen group of plasmids based on compatibility. They carry markers
60
conferring resistance to various antibiotics as well chemicals like mercury,

borate and tellurite (Gransden, 1997).
Of all available -lactams, the most potent against ESBL-producing
Enterobacteriacae are carbapenems (imipenem and meropenem) MK- 0826
is a novel carbapenem with clear pharmacokinetic as vantages over
currently available carbapenems and with excellent antimicrobial activity
against multiple drug resistant isolates of E. coli and Klebsiella spp.
(Kohler et al., 1999).
Resistance of Enterobacteriacae to -lactams is mediated mainly by
penicillin-interactive, active site serine proteins which include -lactamases
and penicillin binding proteins (PBPs), and secondly by changes in the
outer membrane proteins decreasing the intracellular uptake of the drug
(Mandomando et al., 2009).
8. Bactericidal action of different antimicrobial agent
Antiseptics are chemical agents that slow or stop the growth of
micro-organisms (germs) on external surfaces of the body and help prevent
infections. Antiseptics should be distinguished from antibiotics that destroy
micro-organisms inside the body, and from disinfectants, which destroy
micro-organisms found on inanimate (non-living) objects. However,
antiseptics are often referred to as skin disinfectants. Most chemical agents
can be used as both an antiseptic and a disinfectant. The purpose for which
it is used is determined by its concentration. For example hydrogen
peroxide 7% solution is used for cleansing wounds, while stronger
solutions (>30%) are used in industry as a bleach and oxidizing agent
(Kuyyakanond & Quesnel, 1992).
Many disinfectants are used alone or in combinations in the healthcare setting. These include alcohols, chlorine and chlorine compounds,
hydrogen peroxide, iodophors, peracetic acid, phenolics, and quaternary
ammonium compounds. All chemicals used to disinfect can be harmful and
61
need to be used with great care. Some chemicals commonly used to

disinfect include hydrogen peroxide (7%), chlorine bleach, ethanol (70%),
and isopropyl alcohol (70% to 90%). Many common cleaning and
disinfecting products contain glutaraldehyde or formaldehyde. Regular
exposure to glutaraldehyde and formaldehyde can cause cancer and death.
These chemicals should not be used (Marian, 2004).
Antiseptics are medicines that slow or stop the growth of germs and
help prevent infections in minor cuts, scrapes, and burns. Antiseptics are
applied to the skin to keep bacteria from getting into wound sand causing
infection. Although antiseptics do not usually kill bacteria, they do weaken
them and slow their growth (NCC LS, 2000).
Simply applying an antiseptic to a wound of burns is not adequate
treatment. The wound should be cleaned first, and in most cases it should
be covered with a bandage or other type of dressing to keep it clean and
moist while it heals. However, some antiseptics, such as phenol, can
damage the skin if the wound is covered after they are applied. Others, such
as hydrogen peroxide and iodine, should be allowed to dry completely
before the wound is covered. Because antiseptics can irritate the skin and
even interfere with the healing process, they should be used sparingly.
Some medical experts advise people to use antibiotic ointments instead of
antiseptics because they can actually kill the bacteria that may cause a
wound to become infected. Rule of thumb: if hydrogen peroxide or another
antiseptic is the only thing available to use at the time of injury, use it. If an
antibiotic ointment or cream is available, use one of them instead (Klotz et
al., 2003).
Some commonly used antiseptics are isopropyl alcohol, hydrogen
peroxide, iodine, methyl salicylate, and thymol. Most of the antiseptic
products on the market contain one or more of these ingredients (Marian,
2004).
62
Medical wipes clean the skin before a medical procedure or in the

treatment of minor burns. There are various ingredients which may be used
as disinfectant for these purposes, including ethanol alcohol, iodine, or
hydrogen peroxide (Ali et al., 2001).
Use mild soap and boiled, cooled water on sterile cotton or gauze, or
disinfected cloth, to gently clean the burn. You can also use hydrogen
peroxide or ethanol. Remove any remaining burned skin on and around the
burn. Cover this fresh skin with a piece of sterile gauze or disinfected cloth.
If the cloth sticks to the burn when you want to remove it, wet it with water
that has been boiled and cooled. To prevent infection in the burn area,
apply a sterile gauze or disinfected cloth that has been soaked in hydrogen
peroxide or ethanol or a salt water solution for 15 minutes, 3 times a day.
Each time you change the cloth, remove the dead skin and flesh carefully
with very clean tweezers, until you see fresh pink skin (Mayaudon & ELZayat, 2005).
9.1. Hydrogen Peroxide.
Overview.
The literature contains several accounts of the properties, germicidal
effectiveness, and potential uses for stabilized hydrogen peroxide in the
health-care setting. Published reports ascribe good germicidal activity to
hydrogen peroxide and attest to its bactericidal, virucidal, sporicidal, and
fungicidal properties. The FDA website lists cleared liquid chemical
sterility and high-level disinfectants containing hydrogen peroxide and
their cleared contact conditions (Kuyyakanond & Quesnel, 1992).
When hydrogen peroxide is applied to a wound, it fizzes as oxygen is
released. This fizzing action helps loosen and remove dead tissue and is not
harmful. After the fizzing stops, the edges of the wound may be white.
When treating children or others who might be frightened by these effects,
63
explain what is going to happen and assure them that the fizzing will not be
painful (Mayaudon & EL-Zayat, 2005).
Hydrogen peroxide solutions that contain more than 20% hydrogen
peroxide can damage the skin and mucus membranes and can even lead to
infection, rather than preventing it. Solutions this strong should be used
only occasionally and in small amounts, if at all. Care should also be taken
if hydrogen peroxide is used in the mouth, as a mouthwash or a gargle. Spit
out the hydrogen peroxide after gargling (Mertens et al., 2000).
Mode of Action.
Hydrogen peroxide works by producing destructive hydroxyl free
radicals that can attack membrane lipids, DNA, and other essential cell
components. Catalase, produced by aerobic organisms and facultative
anaerobes that possess cytochrome systems, can protect cells from
metabolically produced hydrogen peroxide by degrading hydrogen
peroxide to water and oxygen. This defense is overwhelmed by the
concentrations used for disinfection (Mertens et al., 2000).
Microbicidal activity.
Hydrogen peroxide is active against a wide range of microorganisms,
including bacteria, yeasts, fungi, viruses, and spores. A 0.5% accelerated
hydrogen peroxide demonstrated bactericidal and virucidal activity in 1
minute and mycobactericidal and fungicidal activity in 5 minutes.
Bactericidal effectiveness and stability of hydrogen peroxide in urine has
been demonstrated against a variety of health-careassociated pathogens;
organisms with high cellular catalase activity (e.g., S. aureus, S.
marcescens, and Proteus mirabilis) required 3060 minutes of exposure to
0.6% hydrogen peroxide for a 108 reduction in cell counts, whereas
organisms with lower catalase activity (e.g., E. coli, Streptococcus species,
and Pseudomonas species) required only 15 minutes exposure. In an
investigation of 3%, 10%, and 15% hydrogen peroxide for reducing
64
spacecraft bacterial populations, a complete kill of 106 spores (i.e., Bacillus

species) occurred with a 10% concentration and a 60-minute exposure time.
A 3.0% hydrogen peroxide solution was ineffective against VRE after 3
and 10 minutes exposure times. A 7% stabilized hydrogen peroxide proved
to be sporicidal (6 hours of exposure), mycobactericidal (20 minutes),
fungicidal (5 minutes) at full strength, virucidal (5 minutes) and
bactericidal (3 minutes) at a 1:16 dilution when a quantitative carrier test
was used (Mayaudon & EL-Zayat, 2005).
The 7% solution of hydrogen peroxide, tested after 14 days of stress
(in the form of germ-loaded carriers and respiratory therapy equipment),
was sporicidal (>7 log10 reduction in 6 hours), mycobactericidal (>6.5
log10 reduction in 25 minutes), bactericidal (>6 log10 reduction in 5
minutes). Synergistic sporicidal effects were observed when spores were
exposed to a combination of hydrogen peroxide (5.9%23.6%) and
peracetic acid (Mertens et al., 2000 and Ali et al., 2001).
Concentrations of hydrogen peroxide from 6% to 25% show promise
as chemical sterilants. The product marketed as a sterilant is a premixed,
ready-to-use chemical that contains 7.5% hydrogen peroxide and 0.85%
phosphoric acid (to maintain a low pH). A new, rapid-acting 13.4%
hydrogen peroxide formulation (that is not yet FDA-cleared) has
demonstrated sporicidal, mycobactericidal, fungicidal, and virucidal
efficacy. Manufacturer data demonstrate that this solution sterilizes in 30
minutes and provides high-level disinfection in 5 minutes. Under normal
conditions, hydrogen peroxide is extremely stable when properly stored
(e.g., in dark containers). The decomposition or loss of potency in small
containers is less than 2% per year at ambient temperatures 670 (Hegde et
al., 2008).
65
9.2. Alcohol
Overview.
In the healthcare setting, "alcohol" refers to two water-soluble
chemical compoundsethyl alcohol and isopropyl alcoholthat have
generally underrated germicidal characteristics. FDA has not cleared any
liquid chemical sterilant or high-level disinfectant with alcohol as the main
active ingredient. These alcohols are rapidly bactericidal rather than
bacteriostatic against vegetative forms of bacteria; they also are
tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores.
Their cidal activity drops sharply when diluted below 50% concentration,
and the optimum bactericidal concentration is 60%90% solutions in water
(volume/volume) (Rutala, 1999).
Mode of Action
The most feasible explanation for the antimicrobial action of alcohol
is denaturation of proteins. This mechanism is supported by the observation
that absolute ethyl alcohol, a dehydrating agent, is less bactericidal than
mixtures of alcohol and water because proteins are denatured more quickly
in the presence of water. Protein denaturation also is consistent with
observations that alcohol destroys the dehydrogenases of E. coli, and that
the lag phase effect could be reversed by adding certain amino acids. The
bacteriostatic action was believed caused by inhibition of the production of
metabolites essential for rapid cell division (Pitts et al., 2003).
Microbicidal activity
Methyl alcohol (methanol) has the weakest bactericidal action of the
alcohols and thus seldom is used in healthcare. The bactericidal activity of
various concentrations of ethyl alcohol (ethanol) was examined against a
variety of microorganisms in exposure periods ranging from 10 seconds to
one an s hour. P. aeruginosa was killed in 10 seconds by all concentrations
of ethanol from 30% to 100% (v/v), and Serratia marcescens, E, coliand
66
Salmonella typhosa were killed in 10 seconds by all concentrations of

ethanol from 40% to 100%. The Gram-positive organisms S. aureus and
Streptococcus pyogenes were slightly more resistant, being killed in 10
seconds by ethyl alcohol concentrations of 60%95%. Isopropyl alcohol
(isopropanol) was slightly more bactericidal than ethyl alcohol for E. coli
and S. aureus (Reckitt and Colman, 1997).
9. Antimicrobial agents of plant origin
Plants and their extracts have been used many centuries ago as
remedies for the cure or alleviation of disease. Many plants to contain
active constituents in vitro against Gram-negative, Gram-positive, and
fungi. The antibacterial substances in certain plants are always found in
higher concentration in certain organs in the plant, e.g.: leaves, flowers,
roots, rhizomes ... etc (Bakkali et al., 2008).
Volatile oils had been widely used for bactericidal, virucidal,
fungicidal,
antiparasitical,
insecticidal,
medicinal
and
cosmetic
applications, especially nowadays in pharmaceutical, sanitary, cosmetic,

agricultural and food industries. Because of the mode of extraction, mostly
by distillation from aromatic plants, they contain a variety of volatile
molecules such as terpenes and terpenoids, phenol-derived aromatic
components and aliphatic components (Bakkali et al., 2008).
9.1. Essential oils as antibacterial agents:
The preservative properties of thyme essential oil (3%) with a known
composition were evaluated in two types of final formulations, suitable for
use as pharmaceutical or cosmetic vehicles, by means of a standard
challenge test proposed by the latest European Pharmacopoeia (Manou et
al., 1998).
9.2. Antimicrobial activity of essential plant oils against resistant
bacteria.
67
Volatile oils and their related compounds affect on bacterial cells by

different modes of action. They can degrade the cell wall; disrupt the
cytoplasmic membrane, cause leakage of cellular compounds, change fatty
acids and phospholipids constituents and influence the synthesis of DNA,
RNA and cell protein (Shan et al., 2007).
The Gram-negative bacteria were generally more resistant than
Gram-positive bacteria to the oil treatment with P. aeruginosa being the
most resistant bacteria (Sue et al, 2000).
The essential oils of thyme and others were effective against S.
aureus, P. aeruginosa, E. coli, and other and other bacteria at different
concentrations from/and 2% of oil, also marjoram and cumin oils showed
antibacterial activities against S. aureus (Ozkan et al., 2003).
Antimicrobial drug-resistant bacteria achieved a significant risk to
public health, and consequently there was great interest in reducing the
prevalence of antimicrobial resistance (AR) genes in both commensal and
pathogenic bacteria. One strategy to decrease the prevalence of (AR)
bacteria was to discontinue using antimicrobial drugs for growth promotion
and prophylaxis in food animals (Kelly et al., 2004).
The
essential
oil
of
Thymus
leptophyllus
showed
higher
antimicrobial activity against E.coli, K.pneumoniae, P. aeuroginosa,

S.aureus, C. albicans and Mycobacterium phlei than Thymus webbianus.
The strongest antibacterial activity of Thymus vulgaris and Pimpinella
anisum seeds volatile oils against Bacillus cereus, S. aureus and Proteus
vulgaris with MIC values 15.6 & 31.2 & 125.0 and 31.2 & 62.5g/ml
respectively (Dorman and Deans, 2000). While the combination between
the two oils showed the most antibacterial activity with MIC values 62.5,
62.5and 15.6 g /ml respectively (Al-Bayati, 2008).
9.3. Mechanism of action of essential oil against pathogenic bacteria.
68
Voaltile oil can affect bacteria in ways other than the expected
bactericidal or bacteriostatic action. Volatile oils cause alteration in the cell
morphology (Caelli, et al., 2000).
The thymol from thyme oil (Thymus vulgaris) and carvacrol from
oregano oil (Origanum vulgaris) both disrupt the cell membrane thereby
decreasing the intracellular ATP pool and increasing the extracellular ATP
pool in S. aureus (Dorman and Deans, 2000).
The essential oil of Melaleuca alternifolia (tea tree) has broadspectrum antimicrobial activity. The mechanisms of action of tea tree oil
and three of its components, 1,8-cineole, terpinen-4-ol, and -terpineol,
against S. aureus were investigated. Treatment with these agents at their
MICs and two times their MICs, particularly treatment with terpinen-4-ol
and -terpineol, reduced the viability of S. aureus. None of the agents
caused lysis, as determined by measurement of the optical density at 620
nm, although cells became disproportionately sensitive to subsequent
autolysis. Loss of 260-nm-absorbing material occurred after treatment with
concentrations equivalent to the MIC, particularly after treatment with 1,8cineole and -terpineol. S. aureus organisms treated with tea tree oil or its
components at the MIC or two times the MIC showed a significant loss of
tolerance to NaCl. When the agents were tested at one-half the MIC, only
1,8-cineole significantly reduced the tolerance of S. aureus to NaCl.
Electron microscopy of terpinen-4-ol-treated cells showed the formation of
mesosomes and the loss of cytoplasmic contents. The predisposition to
lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl,
and the altered morphology seen by electron microscopy all suggest that tea
tree oil and its components compromise the cytoplasmic membrane (Ela et
al., 1996).
Some abnormalities in E. coli and S. aureus cells treatment with
cinnamon and oregano oils (Rios et al., 1998). The mechanism of the
69
antimicrobial action of Spanish oregano (Corydothymus capitatus),

Chinese cinnamon (Cinnamomum cassia), and savory (Satureja montana)
essential oils against cell membranes and walls of bacteria was studied by
the measurement of the intracellular pH and ATP concentration, the release
of cell constituents, and the electronic microscopy observations of the cells.
Treatment with these essential oils at their MICs affected the membrane
integrity of bacteria and induced depletion of the intracellular ATP
concentration. Spanish oregano and savory essential oils, however, induced
more depletion than Chinese cinnamon oil. An increase of the extracellular
ATP concentration was observed only when Spanish oregano and savory
oils were in contact with E. coli and S. aureus. These results suggest that
the cytoplasmic membrane is involved in the toxic action of essential oils
(Sagdic, 2003).
The volatile oils can act as prooxidants affecting inner cell
membranes and organelles such as mitochondria. Depending on type and
concentration, they exhibit cytotoxic effects on living cells but are usually
non genotoxic (Bakkali et al., 2008).
9.4. Combination of antimicrobial agents.
The plant essential oils, alone or in combination with other therapies,
may be beneficial in treating a number of health conditions (Halcon, 2002).
Combination of antimicrobial agents are considered to be synergetic
if the effect of combination is greater than the effect of either agent alone
or greater than the sum of the effect of individual agents. Antagonism result
occurred if the combination provides an effect worse than the effect of
either agent alone or worse than the sum the effect of individual agents
(Cappelletty and Rybak, 1996).
The effect of different combinations of terpenes and penicillin
studied on S. aureus MRSA, they found that, carvone/penicillin was totally
synergistic with Fractional Inhibitory Concentration (FIC) (0.0078), while
70
thymol/penicillin was antagonistic with S. aureus MRSA. The associations

of citronellol, eugenol, geraniol, menthol, menthone and myrcene, each one
combined with penicillin showed to be indifferent, independently of each
antimicrobial activity when they were used alone. Associations between
thymol/penicillin and eugenol/penicillin showed excellent synergistic
activities with FIC (Fractionation Inhibitory Concentration) values of 0.15
and 0.168, respectively, while the association myrcene/penicillin was
antagonistic with FIC (10) against E. coli (Gallucci et al., 2006).
The combination of norfloxacin with either Pelargonium gruveolens
volatile oil or with some of the main components of the latter, in the
treatment of infections caused by some bacterial species was likely to
reduce the side effects of the antibiotic (Rosato et al., 2007).
10. Molecular studies on S. aureus
10.1. Molecular identification
Since conventional identification and antibiotic resistance detection
oftentake
more than 48 h molecular based detection techniques, including
conventionalPCRandreal timePCR, havebeen developedfortherapid
and accurate identification and characterisation of MRSA isolates (Gtz,
2010). Molecular techniques are often applied for the routine diagnostic
MRSAdetectionalongwithantimicrobialsusceptibilitytestingmethods,
partly becausesusceptibilitytestingaloneisnotenoughtoconfirmMRSA
presenceduetothe sensitivityofthetestconditions.Theidentificationof
MRSA wassimplified by the polymerase chain reaction (PCR)
techniquePolymerase chain reaction is a process that allows amplification
ofdetermineDNA(genes)bytheuseofsmalltargetspecificDNAprimers
(Van PeltVerkuiletal.,2008).ThePCRamplificationsystemcanturnafew
molecules of specific target nucleic acid into as much as a microgram of
DNA(Hoffmannetal.,2009).
71
Polymerase chain reaction based methods have been shown to have
shortened the turn-around time (2 h to 4 h) in identifying MRSA isolates
resulting inprompttreatmentforMRSA associatedinfection(Van Halet
al., 2007). These PCR-based methods are able to detect multipleother S.
aureusspecificgenesincludingthe16SrRNAandfemgenes(McClureet
al., 2006). This PCR method of detecting multiple genes in S. aureus
simultaneouslyiscalledmultiplexPCR(M-PCR).
The detection and identification of microorganisms based on their
16S rRNAs have many advantages. First, each bacterial cell contains
multiple copies of the 16S rRNA in its ribosomes. Hence, the technique is
sensitive enough to detect single bacterial cells. Second, 16S rRNA genes
are highly conserved throughout bacterial evolution. They consist of
regions which are common to all eubacteria and other regions which are
extremely species specific. By using the appropriate gene probes, it is
possible either to detect any bacterial pathogen or, when highly specific
probes are used, to identify single bacterial species. Also, this technique
allows for the identification of microorganisms independently of bacterial
growth rates and metabolic activities. This is a special advantage for the
detection of dormant and metabolically inactive bacteria, since the number
of ribosomes is not significantly affected in such organisms (Walsh and
Howe, 2002).
16S rRNA gene sequencing will continue to be the gold standard for
the identification of bacteria, and the automation of the technique could
enable it to be used routinely in clinical microbiology laboratories, as a
replacement of the traditional phenotypic tests. Modern technologies have
made it possible to construct a high density of oligonucleotide arrays on a
chip with oligonucleotides representing the 16S rRNA gene sequence of
various bacteria. Such a design will facilitate automation of the annealing
and detection of the PCR products of 16S rRNA gene amplification, and
hence routine identification of most clinical isolates will be possible
72
(Arthur et al., 1993). The use of 16S rRNA gene sequencing has several
advantages. First, the turnaround time is short. Because amplification of the
16S rRNA gene takes only four to six hours, and the annealing and
detection of PCR products takes only another few hours, theoretically the
identification can be completed within one day. Second, it can be used for
slow growing bacteria, unlike most commercially available kits that are
based on phenotypic tests that require the detection of growth of the
organism in the presence of certain specific substrates, and hence the slow
growing bacteria are usually unidentified when the growth control shows
a negative result. Third, the problem of unidentifiable strains will be
overcome and there would be minimal misidentificationthe identification
of a clinical strain is clearly defined by the number of base differences
between it and the existing species. Fourth, oligonucleotides representing
all bacterial species, including those rarely encountered clinically, can be
included in the array, making it easy to identify the rare species. Lastly,
such a technique will be applicable not only to gynogenic bacteria, but also
to other organisms such as mycobacteria, which are not identified in most
clinical microbiology laboratories because special expertise and equipment,
such as gas liquid chromatography, are required. Therefore, eventually it
should reduce not only manpower, but also capital costs and costs of
consumables. Although the cost effectiveness of using 16S rRNA gene
sequencing in routine clinical microbiology laboratories remains to be
evaluated, our study reports the first case of tube coagulase negative S
aureus bacteremia identified by 16S rRNA gene sequencing(Sudagidan et
al., 2010).
Gene amplification techniques are increasingly used in diagnostic
microbiology and the first applications for the detection of antibiotic
resistance have been described. Studies comparing classical methods such
as disc diffusion and MIC determinations with PCR for the detection of
resistance have been performed for a number of antibiotics or specific
73
organisms. The detection of Methicillin resistance in S. aureus is one of

the best studied applications of PCR for the detection
of antibiotic
resistance. The problems caused by multiple genes leading to the same

resistance
phenotype are illustrated by aminoglycoside
resistance
detection by PCR. Finally, the detection of vancomycin resistance in

enterococci shows the importance of PCR in monitoring emerging
antibiotic resistance. In conclusion it can be said that PCR techniques have
a potential in the field of antibiotic
resistance detection,
but many
problems have to be overcome before gene amplification techniques will

find a widespread use in this field (Altoparlak et al., 2004).
Analysis of specific regions of genomic DNA, on the other hand, has
produced much more discriminative data. For example, several genomic
targets have been effectively used for the identification of Staphylococcus
species, including the 16S rRNA gene, the tRNA gene intergenic spacer, the
internal transcribed spacer, the heat-shock protein 60 (HSP60) gene, the
chaperonin 60 gene, the femA gene, the sodA gene, the gap gene and the
nuc gene (Gtz, 2010).
10.2. Detection van A and mec A gene in vancomycin and mecithillin
resistant bacteria
The genetic mechanism of vancomycin resistance in VRSA is not
well understood. Several genes have been proposed as being involved in
certain clinical VRSA strains (Jansen et al., 2007)
Different clones of VRSA with different locations of the van A gene
indicate the instability of genetic element that carries the van A gene
(Clark et al., 2005). Vancomycin over use in the community may play a
role in maintenance of an unstable van A determinant in VRSA clones.
Vancomycin-resistant S. aureus isolate contained two plasmids of
120 and 4 kb and was positive for mec A and van A by PCR amplification.
The van A sequence was identical to the vanA sequence present in Tn1546.
74
A DNA probe for van A hybridized to the 120-kb plasmid. This is the
second VRSA isolate reported in the United States (Biswajit et al., 2008).
Thus, it surprised many scientists that the first S. aureus isolate
reported to manifest reduced susceptibility to vancomycin did not contain
vanA or any of the other known vancomycin resistance determinants.
Instead, the reduced susceptibility has been attributed to unusually
thickened cell walls containing
D-alanyl-D-alanine
targets capable of
binding vancomycin. The appearance of similar vancomycin-intermediate

S. aureus strains in the United States and elsewhere during the next several
years fueled speculation that the transfer of van A from enterococci to
staphylococci may not occur in nature. Such speculations were discarded in
June 2002 when a van A-containing VRSA isolate was obtained from a
dialysis patient in Michigan. The isolate was also resistant to oxacillin,
levofloxacin, and rifampin. Only 2 months later, a second VRSA isolate
was obtained from a patient in Pennsylvania(Askarian et al., 2009).
The transfer of the genetic element containing the van A vancomycin
resistance gene from E. faecalis to S. aureus was demonstrated in the
laboratory in the first clinical infection with VRSA was reported in
July 2002. This report describes the second documented clinical isolate of
VRSA from a patient (Chang et al., 2003).
The primer synthesis and the PCR amplification of the vanA were done at
Bioserve Biotechnologies, Hyderabad, using Gene Amp (Applied
Biosystems, USA) thermocycler. The primers and the PCR conditions were
as described (Biswajit et al., 2008). The primers for amplification of vanA
were;
Forward
ATGAATAGAATAAAAGTTGC
and
Reverse
TCACCCCTTTAACGCTAATA. The amplification conditions were initial

denaturation at 98oC for 10 sec, annealing at 50oC for 1 min,
polymerization at 72oC for 1 min; and final extension at 72oC for 5 min.
75
The experimental transfer of the vanA gene cluster from E. faecalis

to S. aureus has raised fears about the occurrence of such genetic transfer
in clinical isolates of methicillin resistant S. aureus. In this study, all the
VRSA isolates contained mecA, but only six contained vanA. Tiwari &
Sen8 have also reported a van gene-negative VRSA (Jansen et al., 2007).
DNA of the resistant isolates was subjected to multiplex PCR to
detect the presence of vanA, vanB, vanC1, and vanC2/vanC3genes. Primers
and sequence of primers and size of the expected amplicons are shown in
the following table. The primers were obtained from TIB MOLBIOL
Syntheselabor GmbH (Berlin, Germany), 1.5 mM MgCl2; 200 M each
dATPs, dCTPs, dGTPs, and dTTPs; 50 mM KCl, 10 mM Tris-HCl, 1 U
Taqpolymerase (Fermentas, Lithvania), 2500 ng DNA in 5 L volume were
added to the reaction mixtures as follows: 5 pmol of each VanA primer, 2.5
pmol of the VanB, VanC1, VanC2/C3 and rrsprimers; 5 pmol of the E.
faecalis-specific primers and 1.25 pmol of the E. faecium-specific primers
in total 25 L volume. (Eliopoulos, 1997; Schaaff et al., 2002).
Polymerase chain reaction was optimized under pre-denaturation at 94C
for 8 min followed by 30 cycles at 94C for 70 sec, 54C for 65 sec, 72C
for 95 sec and a final extension step at 72C for 10 min. A VanA-positive
strain (E. faecium ATCC 51559) and E. faecalis ATCC 51299 (Van B)
were used as positive genotypes(Chou et al., 2008).
In each set of PCR reactions, the rrs gene (16S rRNA) with 320 bp
in size was also included as an internal control. Products were
electrophoresed in 1.5% agarose, stained by ethidium bromide and video
image were obtained by gel documentation Uvtec, Sigma, Germany
system. Several PCR protocols have been developed to identify
enterococcal species and to detect glycopeptide resistance genotypes
(Dutka-Malen et al., 1995; Woodford et al., 1997). They found 95%
agreement between genotypic and phenotypic methods. In some
investigations, primers concentration, annealing temperature, amplification
76
cycles with pure and defined concentration of template DNA were

carefully adjusted in order to optimize a multiplex PCR assay that allows
simultaneous detection of Van genes and the bacteria species. The
optimized PCR method has 100% agreement in term of species
identification with phenotypic method and could detect two main human
species and their associated Van genes. The only limitation was the absence
of primers to detect S. aureus. Precise and quick identification of resistant
S. aureus could help clinicians to timely administer appropriate antibiotics
which may be lifesaving (Wu et al., 2004).
Mutant isolates, therefore, may not produce very strong D-alanyl-Dalanine termini where it is the target site for vancomycin to block cross-link
formation in sensitive isolates. Alternatively, mutant isolates could emerge
due to produced remarkably thickened cell wall with an increased
proportion of glutamine nonamidiated mucopeptides. Presence or absences
of glucose or glutamine that influence cell wall thickness have been proven
in vancomycin intermediate resistant Staphylococcus (Gemmell, 2004).
Similar mechanism of resistance may contribute to the emerging
vancomycin intermediate resistant entrococci. Continuous administration of
vancomycin for prophylactic, empiric or treatment purposes could exert
high pressure on sensitive isolates. As a consequence, this pressure can
accelerate selection of both van-positive (acquired resistant) and Vannegative (resistant mutant) entrococci from the pool of microflora of
urogenital and gastrointestinal organs of colonized and other infected sites
of patients (Talon et al., 2001). It has been well established that the
transfer of van A genes could happen via gene exchange through of mobile
genetic elements such as transposon, plasmid or integrons (Arthur et al.,
1993; Arthur and Courvalin, 1993).
Emerging of VRSA identical to van A of E. fecalissuggests the transfer of
vanA gene from E. fecalis to S. aureus (Chang et al., 2003). Unfortunately,
the transfer of van A gene to MRSA could have adverse clinical and
77
nosocomial consequences. In the present study, van A isolates mainly

consisted of E. feceium.Similar data were reported earlier in other parts of
Iran (Emaneini et al., 2008). Nevertheless, in consistent with some earlier
international reports, E. feceium acquired resistance was of higher
frequency as compared to E. faecalis (Wu et al., 2004). Difference in rates
of acquisition of van resistance genes may lead us to speculate that E.
feceium might have more efficient gene capturing system such as integron.
These types of integron are well known in acinetobacter (Wu et al., 2004
and Turton et al., 2005).
Tiwari and Sen. (2006) reported that from 12 VRSA isolate
contained vanA gene, six isolates were negative for vanA by PCR whereas
all the isolates had not expressed mecA as showed in the following figure.
PCR amplification of the vanA gene for vancomycin resistance in methicillin resistant S.
aureus. Lanes 1-6 van A positive VRSA; Lane 7-12: van A negative (Tiwari and Sen, 2006)
The results of multiplex PCR assay revealed the isolates with van Apositive genes among others (van A, van B, van C1, van C2/C3) are
predominant. van B and Van C2/C3 were not detected while van C1 and
van C1+B were each observed in only 1 (1%) of vancomycin resistant
isolates (Emaneini et al., 2008).
78
79
Materials and Methods

1. Patients
This study was conducted on seventy patients. Forty patients
admitted to Sidnawy Hospital, Zagazig University, Zagazig, Egypt in
period from February to August 2009 and 30 patients from king Fahd
Hospital, Al Qassim, Saudi Arabia in period from March to September
2010.
The following patient data were collected:
1. Type of injury
2. Age
3. Gender
4. TBSA% (total body surface area burns)
5. Antibiotic intake
6. White blood corpuscles (WBCs) count
7. Platelets counts
2. Materials Equipment for samples collection and processing:

Sterile cotton swabs, collecting clean dry cups, syringes, pipettes and
blood culture bottles.
3. Isolation and purification of pathogenic bacteria isolated from
medical specimens:
3.1. Collection of samples:
Several medical specimens of pus, sputum and urine were collected
from patients suffering from different types of burn injury at Sidnawy
Hospital, Zagazig University, Zagazige, Egypt and king Fahd Hospital, Al
Qassim, Saudi Arabia.
80
3.2. Isolation:Samples were collected from wounds, blood, respiratory tract

infections and urinary tract infections as pus, blood, sputum and urine
samples, respectively.
Pus was handled by sterile swab. Also five ml. blood samples were
drawn for blood culture at completion of the procedure, while sputum
collected by clean dry cups or aspiration of end tracheal tubes and fresh
uncentrifuged urine samples collected in sterile disposable container. All
collected samples were transferred to laboratory within two hours. Sample
streaked on nutrient agar for obtaining single colony.
3.3. Purification:The swabs were streaked on nutrient agar surface and different
diagnostic and selective media namely C.L.E.D agar (cystine lactose
electrolyte deficient medium), MacConkey agar, mannitol salt agar and
blood agar. Plates were incubated aerobically at 37oC for 24 h.. Growing
colonies were purified and examined for their systematic position using
cultural morphological and Grams stain preparation.
3.4. Media used for isolation and purification:A- General media for culturing, purification and isolation of bacteria:-Nutrient agar -medium (Oxoid Ltd., England)
(g /L):
Peptone
5.0
Yeast extract
2.0
Lab-lemco powder
1.0
Sodium chloride
5.0
Agar
15
Distilled water
1Liter
pH value was adjusted at 7.1 - 0.2

81
The ingredients were mixed well before pouring then sterilized by

autoclaving at 121C for 15 minutes.
B- Selective media
I -MacConkey agar medium (Oxoid Ltd., England): (g/L):
Peptone
20.0
Lactose
10.0
Bile salts No. 3
1.50
Sodium chloride
5.00
Neutral red
0.03
Crystal violet
0.001
Agar
15.0
Distilled water
1Liter

The ingredients were mixed well before pouring then sterilized by
autoclaving at 121C for 15 min.
II- Blood agar medium (Oxoid Ltd., England):
(g/L):
Peptone
5.0
Yeast extract
2.0
Lab-lemco powder
1.0
Sodium chloride
5.0
Agar
15
Distilled water
1Liter
Blood
100 ml
82
III -Mannitol Salt Agar

A selective medium for the isolation of presumptive pathogenic
staphylococci. Most other bacteria are inhibited.
Mannitol Salt Agar
(g/L):
`Lab-Lemco powder
1.00
Peptone
10.0
Mannitol
10.0
Sodium chloride
7.50
Phenol red
0.025
Agar
15.0

IV-A dehydrated Cystine - Lactose Electrolyte Deficient medium
(C.L.E.D.) agar media for diagnostic urinary bacteriology. The
medium supports the growth of all urinary potential pathogens giving good
colonial differentiation and clear diagnostic characteristics.
(C.L.E.D.) agar medium (Oxoid Ltd., England):
(g/L)
Peptone
4.0
`Lab-Lemco powder
3.0
Tryptone
4.0
Lactose
10.0
L-cystine
0.128
Bromothymol blue
0.02
Agar
15.0

V- Muller-Hinton agar medium (Oxoid Ltd., England) (g /L)
Beef dehydrated infusion
30.0
Casein hydrolysate
17.5
83
Starch
1.50
Agar
17.0
Distilled water
1000 ml
pH adjusted at 7.3 0.2

The ingredient were mixed well before pouring, and then sterilized by
autoclaving at 121C for 15 min.
4. Identification for bacterial isolates

Identification involved the following steps
4.1.Morohological examination:
To observe the colonies morphology, including shape, size, surface
strucrure, edge, color and opacity.
4.2.Microscopic examination:
Grams stain is the most widely used staining procedure in
bacteriology. It divided bacteria in two great categories Gram-positive and
Gram-negative, moreover the gram reaction is correlated with certain other
cell properties of an organism, e.g: shape , size, arrangement and staining
reaction.
Grams staining (Harrigan and McCance, 1966)
Reagents:
Crystal violet solution.
Crystal violet
2g.
Ethyl alchol,95%
20 ml.
Ammonium oxalate
0.8 ml.
Distilled water
100 ml.
Iodine solution
2g.
Potassium iodide
Iodine crystals
84
Distilled water
1g.
Decolerized:
100 ml
Acetone
50 ml
Ethyl alcohol, 95%
20 ml
Counter stain:
Safranine
2.5 g
Ethyl alcohol, 95%
100 ml
Distilled water
100 ml
Procedure:
A heat-fixed smear from an 18-24 h. bacterial culture was prepared
on a clean slide. Smear was stained with crystal violet solution for 30 sec.
and then washed with distilled water. The prepared film was covered with
Grams iodine solution for 10 sec.
The iodine was poured off and the slide was washed with a
decolorized until no more purple stain run from the slide. The slide was
gently washed with distilled water, stained with the counter stain (safranin)
for 30 sec. and then washed with distilled water and dried. The dried slide
was examined under the oil immersion lens of the light microscope.
4.3. Biochemical tests for identification of the bacterial isolates:

a- Oxidation fermentation test:
Nutrient broth
1000 ml.
85
Sugar
10.0 g.
Bromothymol blue
1.00 ml.
pH value was adjusted at 7

*
Sugar as glucose, maltose, arabinose, mannitol, maltose, xylose, lactose,
sorbitol, sucrose
Procedure:
Two tubes containing the OF medium were required for each sugar.
Test tubes contain inverted Durhams tube were inoculated with the tested
organisms. One tube of each pair was covered with 1 cm. of sterile mineral
oil (fermentation test), while the other not covered (oxidation test). Tubes
were incubated at 37C and examined daily for gas formation and change
in medium color (yellow and blue color represented positive and negative
oxidativefermentation tests, respectively).
b-Nitrate reduction test: Nitrate broth medium
Beef extract
Peptone
Potassium nitrate
Distilled water
pH value was adjusted at 7.1
Reagents of nitrate reduction test
(g/L):
3.0
5.0
1.0
1liter
Reagent A
A-Naphthylamine
5.0 g.
Acetic acid (5N), 30%

Reagent B
1liter
Sulfanilic acid
Acetic acid (5N),30%
8.0 g.
1 liter
Procedure:
Sterile test tubes each contained the same volume of nitrate broth
medium were inoculated with bacterial culture (24h. age) and incubated at
86
37 C for 24 h.. After incubation period, 1ml. of reagent A was added to

each tube followed by reagent B. The positive reaction was recorded by
formation of stable deep red dye immediately.
c- Motility test:
(g/L):
Motility test medium

Gelatin
80
Agar-Agar
15
Peptone
10
Sodium chloride
5.0
Beef extract
3.0
pH was adjusted at
7.1
Detection of the motility was made using the stab inoculation technique
according to Tittsler and Sandholzer (1936).
d- Arginine hydrolysis test:Arginine broth medium
(g/L):
Peptone (tryptone)
5.0
Yeast extract
5.0
K2HPO4
2.0
Glucose
0.5
Arginine monohydrochloride
3.0
Distilled water
1 liter
pH value was adjusted at
7.0
The ingredients were dissolved by boiling; the mixer was filtered and
sterilized at 115 C for 20 minutes by autoclaving.
87
Reagent of arginine hydrolysis test

Nessler's reagent
Five g of potassium iodide were dissolved in 5 ml. freshly distilled
water. Cold saturated mercuric chloride solution was added until a slight
precipitate remains permanently after shaking then 40ml. NaOH were
added. The reagent was diluted to 100 ml. with distilled water then stand
for 2 h.
Procedure:
Five ml. arginine broth was inoculated with the tested bacterial
suspension (24 h. age) and incubated at 37
C for 24 h. then 0.25 ml. of
'
Nessler s reagent was added. The positive reaction was indicated by

development of a brown color.
e- Urease test:
Christensens urea agar
(g/L):
Peptone
1.0
NaCl
5.0
KH2PO4
2.0
Glucose
1.0
Urea
20
Phenol red
0.012
Agar
20
1Liter
Distilled water
pH was adjusted at 6.8
Procedure:
Sterilize Christensens urea agar tubes was inoculated by streaking

the tested organism (24h.age) and incubated at 37C for 24 h. The positive
reaction was recorded by the formation of pink or red color.
88
f-Blood hemolysis test:

Nutrient agar medium
900 ml
Defibrinated blood
100 ml
Procedures:
Melted nutrient agar medium was cooled to 50 C and the blood was
added aseptically, and then mixed in plates. Plates were inoculated by the
tested strain by streaking, then incubated at 37 C for 24-48 h. the positive
results were indicated by formation of clear colourless (-hemolysis) or
clear greenish (-hemolysis) zones around the bacterial growth. Non
hymolytic microorganism was not reflected any change in the medium
(negative reaction).
g-Gelatin liquification test:
Nutrient gelatin medium
(g/L):
Beef extract
3.0
Peptone
5.0
Gelatin
120
Distilled water
1Liter
pH value was adjusted at 6.8

Procedures:
The prepared gelatin tubes were kept in the refrigerator until just
prior to use. The medium was inoculated by stabbing with heavy inoculums
(24h.age) to a depth of 0.5-1 inch. Tubes were incubated at 37C and tested
daily for liquifaction. The positive reaction was determined by liquifaction
of gelatin medium after refrigerator for 4 h.
89
h- Oxidase test:
Reagents:
One gram of P- amino dimethyl alanilin oxalate was dissolved in 100
ml of distilled water (1% solution).The reagent was freshly prepared.
Procedure:
A filter paper strip-moistened with freshly prepared 1% aqueous
solution of P- amino dimethyl alanilin oxalate, was rubbed with colony
from a culture grown on nutrient agar. A positive reaction was indicated by
intense deep purple color appearing with 5-10 sec. A delayed positive
reaction may appeare within 60 sec. Negative organisms dont produce any
changes.
i- Citrate Utilization test: Simmon's medium:
(g /L):
Sodium chloride
5.00
MgSO4.7H2O
0.20
NH4H3PO4
1.00
KH3PO4
1.00
Sodium Citrate
2.00
Agar
20.0
Bromothymol blue 0.2% solution
40 m1
Distilled water
1 Liter
pH was adjusted at
6.8
The ingredients were sterilized by autoclaving at 121C for 15 min. and

poured as slopes.
Procedure:A well-isolated colony is picked from the surface of primary
isolation medium and inoculated as a single streak on the slant surface of
the citrate agar tube. The tubes were incubated at 35C for 24 to 48 h.. A
positive test is represented by the development of a deep blue colour within
90
24 to 48 h. A positive test may also be read without a blue colour if there is

visible colonial growth along the inoculation streak line.
j- Methyl red test (MR):
(g /L):
Peptone
5.0
K3HPO4
5.0
Distilled water
1Liter
The solids were steamed until dissolved and filtered; pH value was
adjusted at 7.5.
Five g. of glucose were added, mixed and distributed 1.5ml volumes
into tubes, then sterilized at 121oC for 15 min.
Procedure:
The MR/VP broth was inoculated with a pure culture of the test
organism. The broth incubated at 35C for 48 to 72 h. At the end of this
time, 5 drops of the methyl red reagent added directly to the broth. Pink
color indicates positive result.
k- Vogas-Proskauer test (VP):
It consists of Glucose Phosphate Peptone water, as for the methyl red
test.
Procedure:The MR/VP broth inoculated with a pure culture of the test
organism. The broth incubated for 24 h. at 35c. At the end of this time,
aliquot 1 ml of broth was added to a clean test tube. 0.6 ml of 5% alfa
naphthol was added followed by 0.2 ml of 40% KOH. The tube was
shacked gently to expose the medium to atmospheric oxygen and the tube
remains undisturbed for 10 to15 min. A positive test is represented by the
development of a red colour in 15 min or more after addition of the reagent.
91
l- Triple sugar iron agar test (H2S production test):
(g /L):
Lab-lemco powder
3.0
Yeast extract
3.0
Peptone
20
Sodium chloride
5.0
Lactose
10.0
Sucrose
10.0
Glucose
1.00
Ferric citrate
0.30
Sodium thiosulphate
0.3
Agar
12
Phenol red 0.2% solution
12 ml
Distilled water
1Liter
The ingredients were sterilized by autoclaving at 121C for 15 minutes and

poured as slopes.
Procedure:Triple sugar iron agar was inoculated with a long straight wire which
introduced into the surface of the tube, extending to within 3-5 mm of the
bottom of the tube. Upon removing the inoculating wire from the depth of
the tube, the slant surface streaked with a back and forth motion. Incubated
at 37C for 24 h and then were examined. Yellow color but indicates acid
production while black colour indicated H2S production.
m- Indole production test (Peptone water medium):
(g / L):
Peptone
10.0
NaCl
5.00
Distilled water
1 Liter
92
The solids were dissolved by heating in the water. pH adjusted to 88.4 and boiled for 10 minutes then filtered, pH adjust to 7.2 - 7.4 and
sterilized at 121C for 20 min.
Procedure:
Tryptophan broth was inoculated with the test organism then
incubated at 35c for 18 to 24 h. After incubation, 20 drops of fresh kavac's
reagent added to the tube. The upper layer of the broth was observed for a
red ring. This ring indicates that indole was produced in the tube and that
tryptophan was digested. If the ring remains yellow or light brown, Indole
was not produced and tryptophan digestion failed to occur and this mean
Negative test.
n- Catalase activity:
Most bacteria that utilize O2 in their respiration produce hydrogen
peroxide which is toxic to their own enzyme system. Their survival in the
presence of hydrogen peroxide is possible because they produce catalase
enzyme which converted hydrogen peroxide into water and oxygen. This
test was used to differentiate between producing catalase enzyme (catalase
+ ve) and other non producing catalase (catalase -ve) organisms.
Procedure (Tube or agar plate method of catalase activity):
A few drops (about 1 ml) of 3% H2O2 reagent were added directly to
the surface of the growth on agar plate or slant. The evolution of bubbles of
gas indicating a positive test.
o- Coagulase test
Tube test (free coagulase): a small amount of the colony growth of
the organism were emulsify in a tube containing 0.5 ml of citrated rabbit
plasma diluted 1:10 in sterile saline. The tube was incubated at 35C for 4
h. and observed for clot formation by gently tilting the tube. The clot was
93
observed at that time, the tube was reinsulated at room temperature and the
result read again after 18 h. if the clot not observed.
Controls
The test was considered positive if any degree of coagulum
formation appeared.
The test was considered negative if the plasma remained wholly liquid or
showed only a flocculent precipitate.
5. Antibiotic susceptibility test.
5.1. Antibiotic disks:
Sixteen of different antibiotics were selected for carrying out the
antimicrobial susceptibility test. The antibiotic disks used in this research
were purchased from Oxoid Ltd., England. The evaluation of inhibition
zone diameter (mm) in antibiotic susceptibility test indicated in Table (1).
The evaluation of inhibition zone diameter (mm) in antibiotic susceptibility
test according to Jacques (1980) and NCCLS (1999).
Disc conc.
Susceptible Intermediate Resistant
by ug/ml
Imipenem (IPM)
10 g
16
14-15
13
Rifampicin (RF)
5 g
17
16-15
14
Tetracyline (TE)
30 g
19
15-18
14
Oxycilline (OX)
10 g
14
11-12
10
Vancomycin (VAN)
30 g
12
10-11
9
Chloromphenicol (C)
30 g
18
13-17
12
Cefotaxime (CTX)
10 g
23
15 22
14
Cefepime (FEP
30 g
18
15 17
14
Methicillin (MET)
5 g
20
17 19
16
Cephardin (CE)
30 g
18
15 17
14
Amoxycillin (AX)
20 g
14
12 13
11
Ampicillin\sulbactam
30 g
15
12 14
11
(SAM)
Ampicillin (AM)
30 g
17
14 16
13
Ciprofloxacin (CIP)
10 g
21
16 20
15
Erythromycin (E)
15 g
23
14 22
13
Tobramycin (TOP)
10 g
20
16 19
15
No. Types of antibiotic

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
94
5.2. Disk diffusion agar method:

Antibiotic susceptibility test for the bacterial isolates was carried out
by disk diffusion technique according to Bauer et al (1966). The technique
was done by inoculation of pure colonies of the tested organism into 5 ml
of sterile nutrient broth and incubated at 37C for 24 h. Then 0.1ml of
bacterial suspension (0.5 McFarland turbidity) was spreading by sterile
swabs on nutrient agar plates. Duplicate plates were prepared for the strain.
Antibiotic disks were applied to the surface of plates at constant distances.
The plates were incubated at 37C for 24 h. At the end of incubation period
zones of inhibition were measured as (mm.).The entire diameter of the zone
was measured including the diameter of the disk. The end point of the
reading was taken as complete inhibition of the growth to the naked eye.
6. Determination of the Minimum Inhibitory Concentrations (MICs)
and Minimum Bactericidal Concentrations (MBCs) of different
antibiotic against the selected S. aureus isolates.
For this experiment four antibiotics were used, vancomycin 250mg/L
(a member of glycopeptide family) & oxycilline 250mg/L (a member of lactam family) & Imipenem 250mg/L (a member of carbapenem family),
and ciprofloxacin 250mg/L (a member of quinolones family) purchased
from Oxoid Comp. MICs and MBCs were determined by using the
standard broth dilution technique (Washengton and Sutter, 1980). The
stock solution of four antibiotics was diluted with sterile double strength
broth to obtain an appropriate dilution needed for each experiment (as
micrograms per milliliter).
In Wassermann tubes two fold serial dilutions of one of the
antibiotics were made from the diluted stock solution using broth as
diluents. To each tube 0.5 ml nutrient broth microbial culture (24 age) was
added.
95
With each group of tests, tubes +of uninoculated medium with and
without antibiotic were included to act as a control to ensure sterility and
clarity of the medium. A third control tube containing inoculated medium
but without antibiotic was also included to ensure the ability of the
organism to grow in the medium. All the tubes were incubated at 37C for
24 h. and examined for turbidity as an indicator of bacterial growth.
The MICs of the resistant isolates to vancomycin were also
determined by E-test (AB Biodisk, Sweden). MICs breakpoints for
vancomycin
were
determined
according
to
the
manufacturers
recommendation.
The minimum inhibitory concentration (MIC) is defined as the
lowest concentration that inhibits a visible growth in liquid media
(Washington & Sutter, 1980). One hundred micro liters (l) were taken
from each MIC concentration as well as the lower concentrations and
introduced onto nutrient agar to determine the MBC. The plates were
incubated at 37C for 24 h. The Minimum bactericidal concentration
(MBC) is the lowest concentration at which no growth occurs on solid
media (Washington & Sutter, 1980).
7. Bactericidal activity of different antimicrobial agent.
Different concentration of the bactericidal usually used in burn units
as chemical disinfects were tested for its effect on the viability of the tested
organisms.
Ethanol produced by EL NASR Company for chemical manufacture, Cairo,
Egypt. Ethanol was diluted to different concentration (50, 60 and 70%)
using sterile distilled water.
Hydrogen peroxide (10%) manufacture by El Ameria Company for
chemical manufacture, Cairo, Egypt. Hydrogen peroxide was diluted to
different concentration (6, 7 and 8%) using sterile distilled water.
96
Procedure:
Sterile test tubes containing 9 ml required certain concentration of
disinfectants were prepared, then each tube was inoculate with 1 ml of
tested bacterial suspension for different period (5, 10, 15, 20 & 30 min.).
The most resistant bacterial isolates in this study were tested for each
disinfectant concentration and certain exposure time. Sterile swab was
dipped into tested tube and gently pressed against inside the tube to get rid
of any excess, then gently the wet swab was rubbed against the surface of
nutrient agar plate, incubate for 24hr. at37 C, then the numbers of bacterial
isolates was observe.
8.
Antimicrobial activity of some essential oils against the tested S.

aureus isolates.
8.1. The essential oils sources.

The standard 9 volatile plant oils used in this research were
purchased from (Sekam Company, Egypt).
The essential plant oils used for antibacterial activities.
No.
English name
Scientific name
Family
Thyme oil
Thymus vulgaris
Labiatae
Cinnamon oil
Cinnamomum zeylanicum,
Lauraceae
Lemmon grass oil Cymbopogon citratus.
Clove oil
Syzygium aromaticum
Myrtaceae
Garlic oil
Allium sativum
Alliaceae
Olive oil
Oleum olivae
Oleaceae
Tea tree oil
Melaleuca alternifolia
Myrtaceae
Peppermint oil
Mentha piperita.
Lamiaceae
Nigella sativa oil
Nigella sativa (Black cumin)
Ranunculaceae
97
Poaceae
8.2. Susceptibility of S. aureus (12, 30, 50 and 95) to certain essential

oils.
All oils were tested for their antimicrobial activity using agar well
diffusion technique (Dorman and Deans, 2000). Tested organisms mixed
in 5 ml sterile nutrient broth and incubated at 37 oC for 24 h. The
suspension was diluted if necessary with sterile nutrient broth to a density
visually equivalent to that of standard barium sulfate (0.5 McFarland).
Inocula were spread by sterile swabs on nutrient agar plates. Triplicate
plates were prepared for each strain. Cork porer wells were made at
constant distances. Oils with concentration (100 %) were prepared and
spotted in each well. Control was made by spotting same volume of tween
80. Plates incubated at 37 oC for 24 h.. At the end of incubation period the
inhibition zones measured as mm. The entire diameter of zone was
measured include the diameter of the well. End point of reading was taken
as complete inhibition of the growth.
8.3. Determination of the Minimum Inhibitory Concentrations (MICs)
and Minimum Bactericidal Concentrations (MBCs) of thyme oil
and tea tree oil against the tested S. aureus isolates.
The MBCs and MBCs were determined using double fold dilution
method in nutrient broth. Few colonies of similar appearance of the test
organism were emulsified in a small volume of sterile nutrient broth and
LQFXEDWHG IRU K DW & WKH VXVSHQVLRQ LV WKHQ GLOXWHG LI QHFHVVDU\
with sterile nutrient broth to a density visually equivalent to that of a
standard barium sulphate (0.5 Macfarland).
In this experiment 50l of bacteria suspension adjusted to 0.5
McFarland was added to 2 ml broth containing 1 ml oil in glass test tubes
and make 10 serial dilutions were made. All tubes were incubated at 37C
for 24 h. The Minimum Inhibitory Concentrations (MIC) was defined as
the lowest concentration of the drug which completely inhibited the
98
bacterial growth disregarding a single colony or faint haze of growth

(Goldstein et al., 1978). Whereas the bactericidal concentration were
determined by (solid agar diffusion method) subculturing l00l of each
negative tube and from the positive control. MBC was defined as the
lowest concentration that yielding negative subculture or only one colony.
8.4. Influence of combination between vancomycin antibiotics and
(thyme or tea tree oil) on the tested S. aureus isolates.
The effect of combination between thyme oil or tea tree oil and
vancomycin antibiotics was measurement by absorbance (10ul) of thyme
oil and tea tree oil (which gave the best antimicrobial activity against S.
aureus no.(12, 30, 50 and 95) each alone and placed on the surface of seeded
plates. The plates were incubated at 37 oC for 18-24 h. and the inhibition
zone was measured in mm.
9. Screening for the virulence factors and degrading enzymes produced
by tested S. aureus isolates
Agar well diffusion assay described by Klaenhammer, (1988).
9.1. Materials:
Liquid culture of the tested S. aureus isolates (24 age).
9.2. Media:
Hydrolysis of lecithin was detected on egg yolk agar (TSA
supplemented with 10% egg yolk emulsion). Haemolytic activity was
determined on blood agar (TSA supplemented with 10% defibrinated sheep
blood).
Hydrolysis of proteinase was detected on Casein agar (TSA
supplemented with 10% casein or skim milk).The plates were incubated at
37C for 24 hours.
99
Tryptone soya agar (TSA)
(g/L):
Pancereatic digest of casein
17.0
Papaic digest of soyabean meal
3.0
Sodium chloride
5.0
Dipotassium hydrogen phosphate
2.5
Glucose
2.5
Distilled water
1000 ml.
Agar
20
9.3. Methods:
(1) Wells of 5mm diameter were cut into the plates of specific agar media
of each test previously mentioned.
(2) Supernatants of tested isolates were prepared as follows:
Ten ml culture of tested isolates were grown for 7 to 8 hours in
nutrient broth, then centrifuged at 3500 g/15-20 min. neutralized with 5
mol/l NaOH to the final pH= 7.0 and then filter sterilized (Reinheimer et
al., 1990; Misra and Kuila, 1992). Wells previously prepared were
separately filled with 50 l of the supernatants of the tested isolates. Plates
were preincubated at 4 C for about 2h to allow diffusion of any inhibitory
metabolites into the surrounding agar, and then incubated at 37C for 24
hours. The plates were examined for a clear zone in the agar surrounding
the well. The measurements were recorded in three repetitions.
10. Effect of certain bactericidal concentrations on the enzyme
activities (virulence factors).
10.1. Materials:
The Liquid culture of the tested S. aureus isolates (24 h. age).
Different concentration of Ethanol (50, 60 and 70%) and Hydrogen
peroxide (6, 7 and 8%) were prepared.
100
10.2. Media:
Hydrolysis of lecithin was detected on egg yolk agar (TSA
supplemented with 10% egg yolk emulsion). Haemolytic activity was
determined on blood agar (TSA supplemented with 10% defibrinated sheep
blood).
Hydrolysis of proteinase was detected on Casein agar (TSA
supplemented with 10% casein or skim milk).The plates were incubated at
37C for 24 hours.
10.3. Methods:
Determination of the enzymatic activities of different staphyloccoci
were performed using agar diffusion assay as previously mentioned
11. Effect of tea tree oil on the enzyme activities (virulence factors).
The tested S. aureus isolates treated with different concentration of
tea tree oil were examined for their capability of producing extracellular
degrading enzymes and virulence factors. Tween 80 (v/v) was included in
all dilutions to enhance oil solubility.
Determination of the enzymatic activities of different staphyloccoci
were performed using agar diffusion assay according to Klaenhammer,
(1988) as previously mentioned.
12. Molecular identification and characterization assays of van A &
mec A gene.
The molecular ivestigations have been done in sequence unit, city of
scientific research and technological applications, Burg El Arab, Alex,
Egypt.
101
Chemicals and reagents

All reagents used were of analytical grade. PCR and sequencing
chemicals, molecular biology kits used were purchased from MBI,
Fermentas and Fluka.
Agarose gel
- 6X gel loading buffer: 30% (w/v) glycerin, 0.2% of bromophenol blue
(BPB) (w/v) and 25mM EDTA
- Ethidium bromide: 1g ethidium bromide dissolved in 100ml H2O
- 1% Agarose gel: 1g agarose powder dissolved in 100ml 0.5 X TAE
buffer, heat till complete dissolving.
Primers
The primers used throughout the present work were as follows:
For 16S rDNA gene
Forward primer 5-start (20mer) 5AGAGTTTGATCMTGGCTCAG 3
Reverse primer 3-end (21mer) 5TACGGYACCTTGTTACGACTT3
These primer were used for Sequencing
12.1. Molecular identification of the selected bacterial isolates
The bacterial isolates were identified using 16S rDNA sequencing.
Extraction of chromosomal DNA preparation.
The DNA of the selected bacteria were extracted using GenElute
Bacterial Genomic DNA Kit ,sigma Aldrich.
Amplification of the 16S rDNA gene
The 16S rDNA gene was amplified by polymerase chain reaction
(PCR) using universal primers designed to amplify 1500 base pair fragment
of the 16S rDNA region. After completion the PCR reaction, a fraction of
PCR was examined using agarose gel and the remnant mixture was purified
using fermentase PCR purification reagents.
102
The PCRs were carried out in order to amplify specific DNA

fragments. For standard PCR, the components listed below were pipetted in
a PCR test tube.
Component
Final concentration
10x polymerase buffer
1x
DNTP-Mix (each 2.5 mM)
0.2 pM
Primer a
10 pM
Primer b
10 pM
Template DNA
Ca. 0.1 g
DNA-polymerase ( Taq )
1 or 2 U
MgCl2 25 mM
2 mM
The cyclic reaction, composed of 4min at 95C and then 30 cycles of

1min at 94C, 1min at 50-59C and 2min at 72C, followed by an
additional 10min at 72C. The optimal parameters and the annealing
temperature in particular, had to be determined empirically depending on
the fragment to be amplified. For standard PCR the annealing temperature
was set at 52-58C.
12.2. Detection of van A gene & mec A gene
Different primers were used for detection of these genes and
different conditions were used for the same pair of primers.
Detection of mec A gene
mecA F AAAATCGATGGTAAAGGTTGGC
mecA R AGTTCTGCAGTACCGGATTTTGC
Detection of van A
vanA F:
ATGAATAGAATAAAAGTTGCA
vanA R:
CCCCTTTAACGCTAATACGAT
103
After using different condition for PCR there are not give any PCR
product..so we make another design for primers.
NEW DESIGN
Forward primer TGGCTCAGGTACTGCTATCCACCC
Reverse primer ACCACCCAATTTGTCTGCCAGTT
This pair of primer gives product 818
Forward primer GGCTCAGGTACTGCTATCCACCCT
Reverse primer AACCACCCAATTTGTCTGCCAGT
This pair of primer gives product 818
Forwardprimer
CCAGGAATGCAGAAAGACCAAAGCA
Reverse primer GGGTGGATAGCAGTACCTGAGCCA

Detection of van A gene
van A 1
Forward primer: AGCTCAGCAATTTGTATGGA
Reverse primer: GGGGATAACGACTGTATGAC
van A 2
Forward primer: GCTCAGCAATTTGTATGGAC
Reverse primer: TGTCATATTGTCTTGCCGAT
Amplification of the van A gene
The van A gene was amplified by polymerase chain reaction (PCR)
using primers designed to amplify 945 base pair fragment. After
completion the PCR reaction, a fraction of PCR was examined using
agarose gel.
104

a PCR test tube.
Component
10x polymerase buffer
Final concentration
1x
DNTP-Mix (each 2.5 mM) 0.2 pM

Primer van A 1 F
10 pM
Primer van A 1 R
10 pM
Template DNA
Ca. 0.1 g
DNA-polymerase ( Taq )
1 or 2 U
MgCl2 25 mM
2 mM
The cyclic reaction, composed of 4min. at 95C and then 30 cycles

of 1min at 94C, 1min at 40-55C and 90 seconds at 72C, followed by an
additional 10 min. at 72C. The optimal parameters and the annealing
temperature in particular, had to be determined empirically depending on
the fragment to be amplified. The annealing temperature was set at 46C.
13. Statistical analysis.
The obtained data were subjected to analysis of variance for a
completely randomized design with three replicates in each treatment
(Snedecor and Cochran, 1968). The means were compared by using the
method of new Least Significant Differances (New L.S.D) value at 0.05%
described by Waller and Duncan (1969).
105
106
Results
Results
1- History of burned patients.
Infections are the major cause of morbidity and mortality in burned
patients. Three fourth of deaths in burned patients occur due to infections.
The present investigation covered a tRWDO RI VHYHQW\ SDWLHQWV IURP
Sidnawy Hospital, Zagazig University, Egypt and 30 patients from king
Fahd Hospital, Al Qassim, Saudi Arabia.
Type of injury, age, gender, total body surface area burned and
antibiotic treatment were included in table (1 & 2). In the present study
females (57.1%) were affected more than that of males (42.9%) (Figure1).
Most common age group affected was from 3 up to 64 years. Results in
table (3) revealed that the patients had different causes of burned injury as
exposure to flame which led to thermal injury in 33 patients out of 70 total
patients. This accounted for 47.1%. Hot water thermal injury was
implicated in 16 cases. Electricity revealed 17 patients all of them from
males. Only four patients out of 70 total patients represented in (5.7%)
suffered from chemical burns.
All the patients suffered burn from 7% up to 94% of total body
surface area burned (TBSA). More than 58 % of the patients died either
from infection, by the burn itself or by both of them as represent in table
(3). Data in tables (4 and 5) show the results of blood, swabs and other
culture. Tables (6 and 7) show the results of account of platelets, white
blood cells, the presence or absence of inhalation injury and the final
outcome for each of the seventy patients.
Prophylactic antibiotics were routinely used. Thus peflacin,
sulperazone, fortum, pencillin, flummox, cefotaxim, ciprocin, ampicillin,
cefoxitin, garamycin, amikacin and erythromycin were administrated from
the first day of admission.
107
Results
Table (1): Type of injury- age- gender - total body surface area burned
and antibiotic treatment of burned patients hospitalized at
Sidnawy Hospital, Zagazig, Egypt.
No.
Type of
Injury
Age
Gender
TBSA%
Out come
Antibiotic
intake
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Flame
Hot water
Electricity
Flame
Flame
Flame
Hot water
Electricity
Flame
Flame
Hot water
Flame
Electricity
Flame
Electricity
Flame
Flame
Flame
Electricity
Hot water
Hot water
Hot water
Chemicals
Flame
Flame
Flame
Hot water
Hot water
Hot water
Hot water
Hot water
Flame
Flame
Electricity
Electricity
Flame
Flame
Flame
Flame
Electricity
17
22
45
54
12
50
13
57
48
42
39
10
20
3
50
44
24
64
30
4
56
11
46
43
22
20
53
39
44
58
23
21
38
15
25
50
40
36
45
62
F
M
M
F
F
F
M
M
F
F
F
F
M
F
M
F
F
F
M
F
F
F
M
F
F
F
M
F
M
M
F
M
F
M
M
F
F
F
F
M
35
48
40
71
41
60
18
90
58
53
55
18
15
20
80
40
15
94
7
18
70
16
40
50
18
20
55
40
48
32
16
15
48
20
45
90
49
40
62
90
recovered
recovered
died
died
recovered
died
recovered
died
died
died
died
recovered
recovered
recovered
died
died
recovered
died
recovered
recovered
died
recovered
died
died
recovered
recovered
died
died
died
died
recovered
recovered
died
recovered
recovered
died
died
died
died
died
Peflacin
Sulperazone
Fortum
Penicillin
Fortum
Penicillin
Flummox
Sulperazone
Sulperazone
Fortum
Penicillin
Sulperazone
Sulperazone
Penicillin
Penicillin
Fortum
Cefotaxime
Penicillin
Sulperazone
Penicillin
Penicillin
Erythromycin
Penicillin
Fortum
Penicillin
Sulperazone
Penicillin
Penicillin
Sulperazone
Penicillin
Penicillin
Penicillin
Flummox
Penicillin
Penicillin
Sulperazone
Penicillin
Penicillin
Ciprocin
Penicillin
108
Results
Table (2): Type of injury - age- gender - total body surface area burn
and antibiotic treatment of burned patients hospitalized at
king Fahd Hospital, Al Qassim, Saudi Arabia.
No.
Type of
injury
Age
Gender
TBSA%
Outcome Antibiotic
intake
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
Flame
Electricity
Hot water
Flame
Flame
Chemicals
Flame
Electricity
Electricity
Hot water
Electricity
Electricity
Hot water
Flame
Hot water
Electricity
Flame
Flame
Hot water
Flame
Electricity
Flame
Electricity
Chemicals
Flame
Chemicals
Flame
Flame
Electricity
Flame
26
40
28
18
13
50
40
60
44
37
23
43
11
54
37
53
60
18
45
48
49
5
25
55
60
38
24
15
39
62
F
M
M
F
F
M
F
M
M
F
M
M
F
M
F
M
M
F
F
F
M
F
M
M
F
M
F
F
M
F
20
87
15
22
20
50
60
70
72
50
30
75
40
60
37
50
60
22
35
55
85
20
30
80
24
recovered
died
recovered
recovered
recovered
died
died
died
died
died
recovered
died
recovered
died
recovered
died
died
recovered
died
died
died
recovered
recovered
died
died
40
15
30
7
18
died
recovered
recovered
died
died
TBSA%: percent of total body surface area burned
109
Sulperazone
Sulperazone
Penicillin
Sulperazone
Penicillin
Ampicillin
Penicillin
Penicillin
Cefoxitin
Garamycin
Amikicin
Penicillin
Cefotaxim
Penicillin
Fortum
Penicillin
Penicillin
Flummox
Penicillin
Sulperazone
Erythromycin
Penicillin
Penicillin
Cefotaxim
Penicillin
Fortum
Cefotaxime
Penicillin
Sulperazone
Penicillin
Results
Table (3): Distribution of burned injury types among male and female:
Type of injury
Total no.
Gender
Outcome
Male Female recovery Death
3
30
15
18
Flame
33 patients
Hot water
16 patients
10
Electricity
17 patients
17
11
Chemicals
4 patients
Total
70 patients
30
40
29
41
&
&
,
Type of thermal injury
Fig (1): Distribution of burned injury types among male and female.
5.7%
24.3%
47.1%
flame
hot water
22.9%
Fig (2): Distribution of burned according to type of injury
110
Results
2- Factors affecting mortality:

There are many factors affecting mortality as age, total body surface
area burns, local infection, systemic infection, inhalation injury and its
effect on platelets count and white blood cells count.
2.1. Age.
The results in table (3) clearly indicated a highly significant
correlation between age and death. The 29 patients who were able to
recover were significantly younger than those who died (P < 0.01). Thus
the recovered group had a mean age of 18 years, while the group that
suffered death had a mean age of 48.3 years.
2.2. Total Body Surface Area burns.
The 29 patients who were able to recover with mean TBSA burns of
22% while, the other 41 patients whose mean TBSA burns of 51% died.
This indicated that the death is directly proportional to the total body
surface area.
2.3. Local infection
The results (tables, 4 and 5) showed that from the twenty nine
patients (41.4%) who recovered, and the other 41 patients (58.7%) who
died, and compared the number of positive and negative swab and other
cultures for each patient, it was found that there is no relation between
patient death and number of positive and negative cultures. Also the data
was analyzed by Chi square test (X2 test) and revealed that there was no
significant correlation between patient death and the presence of local
infection (P > 0.05).
2.4. Systemic infection
Blood cultures were done in 58 patients, 31 of them had positive
blood cultures, while no organisms were detected in the other 27 blood
cultures. Only one of the positive blood cultures were able to recover and
111
Results
other 30 died. From the 27 patients who had no organisms in their blood
cultures, 24 recovered and 3 died. The above data were analyzed by Chi
square test (X2 test) and revealed that there was a significant correlation
between patient death (P < 0.01) and the presence of systemic infection.
2.5. Inhalation injury
In the present investigation 25 patients had inhalation injury, while
the other 45 patients did not show inhalation injury. The inhalation injury
had its effect on platelets count and white blood cells count as follows:
Platelets:
The results in table (6 and 7) had proved that the patients with
inhalation injury had mean platelets count 185.03 * 1000/ cmm. The others
who had no inhalation injury and give a mean platelets count of 296.6 *
1000/ cmm. This finding suggested that the platelets count significantly
decreased in patients with positive inhalation than those with no inhalation
injury.
White blood cells
The mean of white blood cells (WBCs) count in patients with
inhalation injury was 12.6* 1000 / cmm, while in patients with no
inhalation injury the mean of white blood cells count were 14.08 * 1000 /
cmm as showed in tables (6 and 7). Thus no significant relationship
between white blood cells count and the presence of inhalation injury.
2.6. Effect of total body surface area burn on the kind of organisms
The data demonstrated that there was a significant relationship
between the organisms that appeared in the swab cultures and the size of
burn. Burned patients with large TBSA burns were more liable to be
infected with mixed organisms. On the other hand, burned patients with
small TBSA burns showed absence of contaminating organisms. The mean
of TBSA of the recovered patients were 24.7%, while the mean of TBSA of
died patients were 55.7%.
112
Results
Table (4): Relation between total body surface area burned, tested
cultures and outcome of patients hospitalized at Sidnawy
Hospital, Zagazig University, Egypt.
No.
TBS
A%
35
48
40
71
41
60
18
90
58
10
53
11
55
12
18
13
14
15
20
15
80
16
40
17
18
15
94
19
20
7
18
21
70
22
16
23
40
24
50
Swabs
+ve gave1
organism
+ve gave1
organism
+ve gave 2
organisms
+ve
gave1organism
+ve gave1
organism
+ve gave 2
organisms
+ve gave 1
organism
+ve gave 3
organisms
+ve gave1
organism
+ve gave2
organisms
+ve gave1
organisms
+ve gave1
organism
-ve no growth
+ve gave1
organism
+ve gave1
organism
+ve gave1
organism
-ve no growth
+ve
gave1organism
-ve no growth
-ve no growth
Cultures
Blood
Outcome
Other
cultures
ND
ND
Recovered
-ve no growth
+ve gave1
organism(S)
ND
Recovered
+ve gave1
organism(S)
ND
Died
Recovered
ND
Died
ND
Recovered
+vegave1organis
m
+vegave1
organisms
+vegave2
organisms
+vegave1
organism
ND
+ve gave2
organisms(S)
+ve gave1
organism(U)
ND
Died
+vegave1
organism(S)
ND
Died
-ve no growth
-ve no growth
ND
-ve no
growth(U)
ND
Recovered
Recovered
+ve gave 1
organism(S)
ND
+ve gave1
organism(S)
ND
+ve gave1
organism(S)
+ve gave1
organism(U)
+ve gave1
organism(U)
ND
Died
+ve gave1
organism
+vegave2
organisms
+ve gave1
organism
+vegave1
organism
ND
+ve gave1
organism
+ve gave1
organism
-ve no growth
+vegave1
organisms
-ve no growth
-ve no growth
+ve
gave1organism
-ve no growth
+vegave2
organisms
-ve no growth
+ve gave2
organisms
+ve gave2
+vegave2
organisms
+vegave2
113
ND
Died
Died
Died
Recovered
Died
Recovered
Died
Recovered
Recovered
Died
Recovered
Died
Died
Results
organisms
-ve no growth
-ve no growth
+ve gave1
organism
+ve gave1
organism
+ve gave2
organisms
+ve gave1
organism
-ve no growth
-ve no growth
organisms
-ve no growth
-ve no growth
+ve gave2
organisms
+ve
gave1organism
-ve no growth
ND
ND
ND
Recovered
Recovered
Died
-ve no
growth(U)
ND
Died
-ve no growth
ND
Died
ND
-ve no growth
Recovered
Recovered
+vegave2
organisms
-ve no growth
ve no growth
+ve gave 1
organism
ND
-ve no growth
ND
+ve gave1
organism(S)
ND
ND
-ve no
growth(S)
+ve
gave2organisms
(U)
+ve gave2
organisms(S)
ND
Recovered
Recovered
ND
Died
ND
Died
25
26
27
18
20
55
28
40
29
48
30
32
31
32
16
15
33
48
34
35
20
45
36
90
+ve gave2
organisms
+ve gave1
organism
37
49
38
40
39
62
+ve gave1
organism
+ve gave1
organism
ND
40
90
+ve gave2
organisms
+ve gave1
organism
+ve gave 1
organism
+ve gave1
organisms
+ve gave1
organism
Died
Died
Died
Died
Died

(S) Sputum culture (U) Urine culture (ND) not detected
Table (5): Relation between total body surface area burned, tested
cultures and outcome of patients hospitalized at king Fahd
Hospital, Al Qassim, Saudi Arabia.
No. TBSA
%
41
42
20
87
43
15
44
22
45
20
Swabs
-ve no growth
+ve gave2
organisms
+ve gave1
organism
+ve gave1
organism
+ve gave1
Cultures
Blood
Other
cultures
Outcom
e
-ve no growth
+vegave2
organisms
-ve no growth
ND
+ve gave 1
organism(S)
ND
Recovered
Died
-ve no growth
+ve gave1
organism(U)
ND
Recovered
-ve no growth
114
Recovered
Recovered
Results
46
47
50
60
48
70
49
72
50
50
51
30
52
75
53
54
40
60
55
56
37
50
57
60
58
59
22
35
60
55
61
85
62
63
20
30
64
80
65
organism
-ve no growth
+-ve no
growth
+ve gave2
organisms
+ve gave2
organisms
+ve gave2
organisms
ve no growth
+ve gave1
organism
-ve no growth
+ve gave2
organisms
-ve no growth
+ve gave1
organism
+ve gave1
organism
-ve no growth
+vegave1
organism
+vegave1
organism
-ve no growth
+vegave1 organism
+vegave1 organism
ND
ND
Died
Died
+vegave1 organism
ND
Died
ND
ND
Died
ND
ND
Died
-ve no growth
+ve gave1
organism(U)
ND
Recovered
ND
+ve gave1
organism(S)
ND
+ve gave1
organism(U)
+ve gave1
organism(S)
ND
+ve gave1
organism(U)
ND
Recovered
Died
Died
Recovered
Recovered
ND
-ve no growth
+vegave1 organism
-ve no growth
-ve no growth
+vegave1 organism
+vegave1 organism
+vegave1 organism
+vegave1 organism
ND
Died
Recovered
Died
Died
Recovered
Died
Died
-ve no growth
-ve no growth
ND
ND
Died
24
-ve no growth
+vegave1
organism
+ve gave1
organism
-ve no growth
+ve gave1
organism(S)
ND
ND
-ve no growth
Recovered
66
67
40
15
-ve no growth
-ve no growth
+vegave1 organism
-ve no growth
68
30
-ve no growth
-ve no growth
69
-ve no growth
70
18
+ve gave1
organism
-ve no growth
-ve no
growth(U)
ND
-ve no
growth(U)
+ve gave1
organism(S)
ND
ND
ND
Recovered

Other cultures: (S) Sputum culture and (U) Urine culture
(ND): not detected
115
Died
Recovered
Recovered
Recovered
Results
Table (6): Result of platelets and white blood corpuscles from Sidnawy
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
1 day
4 day
PLTs
WBCs
PLTs
WBCs
150
319
ND
115
158
ND
ND
216
189
425
235
ND
189
342
254
ND
202
281
204
225
ND
ND
135
95
170
326
152
460
ND
138
ND
481
272
326
143
450
200
ND
310
120
20
12.7
ND
22
4.2
ND
ND
23.8
9
21.7
15
ND
20.3
14
28.9
ND
14.3
41.7
18.1
14
ND
ND
16.2
3.3
23
18.7
5.3
24.5
ND
7.8
ND
9.8
21.8
20.4
22.7
8.7
10
ND
6.5
7.7
200
300
150
ND
ND
131
230
ND
139
265
246
570
152
ND
ND
236
ND
ND
ND
ND
151
450
110
ND
ND
300
ND
ND
344
ND
153
446
ND
ND
107
ND
198
90
310
ND
12
8.9
12
ND
ND
3
4.7
ND
5.9
3
24.9
16.7
8.9
ND
ND
5.3
ND
ND
ND
ND
4.1
7.2
8.2
ND
ND
18
ND
ND
12
ND
14.7
7.8
ND
ND
5.6
ND
3.8
10
2.4
ND
WBCs
PLTs
ND
10 day
PLTs
250
ND
ND
ND
ND
ND
808
ND
ND
344
183
500
291
ND
ND
ND
ND
ND
ND
ND
ND
864
306
ND
ND
250
ND
ND
ND
ND
ND
405
ND
ND
ND
502
ND
63
ND
ND
white blood corpusles

Platelets
116
Inhalation
Outcome
(-)
(-)
(-)
(+)
(+)
(+)
(-)
(+)
(-)
(-)
(+)
(-)
(-)
(-)
(+)
(-)
(-)
(+)
(+)
(-)
(+)
(-)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(-)
(+)
(-)
(+)
(-)
(-)
(-)
(+)
(-)
Recovered
Recovered
Died
Died
Recovered
Died
Recovered
Died
Died
died
died
recovered
recovered
recovered
died
died
recovered
died
recovered
recovered
died
recovered
died
died
recovered
recovered
died
died
died
died
recovered
recovered
died
recovered
recovered
died
died
died
died
died
WBCs
10
ND
ND
ND
ND
ND
14.2
ND
ND
6.8
14.5
4
3.9
ND
ND
ND
ND
ND
ND
ND
ND
24.3
11.4
ND
ND
10
ND
ND
ND
ND
ND
8.6
ND
ND
ND
11.4
ND
4
ND
ND
(+)
(-)
Not detected
Positive inhalation
No inhalation
Results
Table (7): Result of platelets and white blood corpuscles from Fahd
king Hospital, Al Qassim, Saudi Arabia.
No.
1 day
4 day
10 day
inhalation outcome
PLTs WBCs PLTs WBCs PLTs WBCs
41
42
261
100
8.5
5.5
ND
80
ND
3
ND
ND
ND
ND
(-)
(+)
recovered
died
43
414
18.5
ND
ND
ND
ND
(+)
recovered
44
45
372
ND
13.9
ND
230
164
10.3
12.6
ND
ND
ND
ND
(-)
(-)
recovered
recovered
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
167
139
405
ND
236
321
269
ND
157
594
156
345
319
ND
125
ND
189
342
130
425
235
170
340
167
200
8.4
13.8
35.7
ND
15.7
37.1
39.4
ND
16.7
16.3
14.5
20
12.7
ND
7.5
ND
20.3
14
16.0
21.7
15
23
18.5
9
10
93
ND
165
172
100
45
99
ND
51
ND
ND
58
300
150
ND
236
152
ND
110
265
246
ND
300
93
195
5.5
ND
7.5
7.3
10.5
12
3.6
ND
9
ND
ND
5
8.9
12
ND
5.3
8.9
ND
8.0
3
24.9
ND
18
6.2
3.8
ND
ND
ND
200
ND
172
ND
499
ND
ND
ND
ND
ND
ND
ND
ND
291
ND
306
344
183
ND
250
ND
ND
ND
ND
ND
9
ND
19.2
ND
16.4
ND
ND
ND
ND
ND
ND
ND
ND
3.9
ND
11.5
6.8
14.5
ND
10
ND
ND
(+)
(+)
(-)
(-)
(-)
(-)
(+)
(-)
(+)
(-)
(+)
(+)
(-)
(-)
(+)
(-)
(-)
(-)
(+)
(-)
(+)
(-)
(-)
(+)
(+)
died
died
died
died
died
recovered
died
recovered
died
recovered
died
died
recovered
died
died
died
recovered
recovered
died
died
died
recovered
recovered
died
died
WBCs
PLTs
ND
white blood corpusles

Platelets
(+)
(-)
Not detected
117
Positive inhalation
No inhalation
Results
3- Distribution of collected isolates

In this experiment the isolate numbers, source of collected isolates,
cell shapes & arrangements as seen under oil immersed lens of light
microscope and the reaction against Gram's stain are indicated in table: 8
and 9. The data present in table: 8 and 9 were summarized in tables: 10 and
11 according to the Gram's stain reaction and morphological
characteristics, respectively.
3.1. Distribution of collected bacterial isolates according to their
Gram's stain reaction and source of isolation from Sidnawy
One hundred cultures were collected from seventy burned patients;
seventy six cultures were positive for result, while twenty four showed
negative for result. Direct film was performed to show pus cells, epithelial
cells and the presence of bacteria (either Gram-positive or Gram-negative).
The incidence of bacteria isolated from Sidnawy Hospital, Zagazig
University, Egypt in swab culture, blood culture, urinary and sputum
culture in table (10) and figure (3) indicate that Gram-positive bacteria
were the most predominant.
The Gram-positive isolates represent 47 out of 80 total isolates
(58.75 %), while 33 isolates from the total 80 isolates were Gram-negative
bacteria (41.25 %).
3.2. Distribution of collected bacterial isolates according to their
Gram's stain reaction and source of isolation from king Fahd
The incidence of bacteria isolated from Fahd king Hospital, Al
Qassim, Saudi Arabia in swab culture, blood culture, urinary and sputum
culture in table (11) and figure (4) indicate that Gram-positive bacteria
were the most predominant.
The Gram-positive isolates represent 25 out of 41 total isolates
(60.97 %) , while 16 isolates from the total 41 isolates were Gram-negative
bacteria (39.3 %).
118
Results
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
WOUND CULTURE
3.3. Distribution of the total bacterial isolates according to their

Gram's stain reaction and source of isolation.
The incidence of total bacteria isolated from Egypt and Saudi Arabia
in swab, blood, urine and sputum culture in table (12) and figure (5)
showed that Gram-positive bacteria were the most predominant.
The isolates represent 72 out of 121 total isolates (59. 50%) were
Gram-positive bacteria, while 49 isolates from the total 121isolates were
Gram-negative bacteria (40.50%).
Table (8): Shape and arrangement of bacterial isolates collected from
wounds, blood, urine and sputum and their reaction against
Gram's stain from Sidnawy Hospital, Zagazig, Egypt.
Isolate
Source of
Shape of cell and arrangement
Gram
no.
isolates
stain
Cocci in cluster
Cocci in cluster
Bacilli straight thick rods
Cocci in cluster
Bacilli short rods
Cocci singly
Bacilli short rods
Bacilli short rods
Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Cocci in cluster
Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Bacilli short rods
Bacilli short rods
Cocci in pairs
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Cocci in cluster
Cocci in cluster
Bacilli short rods
119
+ ve
+ ve
- ve
+ ve
- ve
- ve
- ve
+ ve
- ve
- ve
+ ve
+ ve
-ve
+ ve
- ve
+ ve
+ ve
+ ve
+ ve
- ve
+ ve
- ve
- ve
- ve
+ ve
+ ve
- ve
+ ve
+ ve
+ ve
+ ve
- ve
Results
Bacilli short rods
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in pairs
Cocci in cluster
- ve
+ ve
- ve
+ ve
+ve
- ve
- ve
+ ve
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80

Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Cocci in cluster
Cocci in cluster
Bacilli short rods
Bacilli short rods
Cocci in cluster
Cocci in cluster
Cocci in cluster
Cocci in pairs
Cocci in cluster
Cocci in cluster
Monococci
Cocci in pairs
Cocci in pairs
Bacilli short rods
Bacilli short rods
Cocci in pairs
Bacilli short rods
Cocci in cluster
Bacilli short rods
Cocci in pairs
Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Bacilli short rods
- ve
+ ve
+ ve
- ve
+ ve
- ve
+ve
- ve
- ve
+ ve
- ve
+ ve
+ ve
+ ve
+ ve
- ve
- ve
+ ve
+ ve
+ ve
- ve
+ve
+ ve
+ ve
+ ve
+ ve
+ ve
-ve
-ve
+ ve
- ve
+ ve
- ve
+ ve
+ ve
+ ve
- ve
+ ve
+ ve
- ve
URINE AND SPUTUM
BLOOD CULTURE
33
34
35
36
37
38
39
40
120
Results
116
117
118
119
120
121
BLOOD CULTURE
URINE AND SPUTUM
CULTURE
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
WOUND CULTURE
Table (9): Shape and arrangement of bacterial isolates collected from

wounds, blood, urine and sputum and their reaction
against Gram's stain from Fahd king Hospital, Al Qassim,
Saudi Arabia.
Isolate Source of
Shape of cell and arrangement
Gram
no.
isolates
stain
Cocci in cluster
Bacilli short rods
Cocci in cluster
Bacilli short rods
Bacilli short rods
Cocci in pairs
Cocci in cluster
Bacilli short rods
Bacilli short rods
Bacilli short rods
Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Cocci in pairs
Cocci in cluster
Cocci in cluster
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Bacilli short rods
Bacilli short rods
Cocci in pairs
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in cluster
Bacilli short rods
Bacilli short rods
Cocci in cluster
Cocci in cluster
Cocci in cluster
+ ve
- ve
+ ve
-ve
-ve
+ ve
+ ve
- ve
- ve
- ve
+ ve
+ ve
-ve
+ ve
+ ve
+ ve
+ ve
+ ve
+ ve
- ve
+ ve
+ ve
- ve
- ve
+ ve
+ ve
- ve
+ ve
+ ve
- ve
- ve
- ve
+ ve
+ ve
+ ve
Bacilli in chain
Cocci in cluster
- ve
+ ve
Cocci in cluster
Bacilli short rods
Cocci in cluster
+ ve
- ve
+ ve
- ve
121
Results
Table (10): Distribution of collected bacterial isolates according to

their Gram's stain reaction and source of isolation from
Sidnawy Hospital, Zagazig, Egypt.
Source of isolation
Wound culture
blood culture
Urinary and sputum
culture
Total
Gram positive
isolates
No.
%
Gram negative
isolates
No.
%
Total
No.
22
17
8
46.8
36.2
17
18
9
6
54.5
27.3
18.2
40
26
14
50
32.5
17.5
47
58.75
33
41.29
80
100
'
'
:RXQGFXOWXUH
EORRGFXOWXUH
8ULQDU\DQG
VSXWXPFXOWXUH
Figure (3): Distribution of collected bacterial isolates

according to their Gram's stain reaction and source of
isolation
122
Results
Table (11): Distribution of collected bacterial isolates according to

their Gram's stain reaction and source of isolation from
king Fahd Hospital, Al Qassim, Saudi Arabia.
Source of isolation
Wound culture
blood culture
Urinary and sputum
culture
Total
Gram positive
isolates
No.
%
Gram negative
isolates
No.
%
Total
No.
13
6
6
52
24
24
8
5
3
50
31.25
18.75
21
11
9
51.22
26.83
21.95
25
60.98
16
39.02
41
100
'
'
:RXQGFXOWXUH
EORRGFXOWXUH
8ULQDU\DQG
VSXWXPFXOWXUH
Figure (4): Distribution of collected bacterial isolates according to their

Gram's stain reaction and source of isolation.
123
Results
Table (12): Distribution of the total bacterial isolates according to

their Gram's stain reaction and source of isolation.
Source of isolation
Gram positive
isolates
No.
%
Gram negative
isolates
No.
%
Total
No.
Wound culture
blood culture
35
23
57.4
62.2
26
14
42.6
37.8
61
37
50.4
30.6
Urinary and sputum

culture
Total
14
60.9
39.1
23
19.0
72
59. 50
49
40.50
121
100
'
'
:RXQGFXOWXUH
EORRGFXOWXUH
8ULQDU\DQGVSXWXP
FXOWXUH
Figure (5): Distribution of total collected bacterial isolates according to

their Gram's stain reaction and source of isolation for all
isolates.
124
Results
4. Morphological and biochemical tests for identification of all

bacterial isolates.
This experiment deals with studying the culture, morphological and
physiological characteristics of the 121 isolates.
The isolated bacteria were divided into 5 species according to
microscopy and cultural characters (their morphological characteristics):
1: Gram +ve cocci, cluster arrangement, coagulase positive, Non-motile,
grow on all ordinary media, grow on mannitol salt agar give yellow
colonies and non-spore forming.
2: Gram + ve cocci, cluster arrangement, coagulase negative, Non-motile,
grow on all ordinary, media grow on mannitol salt agar give pink
3: Gram -ve bacilli, Motile, straight arrangement, grow on all ordinary
media and on nutrient media and non-spore forming.
4: Gram -ve bacilli, Non-motile, monoid or diploid arrangement, grow well
on nutrient and MacConkey media and non-spore forming.
2: Gram + ve cocci, monoid arrangement, coagulase negative, Non-motile,
grow on all ordinary, media grow on mannitol salt agar give pink
Tables: 13 and 14 indicate that 57 isolates were included in group 1, while
12 isolates in group 2, On the other hand there were 14 isolates in group 3.
While 36 Gram-negative isolates in group 4.
125
Results
Table (13): Grouped of bacterial isolates from Sidnawy Hospital,

Zagazig University, Egypt according to morphological and
physiological characteristics.
Species (1) Species (2) Species (3) Species (4) Species (5)
1
2
3
5
8
4
17
6
9
65
11
25
7
10
12
37
15
13
14
62
22
20
16
66
38
23
18
70
39
24
19
74
41
27
21
76
46
32
26
48
33
28
61
35
29
44
30
49
31
51
34
56
36
57
40
68
42
69
43
71
45
73
47
77
50
80
52
53
54
55
58
59
60
63
64
72
75
76
78
79
126
Results
Table (14): Grouped of bacterial isolates from king Fahd Hospital Al

Qassim, Saudi Arabia according to morphological and
physiological characteristics.
Species (1) Species (2) Species (3) Species (4) Species (5)
81
86
110
82
83
96
116
84
87
105
121
85
91
88
92
89
94
90
95
93
97
100
98
103
99
104
101
107
102
111
106
112
108
119
109
113
114
115
117
118
120
127
Results
4.1. Identification of clinical bacterial isolates

All bacterial isolates included in the five Species were identified
manually as detected in table (15), figure (6, 7, and 8) and photo (1, 2, 3
and 4).
The staining reactions the culture characteristics of the isolates on
simple, enriched and selective media were recorded. As follows Gram
positive cocci and Gram negative bacilli were occurred. The biochemical
reactions were tabulated and examined as catalase test, coagulase, oxidase,
indole, methyl red, vogas proskuar , citrate utilization , H2S ,sugar
fermentation as (D-glucose , lactose , sucrose, maltose , D-sorbitol , Dmannose ) , DNase at 25c nitrate reduction and ONPG.
The results of manually identification indicated that,
Species 1: Staphylococcus aureus
Species 2: Staphylococcus
Species 3: Pseudomonas
Species 4: Klebsiella
Species 5: Micrococcus
128
Results
Table (15): Morphological and biochemical reactions of the five

groups of bacterial isolates:
Biochemical tests Species 1
Catalase
+
Coagulase
+
Oxidase
Indole
MR
Vp
Citrate
H2 S
Urease
+
Nitrate reduction
+
D-Glucose
+
Lactose
+
Sucrose
+
Maltose
D-sorbitol
D-Mannitol
D-Xylose
D-Mannose
DNase at 25OC
ONPG
Tentative ID
+
+
+
+
-
Species 2
+
+
+
+
+
+
Species 3
+
+
+
+
+
-
+
+
+
+
-
+
+
+
-
Species 4 Species 5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Staphylococ Staphylococc Pseudomon Klebsiella Microococ

us
as
us
cus aureus
129
Results
Photo no.1: S. epidermidis

and
S. aureus on
mannitol salt agar
Photo no. (2): P. aeruginosa

On nutrient agar
Photo no.(3):Staphylococcusaureusonabloodagarplate.
S.aureuscoloniesappearsmooth,convex,creamwhite Photo no. (4): K. pneumoniae
on MacConkey agar
Figure(6):Indole reaction,
Figure(7):Voges-Proskauer,
130
Figure(8):Methyl red
Results
5. Prevalence of microorganisms in burned patients.

5.1. Swab culture
The incidence of bacteria in swab culture table (16) and figure (9)
indicated that S. aureus were the most predominant. It represented 28 out
of 61total isolates (45.9%). This was followed by P. aeruginosa 19 isolates
(31%). Seven isolates of K. pneumonia were recorded (11.5%). Sex isolates
of S. epidermidis (9.9%) were encountered. Furthermore, one isolates of
Micrococcus (1.7%) was also obtained.
5.2. Blood culture:
Results in table (17) and figure (10) indicated that S. aureus were the
most predominant. It represented 18 out of 61total isolates (48.7%). This
was followed by P. aeruginosa 10 isolates (27%). Five isolates of K.
pneumoniae were recorded (13.5%). Three isolates of S. epidermidis
(8.1%) were encountered. Furthermore, one isolates of Micrococcus (2.7%)
was also obtained.
5.3. Urine and sputum cultures:
Results in table (18) and figure (11) indicated that S. aureus were the
most predominant. It represented 18 out of 61total isolates (47.8%). This
was followed by P. aeruginosa seven isolates (30.5%). Two isolates of K.
pneumoniae were recorded (8.7%). Three isolates of S. epidermidis
(13.0%) were encountered.
5.4. Incidence of bacteria in total cultures:
The above results could be summarized as the following: A total of
cultures were carried out during the study period. These included one
hundred cultures were collected from seventy burned patients; seventy six
were positive result (presence of colony), while twenty four showed
negative result (no growth).
A total of one hundred and twenty one bacterial isolates were
isolated from all the culture specimens. Sixty one isolates were isolated
from swab cultures; thirty seven isolates obtained from blood cultures,
twenty three isolates were isolated from urine and sputum cultures.
Staphylococci were the most predominant bacteria. Thus 57 isolates
were identified as S. aureus. This represented 47% of the total 121 isolates
followed by P. aeruginosa 36 isolates 29.8%, while K. pneumoniae 14
131
Results
isolates represented 11.6% of the total 121 isolates. S. epidermidis 12

isolates represented 9.9%, while only twso isolates of 121 isolates were
Micrococcus as showed in and table (19) and figure (12).
Table (16): Prevalence of bacterial group isolated from wound culture
(swabs of the wound):
Bacterial group
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Total
Number of isolates
28
6
19
7
1
61
Percentages
45.9%
9.9%
31%
11.5%
1.7%
100%
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Figure (9): Prevalence of bacterial group isolated from Swab culture.
132
Results
Table (17): Prevalence of bacterial group isolated from blood cultures:

Bacterial group
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Total
Number of isolates
18
3
10
5
1
37
Percentages
48.7%
8.1%
27.0%
13.5%
2.7%
100%
Prevalence
percentage%
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Figure (10): Prevalence of bacterial group isolated from blood cultures
133
Results
Table (18): Prevalence of bacterial group isolated from urine and

sputum cultures:
Bacterial group
Number of isolates Percentages
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Total
11
3
7
2
0
23
47.8%
13.0%
30.5%
8.7%
0%
100%
W
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Figure (11):Prevalence of bacterial group isolated from urine and

sputum cultures.
134
Results
Table (19): Incidence of bacterial group in total cultures:

Bacterial group
Number of isolates
Percentages
57
12
36
14
2
121
47%
9.9%
29.8%
11.6%
1.7%
100%
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Total
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Figure (12): Incidence of bacterial group in total cultures.
135
Results
6. Antibiotic susceptibility test.

The antibiotic resistance profile was determined by the disc agar
diffusion (DAD) technique using different antibiotic discs against the
different bacterial isolates collected from different clinical specimens from
burned patients.
6.1. Antibiotic resistance of Staphylococcus.
The activity of different types of antibiotic against 57 isolates of
Staphylococcus isolates recovered from different clinical specimens was
shown in table (20).
The antibiotic susceptibility of Staphylococcus is represented in table
(21) and figure (13). The activity of ciprofloxacin, cefotaxime, amoxycillin,
ampicillin\sulbactam,
chloromphenicol,
rifampicin,
erythromycin,
tetracyline, oxycilline, cephardin, vancomycin, ampicillin, imipenem,

methicillin and cefepime were determined against Staphylococcus. The
result suggested high resistant to ampicillin and methicillin, while
susceptible to imipenem and vancomycin except the isolate no. 12, 30, 50
and 95 which showed resistance to vancomycin. S. aureus isolates showed
high intermediate to Amoxicillin.
6.2. Antibiotic resistance of Micrococci
The activity of different types of antibiotic against 2 isolates of
Micrococcus recovered from different clinical specimens were shown in
table (22) and figure (14).
The result suggested high resistant to many antimicrobial agent as
amoxicillin oxycilline and methicillin, while Micrococcus isolates showed
high susceptibility to imipenem, erythromycin, ciprofloxacin and
vancomycin.
136
Results
6.3. Antibiotic resistance of P. aeruginosa

The activity of different types of antibiotic against 36 isolates of P.
aeruginosa recovered from different clinical specimens was shown in table
(24).
The antibiotic susceptibility of Gram-negative bacteria is represented
in table (25) and figure (15) the activity of 12 antibiotics (ciprofloxacin,
cefotaxime,
amoxycillin,
chloromphenicol,
rifampicin, erythromycin, tetracyline, tobramycin, cephardin, imipenem

and cefepime) were determined against P. aeruginosa and K. pneumoniae.
The result of P. aeruginosa suggested high resistant to many
antibiotic as amoxicillin and rifampicin, while showed high susceptibility
to imipenem and tobramycin. P. aeruginosa isolates showed high
intermediate to cephardin.
6.4. Antibiotic Resistance of Klebsiella isolates.
The activity of different types of antibiotic against 20 isolates of K.
pneumoniae recovered from different clinical specimens was shown in
table (26).
The result of K. pneumoniae suggested high resistant to many
antibiotics as amoxicillin and rifampicin, while showed high susceptibility
to imipenem and ciprofloxacin K. pneumoniae showed high intermediate to
cephardin in table (27) and figure (16).
137
Results
Table (20): Evaluation of inhibition zone for S. aureus against

different antibiotic.
Antibiotic
Isolate no.
CIP CTX AX SAM
R
R R
I
1E
S
R R
S
4E
R
R R R
11E
S
R R R
12 E
R
S
S R
14E
S
R R R
16E
S
S R
S
18E
S
R S
I
19E
S
R
I
I
21E
R
I
I
S
26E
S
S
S R
28E
S
R S
S
29E
R
S R R
30E
S
S R R
31E
R
S
S
S
34E
S
R R
S
36E
S
R R
S
40E
S
R S R
42E
S
R R
I
43E
R
R R R
45E
S
I
S R
47E
R
R R
S
50E
R
I
I
S
52E
R
R
I
I
53E
S
S R
S
54E
S
S
I
S
55E
R
S
S R
58E
I
S R
I
59E
S
I
I
S
60E
S
S R R
63E
R
R R R
64E
S
S R
S
72E
S
R R
S
75E
S
R
I
R
76E
S
R R R
78E
S
R R
S
79E
S
R R
I
81S
S
S R R
83S
R
I
I
S
87S
S
S
S R
91S
S
R S
S
92S
S
S
S
S
94S
C
R
R
S
I
S
S
S
S
S
S
I
R
R
S
R
S
R
S
S
S
I
R
R
S
I
R
S
S
S
S
S
S
S
S
S
S
R
I
R
I
R
S
RF
S
R
S
R
R
R
S
S
S
S
S
R
R
S
R
S
S
R
S
S
S
S
S
S
S
I
R
S
S
R
S
S
S
S
R
S
S
R
S
R
R
S
138
E
I
R
S
R
S
R
R
S
S
S
S
R
R
S
R
S
S
R
S
R
S
R
S
S
S
R
S
S
S
S
S
R
S
S
R
S
S
S
S
R
R
S
TE OX CE VA
R R R S
R S R S
R S R S
R R R R
R R R S
S R R S
S S R S
S R S S
S R R S
R S S S
S S R S
S S R R
R R I R
R R S S
S S R S
I R S S
R S I S
S R R S
S S R S
S S S S
S I I S
S R R R
R R S S
S S R S
R R S I
S S R S
S R R S
I S I S
S R R S
S S I S
R R I S
I S S S
R S I S
R R R S
R R R S
R R S S
S R R S
S S R S
R R S S
S S R S
S S R S
S S I S
AM IPM MET FEP

S
S
R
S
S
R
R
S
S
S
R
S
R
S
R
R
R
S
R
I
R
I
R
R
R
S
S
R
R
S
S
R
I
S
R
I
R
S
R
S
R
S
R
S
S
S
R
R
R
S
R
R
R
I
S
S
S
S
R
S
R
S
R
I
S
S
R
S
R
I
R
R
I
S
R
S
R
S
I
I
R
S
R
R
R R
S
R
R
S
R
S
R
S
R
S
S
S
I
S
R
S
R
S
R
S
R
R
R
S
S
I
R
S
S
R
I
S
S
S
S
S
R
I
R
S
S
S
I
S
S
S
R
S
R
R
I
S
R
R
S
S
I
I
R
S
R
S
S
S
R
S
R
S
R
S
R
S
R
S
S
S
R
R
R
S
R
R
Results
95S
97S
98S
99S
101S
102S
106S
108S
109S
113S
114S
115S
117S
118S
120S
R
R
S
S
S
S
R
S
S
R
R
S
S
S
R
R
S
S
R
R
R
R
I
R
I
I
S
R
S
S
R
S
R
R
S
R
R
S
R
I
I
S
S
S
R
Table continue
R S R S R
S R S R S
S S S S I
S R S S R
R S R R S
I S S S S
R S S R S
R I S S S
S S R S S
S R S S R
S R S S R
R I R R S
S R R R S
S S S S S
R S S S R
R
S
R
S
R
S
S
I
R
R
R
S
S
S
R
R R
R S
S S
I S
S S
R S
S S
I S
R S
S S
S S
R S
R S
I S
S S
R
S
R
S
R
I
R
R
I
R
R
R
S
R
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
I
R
R
R
R
R
R
I
S
S
R
R
R
R
R
S
R
S
I
S
S
S
I
R
R
S
S
S
R
R
S
No. S: Isolates no. from Saudia Arabia.

No. E: Isolates from Egypt.
S: sensitive
R: resistant
I: intermediate
Table (21): Antibiotic susceptibility profile of Staphylococcus isolates.

Staphylococcus
Antibiotic
CIP
CTX
AX
SAM
C
RF
E
TE
OX
CE
VA
Am
IPM
MET
FEP
S
No of
suscepetibile
isolates
37
20
17
23
32
37
36
31
27
17
51
14
49
12
28
%
64.9
35.08
29.8
40.35
56.14
64.91
63.15
54.38
47.36
29.82
89.47
24.56
85.96
21.05
49.12
I
No of
intermediate
isolates
1
8
10
8
8
1
1
4
2
11
1
7
4
4
9
139
%
1.7
14.03
17.5
14.03
14.03
1.75
1.75
7.01
3.50
19.29
1.75
12.28
7.01
7.01
15.78
R
No of
resistant
isloates
19
29
30
26
17
19
20
22
28
29
5
36
4
41
20
%
33.3
50.87
52.6
45.61
29.82
33.33
35.08
38.59
49.12
50.87
8.77
63.15
7.01
71.92
35.08
Results
^
/
/W
y
Ky
s
/WD
&W
Types of Antibiotic discs

Fig (13): Antibiotic susceptibility profile of Staphylococcus isolates.
Table (22): Antibiotic susceptibility of Micrococcus.
Antibiotics
CIP CTX AX SAM C RF E TE OX CE VA AM IPM MET CFP
Isolate no.
8
R I S R R
65
S R S S
Table (23): Antibiotic susceptibility profile of Micrococcus.

Micrococci
Antibiotic
CIP
CTX
AX
SAM
C
RF
E
TE
OX
CE
VA
Am
IPM
MET
FEP
S
No of
suscepetibilty
isolates
2
0
0
1
1
0
2
1
0
1
2
0
2
0
1
%
100
0
0
50
50
0
100
50
0
50
100
0
100
0
50
I
No of
intermediate
isolates
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
140
%
0
0
0
0
0
50
0
0
0
0
0
0
0
0
0
R
No of
resistant
isolates
0
2
2
1
1
1
0
1
2
1
0
0
0
2
1
%
0
100
100
50
50
50
0
50
100
50
0
0
0
100
50
Results
^
/
/W dy
y ^D
Z&
d
Ky

s
D /WD Dd &W

Fig (14): Antibiotic susceptibility profile of Micrococcus.
Table (24): Antibiotic susceptibility of P. aeruginosa.
Antibiotics
Isolate no.
CIP CTX AX SAM C RF E TE TBR CE IPM FEP
10
13
s20
23
24
27
32
33
35
44
49
51
56
57
68
69
71
73
141
Results
77
80
82
84
85
88
89
90
93
100
103
104
107
111
112
119
Table (25): Antibiotic susceptibility profile of P. aeruginosa.

Antibiotic
CIP
CTX
AX
SAM
C
RF
E
TE
TBR
CE
IPM
FEP
S
No of
suscepetibile
isolates
%
16
44.44
14
38.88
12
33.33
17
47.22
12
33.33
4
11.11
15
41.66
22
61.11
36
100
9
25
25
69.44
17
47.22
P.aeruginosa
I
No of
intermediate
isolates
0
5
0
0
6
2
1
5
0
8
5
4
142
%
0
13.88
0
0
16.66
5.55
2.77
13.88
0
22.22
13.88
11.11
R
No of
resistant
isloates
20
17
24
19
18
30
20
9
0
19
6
15
%
55.55
47.22
66.66
52.77
50
83.33
55.55
25
0
52.77
16.66
41.66
Results
^
/
/W
y
dZ
&W

Fig (15): Antibiotic susceptibility profile of P. aeruginosa.
Table (26): Antibiotic susceptibility of K. pneumoniae.
Antibiotics
Bacterial
CIP CTX AX SAM C
RF
TE TBR CE IPM FEP
isolate
15
22
38
39
41
46
48
61
110
116
121
S: sensitive, R: resistant, I: intermediate
143
Results
Table (27): Antibiotic susceptibility profile of K. pneumoniae.

K. pneumoniae
Antibiotic
S
No of
suscepetibile
isolates
12
9
4
3
8
4
4
8
4
8
12
10
CIP
CTX
AX
SAM
C
RF
E
TE
TBR
CE
IPM
FEP
I
No of
intermediate
isolates
0
0
1
0
3
0
0
0
0
2
1
0
%
85.71
64.28
28.57
21.42
57.14
28.57
28.57
57.14
28.57
57.14
85.71
71.42
%
0
0
7.14
0
21.43
0
0
0
0
14.28
7.14
0
R
No of
resistant
isloates
2
5
9
11
3
10
10
6
10
4
1
4
%
14.28
35.71
64.28
78.57
21.43
71.42
71.42
42.86
71.42
28.57
7.14
28.57
^
/
/W
dy
y
^D
Z&
d
dZ

/WD
&W

Fig (16): Antibiotic susceptibility profile of K. pneumoniae isolates.
144
Results
7. Determination of minimum inhibitory concentration (MIC) and

minimum bacterocidal concentration (MBC).
Minimum inhibitory concentration can be defined as: the lowest
csoncentration that inhibit the growth in liquid media while the minimum
bactericidal concentration is defined as the lowest concentration that
inhibits the growth in solid media. The experimented bacteria were treated
separately with different concentration of the antibiotics under test in
Muller-Hinton broth and on solid agar medium. In the present
investigation, MICs and MBCs of vancomycin, imipenem, ciprofloxacin
and oxycillin antibiotics against the selected isolates of S. aureus cultivated
in liquid media were examined. The four antibiotics were observed to be
the most effective antibiotics against the 4 tested bacterial groups. Thus, it
was important to determine the MIC and MBC of this antibiotic. MIC
values were determined visually.
7.1. Determination of minimum inhibitory MICs and MBCs of
vancomycin for multi resistant S.aureus.
The MICs and MBCs of vancomycin against four multiresistant S.
aureus were determined. The obtained data recorded in table (28) and
figure (17).
The results revealed that MICs of S. aureus no. 12 and 50 were 62.5
g/ml.
On the other hand MICs of S. aureus no. 30 and 95 were 31.25 g/ml. The
MBC of S. aureus no. 12, 30 and 95 were 62.5 g/ml., while S. aureus no.
50 was 125 g/ml.
145
Results
7.2. Determination of MICs and MBCs of imipenem for multiresistant

S. aureus.
The MICs and MBCs of imipenem against four multiresistant
isolates were determined. The data were recorded in table (29) and figure
(18).
The results revealed that MICs and MBCs of imipenem antibiotic for
S. aureus no. 12 and 95 were 62. 5 g/ml, also S. aureus no. 50 had the
same MICs and MBCs 15.625 g/ml, while MICs and MBCs of S. aureus
no. 30 were 31.25 and 62.5g/ml, respectively
7.3. Determination of MICs and MBCs of ciprofloxacin for
multiresistant S.aureus.
The MICs and MBCs of ciprofloxacin were determined the obtained
data recorded in table (30) and figure (19).
The results indicated that MICs and MBCs of ciprofloxacin for S.
aureus no. 12 and 30 were 62. 5 and 125 g/ml, respectively. Also S.
aureus no. 50 had the same MICs and MBCs 125 g/ml, while MICs and
MBCs of S. aureus no. 95 were 31.25 and 62.5g/ml, respectively.
7.4. Determination of minimum inhibitory MICs and MBCs of
oxycilline antibiotic for multi resistant S.aureus no. (12, 30, 50 and
95)
The MICs and MBCs of oxycilline were determined the obtained
data recorded in table (31) and figure (20). The results demonstrated that
MICs of oxycilline were 125, 62.5, 31.25 and 15.625 g/ml, for S.aureus
no. 12, 30, 50 and 95, respectively. Also MBCs of Oxycilline antibiotic
were 250, 125, 62.5 and 15.625 g/ml, for S.aureus no. 12, 30, 50 and 95,
respectively.
146
Results
Table (28): Determination of (MIC) and (MBC) of vancomycin

antibiotic against four multiresistant S.aureus.
MIC(g/ml)
MBC(g/ml)
S. aureus (12)
63
63
S. aureus (30)
31.25
63
S. aureus (50)
63
125
S. aureus (95)
31.25
63
Tested bacteria
D/
D
Fig (17): Determination of (MIC) and (MBC) of vancomycin antibiotic

against four multiresistant S. aureus.
147
Results
Table (29): Determination of (MIC) and (MBC) of imipenem against

the four multiresistant S.aureus.
Tested bacteria
MIC(g/ml)
MBC(g/ml)
62.5
S. aureus (12)
62.5
S. aureus (30)
30
62.5
S. aureus (50)
15.625
15.625
S. aureus (95)
62.5
62.5
D/
D
Fig (18): Determination of (MIC) and (MBC) of imipenem against the

four multiresistant S. aureus.
148
Results
Table (30): Determination of (MIC) and (MBC) of ciprofloxacin

against the four multiresistant S. aureus.
MIC(g/ml)
MBC(g/ml)
S. aureus (12)
63
125
S. aureus (30)
63
125
S. aureus (50)
125
125
S. aureus (95)
31.25
63
Tested bacteria
D/
D
Fig (19): Determination of (MIC) and (MBC) of ciprofloxacin against

the four multiresistant S. aureus.
149
Results
Table (31): Determination of (MIC) and (MBC) of oxicillin against the

Tested bacteria
MIC(g/ml)
MBC(g/ml)
S. aureus (12)
125
250
S. aureus (30)
63
125
S. aureus (50)
31.25
63
S. aureus (95)
15.625
15.625
D/
D
Fig (20): Determination of (MIC) and (MBC) of oxicillin against the

150
Results
8. Bactericidal action of different disinfectants against the four

multiresistant S. aureus :
Ethanol and hydrogen peroxide were selected (as the most effective
disinfectants used in burn units)for carrying out this experiment.
8.1. Effect of different concentrations of ethanol against multiresistant
S. aureus.
Ethanol 50, 60 and 70% were tested against the tested bacterial
isolates S. aureus (12, 30, 50 and 95) to detect the required exposure time
needed when ethanol diluted. The obtained results were represented in table
(32) and figure (21). From the results, it was observed that increasing in
exposure time was accompanied with a clear increase in inhibitory effect
against the tested bacterial isolates S. aureus (12, 30, 50 and 95).
The dilution of ethanol to 50% will be ineffective for inhibition of
the tested isolates even exposure period was 30 minutes, while duration at
60% ethanol for 20 minutes was enough to inhibit all tested bacteria. On
the other hand at the highest concentration 70%, the lowest exposure time
was for 15 minutes. It could be concluded that the best concentration of
ethanol to be used DVGLVLQIHFWDQWZDVIRUPLQXWHVLWZDVHQRXJK
to inhibit all associated bacteria.
8.2. Effect of different concentration of hydrogen peroxide against
multiresistant S. aureus
The concentration of 5, 6 and 7 % of hydrogen peroxide for different
exposure time against the most resistant S. aureus of the present work. The
lethal effects depend on both duration time and concentration of
disinfectants as represented in table (33) and figure (22). It was observed
that increasing in exposure time was accompanied with a clear increase in
inhibitory effect against the tested S. aureus (12, 30, 50 and 95).
The dilution of hydrogen peroxide to 6% for inhibition 20 minutes
all S. aureus did not show any growth, while duration at 7% ethanol for 15
151
Results
minutes was satisfied to inhibit all tested bacteria. On the other hand at the
highest concentration 8%, the lowest exposure time was at 10 minutes, so
we could concluded that the best concentration of hydrogen peroxide to be
used as disinfectant was 8 % for 10 minutes it was enough to inhibit all
tested bacterial isolates.
Table (32): Exposure time inhibition of different concentration of
ethanol for multiresistant S. aureus no. (12, 30, 50 and 95).
Concentration of
Exposure time inhibition (minutes)
ethanol % S. aureus12 S. aureus
S. aureus
S. aureus
30
50
95
70
15
13
11
10
60
20
18
13
11
50
28
>30
20
25
S. aureus12
S. aureus30
S. aureus50
S. aureus95
Ethanol concentration %
Figure (21): Exposure time inhibition of different concentration of
ethanol for multiresistant S. aureus no. (12, 30, 50 and
95).
152
Results
Table (33): Exposure time inhibition of different concentration of H2O2

for multiresistant S. aureus no. (12, 30, 50 and 95).
Exposure time inhibition (minutes)
H2O2
S. aureus
concentration % S. aureus12 S. aureus30 S. aureus
50
95
8
7
6
10
11
15
10
15
18
10
11
20
8
11
20
S. aureus12
S. aureus30
S. aureus50
s
S. aureus95
H2O2 concentration %
Figure (22): Exposure time inhibition of different concentration of
H2O2 for multiresistant S. aureus no. (12, 30, 50 and 95).
9. Susceptibility of multiresistant S. aureus (12, 30, 50 and 95) to

certain essential oils.
This experiment was directed to study the effect of different essential
plant oils on the growth of multi resistant S. aureus isolates. All oils were
tested for their antimicrobial activity using the well agar diffusion method.
The antimicrobial activity was expressed as diameter of inhibition zone
(mm). The results in table (34) reveal that, thyme oil and tea tree oil had the
highest antibacterial activity against the four S. aureus (12, 30, 50 and 95)
with inhibition zones (8.2, 7.5, 8.0 and 9 mm), respectively, while tea tree
oil with inhibition zones (8.0, 8.3, 7.5 and 9 mm), respectively followed by
153
Results
clove, lemon grass. Cinnamon oil had the lowest antibacterial activity
against the four S. aureus (12, 30, 50 and 95) with inhibition zones (0, 1.2,
1.3 and 3.5 mm), respectively.
Table (34): Effect of essential oils against the four multiresistant S.
aureus.
Clear zone (mm).
Essential oil
S. aureus12 S.aureus30
Thyme
Cinnamon
Lemmon grass
Clove
Garlic
Olive
Tea tree
Peppermint
Nigella sativa
8.2
-ve
4.5
6.5
-ve
-ve
8.0
2.0
4.3
7.5
1.2
3.0
4.0
1.6
3.5
8.3
2.0
-ve
S. aureus
50
8.0
1.3
2.0
5.5
-ve
1.5
7.5
-ve
5.0
S.aureus
95
9.0
3.5
-ve
4.5
1.2
3.0
9.0
1.6
2.2
^
^
^
^
>
'
d
E
Figure (23): Effect of essential oils against the four multiresistant S.

aureus.
154
Results
9.1. Determination of the MIC and MBC of thyme oil for

multiresistant S. aureus (12, 30, 50 and 95).
The minimum inhibitory concentration and minimum bactericidal
concentration were determined for the selected thyme and tea tree oils
(which gave the best antimicrobial activity). The results in table (35) and
Figure (22 and 23) showed that the MIC and MBC value for thyme oil
against S. aureus12 were 62.5 ug/ml. Also the MIC value of thyme oil for
S. aureus 30 was 15.62 ug/ml and MBC recorded 31.25 ug/ml, while MIC
and MBC value for thyme oil against S. aureus 50 and S. aureus 95 were
7.8 and 15.62 ug/ml, respectively.
The results in table (36) reveal that the MIC and MBC value for tea
tree oil against S. aureus 30 were 31.25ug/ml. Also the same MIC and
MBC for S. aureus (50) 125 ug/ml. The MIC of tea tree oil for S. aureus
(95) was 125 ug/ml and MBC recorded 250 ug/ml.
9.2. Influences of combination between two types of combination
between (vancomycin + thyme oil) & (vancomycin + tea tree oil)
on the growth of S. aureus (12, 30, 50 and 95).
The investigation was extended to include the study of two types of
combination between (vancomycin + thyme oil) & (vancomycin + tea tree
oil) against multi resistance S. aureus (12, 30, 50 and 95).
Different treatments were carried out through this experiment. The
tested bacteria were subjected to the effect of Van disc (30g) loaded with
thyme oil (30l) and VAN (30g) loaded with tea tree oil (30 l).
The results in tables (37 and 38) and figures (24 and 25)
demonstrated that the combination between (vancomycin + thyme oil)
&(vancomycin + tea tree oil) gave a synergetic effect against each isolates
of S. aureus no.(12, 30, 50 and 95) as in plates ( 5& 6), respectively, where
the inhibition zone of VAN disc saturated with 30 l thyme oil or saturated
155
Results
with 30 l tea tree oil were more than that of either VAN disc or thyme oil
or tea tree oil each separately.
Table (35): Determination of the MIC and MBC of thyme and tea tree
oil for multiresistant S. aureus no. (12, 30, 50 and 95).
S. aureus12
Essential
oils
Thyme
oil
Tea tree
oil
MIC
MBC
62.5
7.8
S. aureus 50
S. aureus30
MIC
S. aureus 95
MBC
MIC
MBC
MIC
62.5 15.62
31.25
7.8
15.62
15.62 15.62
7.8
15.62
15.62
31.25
15.62
MBC
7.8
15.62
D/
D
^
^
^
^
Fig (24): Determination of the MIC and MBC of thyme oil for
D/
D
^
^
^
Fig (25): Determination of the MIC and MBC of tea tree oil for
156
Results
Table (36): Influence of combination between VAN antibiotic and

thyme oil.
Bacterial
species
Inhibition zone (mm)

VAN
S. aureus 12
10.0
THYME OIL
(30g)
8.2
S. aureus 30
11.0
7.5
18.5
S. aureus 50
9.0
8.0
17.0
S. aureus 95
10.0
9.0
19.0
VAN + THYME oil (30g)

15.0
^
^
^
^
sE
d,zDK/>
sEd,zD
Figure (26): Influence of combination between VAN antibiotic and

thyme oil.
Table (37): Influence of combination between VAN antibiotic and tea
tree oil.
Bacterial
species
Inhibition zone (mm)

VAN
S. aureus 12
10.0
TEA TREE OIL

(30g)
8.0
S. aureus 30
11.0
8.5
19.5
S. aureus 50
9.0
7.5
16.5
S. aureus 95
10.0
9.0
19.0
157
VAN + TEA TREE OIL

(30g)
18.0
Results
^
^
^
^
sE
d/>
sE
Figure (27): Influence of combination between VAN antibiotic and tea

tree oil.
Vancomycin
VAN+
Thyme oil
Thyme oil
Photo no. (5): Influence of combination between vancomycin and

thyme oil compared with each separately.
158
Results
Tea tree oil

Vancomycin
Van + Tea tree oil

Photo no. (6): Influence of combination between vancomycin and tea
tree oil compared with each separately.
.
10- Screening for the virulence factors and degrading enzymes
produced by multiresistant S. aureus:
The selected bacterial isolates identified as S. aureus were screened
for their capability of producing degrading enzymes and certain virulence
factors by using well agar diffusion method. Data in table (38) revealed that
four examined isolates produced the tested virulence factors including
haemolysin causing -hemolysis, licithinase degrading phospholipids into
insoluble lipids in the form of white ppt. and proteinase degrading casein
protein as extracellular degrading enzymes.
10.1. Studying the virulence factor of selected S. aureus (12, 30, 50 and
95).
Further study was concerned with to determine the activity of
different concentration of crude cell free filtrates 10, 20 & 30 l on Blood
agar, egg yolk agar and casein agar plates. Increase in diameters of
inhibition zones denoting enzymatic activities was directly proportional
with the increase in volume of filtrates.
159
Results
Results in table (39) show that S aureus12 was the highest

concerning hemolytic activity, S. aureus 50 was the highest in lecithinase
activity, S. aureus 30 the highest in proteinase activity, S. aureus 95 was
the second in lecithinase and protease activities.
10.2. The effect of H2O2 and ethanol on virulence factors of tested
isolates:
The capability of tested bacteria to produce its virulence factors after
exposure to hydrogen peroxide (5, 6, and 7%) and ethanol (50, 60, and
70%) as chemical disinfectants was examined. The results in tables (40 and
41) revealed the correlation between zone diameters due to virulence
factors (lecithinase, haemolysin and proteinase (volume 10l)) produced by
tested disinfectant treated isolates. The results revealed that 7% H2O2 was
sufficient to depress lecithinase activity, while 8% H2O2 was sufficient to
depress haemolysin and protease activity. Also the results demonstrated
that 70 % ethanol was sufficient to depress lecithinase, haemolysin and
proteinase activity. The above data was analyzed by Chi square test and
revealed that there was a significant correlation between ethanol & H2O2
concentration and the reduction in virulence factors of tested isolates (P <
0.01).
Table (38): Screening for the virulence factors and degrading enzymes
produced by tested S. aureus isolates.
Bacterial isolates
S. aureus 12
S. aureus 30
S. aureus 50
S. aureus 95
Lecithinase
Haemolysin
+VE
+VE
+VE
+VE
+VE
+VE
+VE
+VE
160
Proteinase
+VE
+VE
+VE
+VE
Results
Table (39): The virulence factors (VFs) of selected S. aureus (12, 30, 50
and 95) using different volumes 10, 20 & 30 l.
Bacterial
isolates
Diameter of inhibition zone (mm)

Lecithinase
Haemolysin
Proteinase
10 l 20l 30l 10l 20l 30l 10l 20l 30l
S. aureus 12
S. aureus 30
20
10
24
12
26
15
24
20
28
19
30
13
15
25
18
30
20
32
S. aureus 50
22
24
28
14
12
20
24
22
S. aureus
95
18
21
27
18
15
12
18
22
24
Table (40): The effect of different conc. of H2O2 (volume 10l) on VFs
of tested isolates.
Lecithinase
H 2O 2
Conc.
0%
6%
7%
8%
Haemolysin
Proteinase
S12
S30
S50
S95
S12
S30
S50
S95
S12
S30
S50
S95
20
10
0
0
10
7
0
0
22
12
0
0
18
0
0
0
24
16
10
0
20
13
8
0
14
8
4
0
18
11
0
0
15
6
0
0
25
18
5
0
20
15
0
0
18
10
0
0
Table (41): The effect of different conc. of ethanol (volume 10l) on

VFs of tested isolates.
ethan
ol
Lecithinase
Haemolysin
Proteinase
Conc.
0%
50%
60%
70%
80%
S12
S30
S50
S95
S12
S30
S50
S95
S12
S30
S50
S95
20
10
8
0
0
10
7
5
0
0
22
12
9
0
0
18
9
5
0
0
24
16
8
0
0
20
13
9
0
0
14
8
5
0
0
18
11
6
0
0
15
12
9
0
0
25
18
10
0
0
20
15
11
0
0
18
10
7
0
0
10.3. Effect of tea tree oil on the enzyme activities (virulence factors).
The effects of tea tree oil on the production of virulence factors
(VFs) by S. aureus (12, 30, 50 and 95) had been investigated.
161
Results
The production of the lecithinase by S. aureus (12, 30, 50 and 95) showed
lower levels when cultured with 0.3% tea tree oil. Four S. aureus (12, 30,
50 and 95) isolates, significantly lower levels of hemolysin and protease
were produced in the presence of 0.6 % tea tree oil when compared to
control cells. In general, these experiments demonstrate significant
reduction in levels of protease, hemolysin and lethinase after growth for 24
h. in the presence of 0.3 and 0.6% tea tree oil.
The above data in table (42) were analyzed by Chi square test and
revealed that there was a significant correlation between oil concentration
and the reduction in virulence factors of tested isolates (P < 0.01).
Table (42): The effect of tea tree oil on virulence factors of
multiresistant S. aureus.
Lecithinase
Hemolysin
Protease
Tea
tree oil
Conc.
S12
S30
S50
S95
S12
S30
S50
S95
S12
S30
S50
S95
0%
0.1%
0.3%
0.6%
20
18
0
0
18
7
0
0
25
12
0
0
20
18
0
0
24
15
5
0
20
11
3
0
12
11
8
0
18
15
7
0
15
7
0
0
25
20
5
0
20
18
8
0
24
22
10
0
11. Molecular identification and characterisation assays of van A &

mec A gene.
11.1. Identification of the selected bacterial isolates
The four multi resistant bacterial isolates were identified using 16S
rDNA sequencing to confirm that the four isolates were S. aureus and were
deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases
(Center for Information Biology and DNA Data Bank of Japan, National
Institute of Genetics, Research Organization of Information and Systems,
Japan), under accession no. 12(ZWFR 12) [AB674512], 30(ZWFR 30)
162
Results
[AB674513], 50(ZWFR 50) [AB674514] & 95(ZWFR 95) [AB674515],

respectively.
11.1.1.Extraction of chromosomal DNA preparation of the selected
bacteria.
The
DNA
of
the
selected
bacteria
was
extracted
using
GenEluteBacterial Genomic DNA Kit, sigma Aldrich as shown in figure

(26).
The 16S rDNA gene was amplified by polymerase chain reaction
(PCR) using universal primers designed to amplify 10 K base pair.
The 16S rDNA gene was amplified by (PCR) using universal primers
designed to amplify 1.5 K base pair fragment of the 16S rDNA region
figure (27).
M: DNA marker
12, 30, 50, 95: bacterial isolates
12 30 50 95
M: DNA marker (10 kb)

(12-95) selected strain
Figure (28): The DNA of the selected

bacteria using GenEluteBacterial Genomic DNA Kit, sigma Aldrich.
163
Results
Figure (29): The 16S rDNA gene amplified by polymerase chain reaction
(PCR) of the selected bacterial isolates no. (12, 30, 50 and 95).
Automated DNA sequencing
The 16S rDNA genes were sequenced using 3130 genetic analyzer at
sequencer unit of city for scientific research and technology application.
Isolate no. 12
CTCCTTCGTTCATATCATGCTAGTCGAGCGACGGACGAGAAGCTTGCTTCTC
TGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAA
ACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTTGAACC
GCATGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCG
CGCTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCAT
AGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGG
TCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAA
GCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAAC
TCTGTTATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGT
ACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACG
TAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGG
TTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGG
Analysis after using ncbi
Sequences producing significant alignments:

Accession
Description
GU459255.1 Staphylococcus aureus isolate K54-2

16S ribosomal RNA gene, partial
sequence
HM311188.1 Uncultured bacterium clone
ncd1164c05c1 16S ribosomal RNA
gene, partial sequence
sequence
164
Max Total
Query
E
Max
score score coverage value ident
1074 1074 97%
0.0
99%
1070
1070
98%
0.0
99%
1068
1068
97%
0.0
99%
Results
DQ170822.1 Uncultured Staphylococcus sp. clone
WS03A_F12 16S ribosomal RNA
ncd948h07c1 16S ribosomal RNA
ncd948g03c1 16S ribosomal RNA
AM945662.1 Staphylococcus equorum partial 16S
rRNA gene, isolate equorum 1
FR821779.1 Staphylococcus aureus subsp. aureus
LGA251 complete genome sequence
JF322979.1 Staphylococcus sp. 1036(2011) 16S
ribosomal RNA gene, partial sequence
JN084556.1 Staphylococcus aureus strain
Pn1016/Pant-Uttar/2007 16S
366/Chen 16S ribosomal RNA gene,
partial sequence
partial sequence
1068
1068
98%
0.0
99%
1066
1066
99%
0.0
98%
1066
1066
99%
0.0
98%
1066
1066
99%
0.0
98%
1066
1066
97%
0.0
99%
1064
6372
98%
0.0
99%
1064
1064
97%
0.0
99%
1064
1064
98%
0.0
99%
1064
1064
98%
0.0
99%
1064
1064
98%
0.0
99%
Phylogenetic analysis
BLAST program www.ncbi.nlm.gov/blast phylogenetic analysis was
used to assess the DNA similarities of obtained 16S rDNA gene sequence.
Multiple sequence alignment and molecular phylogeny were performed
using BioEdit software. The phylogenetic tree was displayed using
TREEVIEW program.
165
Results
JN084556.1| Staphylococcus aureus strain Pn1016/Pant-Uttar/2007
JN084552.1| Staphylococcus aureus strain 366/Chen
JF322979.1| Staphylococcus sp. 1036(2011)
JN652894.1| Uncultured Staphylococcus sp. clone C19
DQ170822.1| Uncultured Staphylococcus sp. clone WS03A_F12
GU459256.1| Staphylococcus aureus isolate K54-3
Isolate no 6
AM945662.1| Staphylococcus equorum partial
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
Isolate no. 30
CCCGGGTCGGTTATGCTCTGCTAGTCGAGCGACGGACGAGAAGCTTGCTTCT
CTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAG
AGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGA
CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGA
CGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTT
ATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTACCTA
ATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT
GGCAAGCGTTAT

Accession
Description
Max Total Query

E
Max Links
929 929
96%
0.0 99%
AM945662.1 Staphylococcus equorum partial

16S rRNA gene, isolate equorum
1
928
ncd1164c05c1 16S ribosomal
RNA gene, partial sequence
GU459256.1 Staphylococcus aureus isolate
928
K54-3 16S ribosomal RNA gene,
partial sequence
166
928
96%
0.0
99%
928
96%
0.0
99%
Results
GU459255.1 Staphylococcus aureus isolate
K54-2 16S ribosomal RNA gene,
partial sequence
GQ467748.1 Uncultured bacterium clone
R4J3C4_B10 16S ribosomal
nbu171g03c1 16S ribosomal
JF411480.1 Bacterium 1H302 16S ribosomal
EU373089.1 Bacterium ACCC1 16S
ribosomal RNA gene, partial
sequence
FR690784.1 Uncultured Staphylococcus sp.
partial 16S rRNA gene, clone
9.32
9.26
3.20
2.28
FR821779.1 Staphylococcus aureus subsp.
aureus LGA251 complete
genome sequence
JF322979.1 Staphylococcus sp. 1036(2011)
sequence
Pn3576/Pant-Orissa/2008 16S
sequence
Pn2087/Pant-Jhark/2008 16S
sequence
sequence
sequence
Pn833/Pant-Jhar/2007 16S
sequence
557/Chen 16S ribosomal RNA
928
928
96%
0.0
99%
928
928
96%
0.0
99%
928
928
96%
0.0
99%
924
924
96%
0.0
99%
924
924
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
5518
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
167
Results
922
922
96%
0.0
99%
922
922
96%
0.0
99%
Phylogenetic analysis
FR690555.1| Uncultured Staphylococcus sp. partial
JF322979.1| Staphylococcus sp. 1036
JF411480.1| Bacterium 1H302
AB674510.1| Staphylococcus aureus
Isolate no 19
HM311188.1| Uncultured bacterium clone ncd1164c05c1
GQ467748.1| Uncultured bacterium clone R4J3C4_B10
GQ001329.1| Uncultured bacterium clone nbu171g03c1
AB674511.1| Staphylococcus aureus gene for
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
Isolate no. 50
TTGCTCGGTTATATCATGCTGTCGAGCGACGGACGAGAAGCTTGCTTCTCTG
ATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAGACT
GGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTTGAACCGCA
TGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGCT
GCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCATAGCC
GACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTC
CAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGC
CTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTC
TGTTATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTAC
CTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA
GGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTT
TTTTAAGTCTGATGTGAAAGCCCACGGC
168
Results

Accession
Description

sequence
Pn2087/Pant-Jhark/2008 16S
partial sequence
partial sequence
FJ899095.1 Staphylococcus aureus strain SC01
sequence
AM945662.1 Staphylococcus equorum partial 16S
rRNA gene, isolate equorum 1
Pn833/Pant-Jhar/2007 16S ribosomal
sequence
nbu170a08c1 16S ribosomal RNA
FN826758.1 Uncultured bacterium partial 16S
rRNA gene, clone
US7.36_V02193C_041
FM163053.1 Staphylococcaceae bacterium ACEMC
3-3 partial 16S rRNA gene, isolate
ACEMC 3-3
WS03A_B09 16S ribosomal RNA
JF228544.1 Uncultured bacterium clone
ncd2613d10c1 16S ribosomal RNA
ncd1300e06c1 16S ribosomal RNA
169
Max Total Query

E
Max
1050 1050 98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1044
1044
98%
0.0
99%
1044
1044
98%
0.0
99%
1044
1044
98%
0.0
99%
1044
1044
97%
0.0
99%
1044
1044
98%
0.0
99%
1042
1042
98%
0.0
99%
1042
1042
97%
0.0
99%
1042
1042
98%
0.0
99%
1040
1040
98%
0.0
99%
1040
1040
97%
0.0
99%
1040
1040
98%
0.0
99%
Results
HM074037.1
HM317980.1
GQ001329.1
FR821779.1
JN084559.1
JN084556.1
JN084552.1
JN084551.1
JN092619.1
JF784041.1
JF513981.1
CP002643.1
JF015377.1

Uncultured Staphylococcus sp. clone
DH06_83 16S ribosomal RNA gene,
partial sequence
Uncultured bacterium clone
ncd331a05c1 16S ribosomal RNA
nbu171g03c1 16S ribosomal RNA
Staphylococcus aureus subsp. aureus
Staphylococcus aureus strain
partial sequence
partial sequence
Staphylococcus aureus strain FFL34
sequence
Staphylococcus aureus strain CIFRI
CH-TSB27 16S ribosomal RNA gene,
partial sequence
Staphylococcus aureus strain sc-sa 16S
Staphylococcus aureus subsp. aureus
T0131, complete genome
Phylogenetic analysis.
170
1040
1040
98%
0.0
99%
1040
1040
97%
0.0
99%
1040
1040
97%
0.0
99%
1038
6217
98%
0.0
99%
1038
1038
97%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
5183
98%
0.0
99%
1038
1038
98%
0.0
99%
Results
FM163053.1| Staphylococcaceae bacterium ACEMC 3-3 partial
DQ170796.1| Uncultured Staphylococcus sp. clone WS03A_B09
JN831370.1| Staphylococcus aureus strain SU-BIO3
JN084555.1| Staphylococcus aureus strain Pn833
FJ899095.1| Staphylococcus aureus strain SC01
JN084557.1| Staphylococcus aureus strain
JN084558.1| Staphylococcus aureus strain Pn2087
JN102552.1| Staphylococcus aureus subsp. aureus strain FUA2076
JN102568.1| Staphylococcus aureus subsp. aureus strain FUA2081
AB674510.1| Staphylococcus aureus
Isolate 30
GQ001181.1| Uncultured bacterium clone nbu170a08c1
0.0008
0.0006
0.0004
0.0002
0.0000
Isolate no. 95
CCCGTCGGCTCATGATCATGCTAGTCGAGCGACGGACGAGAAGCTTGCTTCT
CTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAG
AGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGA
CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGA
CGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTT
ATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTACCTA
ATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT
GGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGGCGGTTTT
TTTAAGTCTGATGTGAAAGCCCCACGGCT

Accession
Description

171
Max Total
Query
E
Max
1046 1046 97%
0.0
99%
Results
ncd920d10c1 16S ribosomal RNA
ncd580a07c1 16S ribosomal RNA
nbw1028e12c1 16S ribosomal RNA
sequence
ncd158b09c1 16S ribosomal RNA
WS03A_F12 16S ribosomal RNA
FR821779.1 Staphylococcus aureus subsp. aureus
partial sequence
partial sequence
JN092619.1 Staphylococcus aureus strain FFL34
sequence
JF784041.1 Staphylococcus aureus strain CIFRI
CH-TSB27 16S ribosomal RNA gene,
partial sequence
JF513981.1 Staphylococcus aureus strain sc-sa 16S
CP002643.1 Staphylococcus aureus subsp. aureus
T0131, complete genome
Phylogenetic analysis.
172
1046
1046
97%
0.0
99%
1046
1046
97%
0.0
99%
1046
1046
97%
0.0
99%
1042
1042
97%
0.0
99%
1042
1042
97%
0.0
99%
1035
1035
97%
0.0
98%
1035
1035
98%
0.0
98%
1033
6183
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
98%
0.0
98%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
5155
97%
0.0
99%
1033
1033
97%
0.0
99%
Results
GQ031979.1| Uncultured bacterium clone nbw1029d06c1

JF228544.1| Uncultured bacterium clone ncd2613d10c1
HM253183.1| Uncultured bacterium clone ncd40d12c1
HM283416.1| Uncultured bacterium clone ncd715b12c1
HM289006.1| Uncultured bacterium clone ncd739f12c1
JF096462.1| Uncultured bacterium clone ncd1300g01c1
JF105744.1| Uncultured bacterium clone ncd1206c07c1
JF108951.1| Uncultured bacterium clone ncd1168h01c1
JF121007.1| Uncultured bacterium clone ncd1385b09c2
GQ033257.1| Uncultured bacterium clone nbw1028e12c1
HM280136.1| Uncultured bacterium clone ncd580a07c1
Isolate no 50
JF096378.1| Uncultured bacterium clone ncd1302g01c1
JF211590.1| Uncultured bacterium clone ncd2439f02c2
JF114021.1| Uncultured bacterium clone ncd1249e12c1
JF096338.1| Uncultured bacterium clone ncd1300d11c2
HM290708.1| Uncultured bacterium clone ncd684f09c1
0.0008
0.0006
0.0004
0.0002
0.0000
11.2. Amplification of target van A & mec A gene using Polymerase

chain reaction
The target van A and mec A genes were detected in four tested
isolates after amplification using PCR in which the primers F: EA1 & & R:
EA2 gave rise to a fragment of about 800 bp size.
The suspected PCR fragments as van A genes were cut from Gel and
purified using Kit EZ-10spin column (BioBasic INC. Canada) for further
cloning.
173
Results
Different primers were used for detection of van A gene & mec A
genes and different conditions were used for the same pair of primers.
11.2.1. Detection of mec A gene
mecA F AAAATCGATGGTAAAGGTTGGC
mecA R AGTTCTGCAGTACCGGATTTTGC
11.2.2. Detection of van A gene
VANA: ATGAATAGAATAAAAGTTGCAATAC
VANA1: CCCCTTTAACGCTAATACGAT
After using different condition for PCR there are not give any PCR
product..so we make another design for primers
NEW DESIGN
Forward primer TGGCTCAGGTACTGCTATCCACCC
Reverse primer ACCACCCAATTTGTCTGCCAGTT
This pair of primer gave product 818
Forward primer GGCTCAGGTACTGCTATCCACCCT
Reverse primer AACCACCCAATTTGTCTGCCAGT
Forward primer CCAGGAATGCAGAAAGACCAAAGCA
Reverse primer
GGGTGGATAGCAGTACCTGAGCCA

All this primers were matched with S. aureus
After using different condition there are not give any PCR product, no mec
A gene in the 4 isolate.
Detection of van A gene
van A 1
Forward primer: AGCTCAGCAATTTGTATGGA
Reverse primer: GGGGATAACGACTGTATGAC
174
Results
van A 2
Forward primer: GCTCAGCAATTTGTATGGAC
Reverse primer: TGTCATATTGTCTTGCCGAT
After using different condition for PCR. Only primer of van A give
positive for S. aureus 12 and S. aureus 50 only and did not present in
another two isolates. May be another gene for vancomycin resistant may
van C or van R or other.
11.2.3. Amplification of the van A gene

The van A gene was amplified by polymerase chain reaction (PCR)
using primers designed to amplify 945 base pair fragment. After
completion the PCR reaction, a fraction of PCR was examined using
agarose gel.
a PCR test tube.
Van A 1 primer give 945 bp as shown in figure 30 ( this analysis
were done by using program for MWanalysis )
Figure (30): Amplification of the van A gene by polymerase chain reaction

(PCR) of the selected bacterial isolates no. (12 and 50).
175
176
Discussion
Discussion
Burn patients are ideal hosts for opportunistic infections. Infections
remain the leading cause of death among patients who are hospitalized for
burns (Tancheva and Hadjiiski, 2005).
In spite of considerable advances in the treatment of burns; infection
continues to pose the greatest danger to burned patients. Approximately 73
per cent of all death within the first five days post-burn have been shown to
be directly or indirectly caused by septic processes.
Different patient
groups such as burned patients and trauma patients have unique

predisposition to different infections (Simon et al., 2009).
The development of antibiotics for the chemotherapy of bacterial
infections represents one of the most remarkable achievements of last
century. The occurrence and spread of antibiotic-resistant bacteria (ARB)
in burn patients are pressing public health problems worldwide. Many
bacteria have become and continue to be resistant nearly against all
antimicrobial agents. The resistance rates are higher in developing
countries (Cochran et al., 2010).
The present investigation covered a total of seventy burned patients;
40 from Sidnawy Hospital, Zagazig University, Zagazig, Egypt and 30
patients from king Fahd Hospital, Al Qassim, Saudi Arabia. The history of
burned patients from type of injury, age, gender, total body surface area
and antibiotic treatment were specified. From static analysis of these
parameters it was found that females (57.1%) were affected more than that
of males (42.9%). This may be because of the reason that accidental burns
are more common in females as they tend to spend more time near fire.
These finding does not correlate with Zaid. (2001) who observed males
(70%) were affected more than that of females (30%).
177
Discussion
Results also revealed that most common age group affected was 20
to 40. This came in concordance with study conducted by Mertens et al.
(2000) who documented that the age group of most burned patients ranged
from 20 50 years.
Data analysis revealed that exposure to flame led to thermal injury in
33 patients out of 70 total patients. This accounted for 47.1%. Hot water
thermal injury was implicated in 16 cases. Electricity revealed 17 patients
all of them from males. Only four patients out of 70 total patients
represented in (5.7%) suffered from chemicals burns. In agreement with
this found, Hill and Bowe (2002) stated that, thermal injury caused by
exposure to flame has the highest percentage among burned patients, then
hot water and the injury caused by electricity.
Moreover, there was a highly significant correlation between age and
death. The 29 patients who were able to recover were significantly younger
than those who died. Thus the recovered group had a mean age of 18 years,
while the group that suffered death had a mean age of 48.3 years. This
matched with (Zaid, 2001) who observed that the twenty three out of 50
patients were able to recover because they were younger than those who
died.
In our study, itisestimatedthat all the patients suffered burn of
7% up to 94% of TBSA burns. More than 58 % of the patients died either
from infection, by the burn itself or by both of them. Also 29 patients who
were able to recover with mean TBSA burns of 22% while, the other 41
patients mean TBSA burns of 51% died. This indicated that the death is
directly proportional to the total body surface area. This clearly matched
with Nichols (2008) who reported that incidence of infection is also
affected by the size of the patients burn injury. At BH, Burn hospital the
incidence of infection was determined for patients with <30% TBSA burn
injury compared to patients with 30% TBSA. The overall incidence of
178
Discussion
infection was low for patients with <30% TBSA burn injuries and generally
associated with the need for invasive devices. Bloodstream infection (BSI)
increases dramatically as burn wound size increases, related to increased
exposure to intravascular catheters and to burn wound manipulationinduced bacteremia. At (BH), Boston from 1996-2000 there were 3 cases of
BSI in 585 patients with <30% TBSA burn injury compared to 55 cases of
BSI in 60 patients with 30% TBSA. In this series, pneumonia occurred
only in ventilated patient, accounting for all 15 cases seen. Similarly,
urinary tract infection (UTI) occurred principally in patients with
indwelling urinary catheters, accounting for 33 of the 36 cases seen.
The present study showed that from the twenty nine patients (41.4%)
were recovered, while the other 58.7% were died. Comparing the number
of positive and negative cultures for each patient, it was found that there is
no relation between patient death and number of positive and negative
cultures. This agrees with Nour El-Dien. (1999) who reported that from
the 30 patients who recovered, five patients had negative swab cultures the
other twenty five had positive culture. On the other hand, among the other
50 patients who died, only three had negative swab cultures, while the
others showed positive results in their swab cultures. So the data revealed
that there was no relation between patient death and the presence of local
infection.
Santucci et al. (2003) reported that infections remain the leading
cause of death among patients who are hospitalized for burns. The risk of
burn wound infection is directly correlated to the extent of the burn and is
related to impaired resistance resulting from disruption of the skins
mechanical integrity and generalized immune suppression
It is quite clear in the present study that the incidence of total
microorganisms isolated from swab, blood, urine and sputum culture from
burned patients indicate that Gram-positive bacteria were the most
179
Discussion
predominant. The Gram-positive isolates represent 72 out of 121 total

isolates (59. 50%) while 49 isolates were Gram-negative (40.50%). Many
investigators have found that initially there is a colonization by Grampositive organisms which is replaced later by Gram-negative organisms
(Ahmad and Aqil, 2006).
In the present study the preliminary differentiation between the
isolates based on staining reactions, cell shape, culture characteristics (on
simple, enriched and selective media) and biochemical reactions were
carried out. The isolated bacteria were divided into 4 main species I: was
Staphylococcus, II: was Micrococcus, III: was Klebsiella, IV: was
Pseudomonas.
The incidence of these groups in tested cultures indicated that S.
aureus was the most predominant (47%) followed by P. aeruginosa
(29.8%), K. pneumonia (11.6%), S. epidermidis (9.9%) and Micrococcus
sp. (1.7%). In this relation Revathi et al. (1998) indicated that the organism
has entered the bloodstream through the wound and is a potential threat for
disseminated infection which can be life threatening. Microorganisms
routinely isolated from burn wounds include aerobic organisms like S.
aureus, P. aeruginosa and Proteus.
Although incidences of mortality and morbidity resulting from burns
have declined over the years, particularly after early excision came into
popular practice, burn wound infections continue to pose a serious threat to
burn victims. The two most prevalent colonizers of burn wounds are S.
aureus and P. areuginosa (Liu et al., 2003).
Also Zaid (2001) reported that Gram-positive organisms of
particular concern include methicillin-resistant S. aureus (MRSA) and
coagulase negative Staphylococcus. MRSA was first seen in the United
States in the late 1960s and has become an endemic organism in many burn
units. It has been argued that no extraordinary efforts be made to control its
180
Discussion
spread; however this view has been increasingly challenged in the era of
vancomycin-resistant Enterococcus (VRE). Also (Tredget, 2004) reported
that the most common organisms isolated from burn patients were S.
aureus and P. aeruginosa.
This on line with Tiwari and Sen. (2006) who found that S. aureus
was the most common isolate from blood followed by P.aeruginosa, and
Coagulase Negative Staphylococci (CONS). In most of the cases the
organisms isolated from blood were the same as isolated from pus.
On the other hand Ahmad and Aqil. (2006) found that P.
aeruginosa (19%) was the commonest isolate from burn wounds followed
by S. aureus (15%), S. epidermidis (11%) and E.coli (10.5%). It is evident
that P.aeruginosa has emerged as a great threat in burn wound infection
and its very important that antibiotic policy is formulated to keep a check
on it. P.aeruginosa was the most common isolate from blood also followed
by S.epidermidis, Klebsiella, S.aureus and E.coli. This in contrary with
Biswajit et al. (2008). They reported that Gram-negative organisms were
found to be more prevalent. P. aeruginosa was found to be the most
common isolate followed by S. aureus, S. epidermidis, Klebsiella and
Salmonella. In most of the cases, same organisms were found in blood and
pus sample.
Antibiotic sensitivity testing (AST) aims to determine the
susceptibility of an isolate to a range of potential therapeutic agents. This
can be with a view to individualizing the antibiotic to be administered or to
monitor resistance patterns developing in that environment, gathering this
information is important for revising and updating the standard antibiotic
prescribing policy for a particular population or institution. The antibiotic
sensitivity against bacteria is assayed by three different methods, liquid
dilution, agar dilution and disc diffusion method by following the reference
standards established by various authorities such as the Clinical and
181
Discussion
Laboratory Standards Institute (CLSI, USA), British Society for

Antimicrobial Chemotherapy (BSAC, UK) (Sahloff and Martin, 2002).
It is worthy to mention here that results in this study showed that the
susceptibility of the selected bacterial isolates were detected toward
different antibiotics. The activity of ciprofloxacin, cefotaxime, amoxycillin,
chloromphenicol,
rifampicin,
erythromycin,
tetracyline, oxycilline, cephardin, vancomycin, ampicillin, imipenem,

methicillin and cefepime were determined against staphylococci. The
results suggested that Staphylococcus sp. showed high resistance to
ampicillin and methicillin, S. aureus isolates showed high intermediate
resistant to amoxicillin, while susceptible to imipenem and vancomycin
except the isolates no. 12, 30, 50 and 95 showed resistance to vancomycin.
Vancomycin- resistant S. aureus tend to be multidrug resistant
against a large number of currently available antimicrobial agents,
compromising treatment options and increasing the likelihood of
inadequate antimicrobial therapy and increase in morbidity and mortality
(Pierard et al., 2004).
In the present study, VRSA showed resistance to a wide range of
antimicrobial agents. This may be due to the misused of antibiotics.
The reduced susceptibility to vancomycin by S. aureus is
hypothesised to be aresult of changes in peptidoglycan synthesis. There is a
visible irregularly shaped and thickened cell wall in these VISA isolates
due toincreased amounts of peptidoglycan. Evidently, there is a decrease in
crosslinking of the peptidoglycan strandsresulting in the exposure of more
D-alanyl-D-alanine residues (Walshand Howe, 2002).
The trend of multidrug resistance in
S. aureus is particularly
alarming because of the severity and diversity of diseases caused by this

pathogen (Bonfiglio et al., 1998). Despite the availability of novel drugs as
an approach to staphylococcal therapy, the bacteria seem to be able to
182
Discussion
rapidly develop resistance to these drugs. Perhaps the most commonly
known resistance of S. aureus, is methicillin resistance, which has caused
alarming reports with regard to the spread of S. aureus in hospitals and the
community (Gtz, 2010).
Moreover, Rybak and Akins. (2001) reported that the emergence of
high levels of penicillin resistance followed by the development and spread
of isolates resistant to the semi synthetic penicillins (methicillin, oxacillin,
and nafcillin), macrolides, tetracycline, and aminoglycosides has made the
therapy of staphylococcal disease a global challenge.
Furthermore, Matsukawa et al. (2001) found that penicillin and
first to second generation cephalosporin in vitro sensitivity tests must be
undertaken as a guide for intelligent antimicrobial therapy.
The emergence of glycopeptide- resistance of S.aureus was
significantly marked in hospital that had been endemic with isolates of
methicilliin resistant staphylococci and followed the policy of empirical
use of glycopeptides. Extensive use of glycopeptides (vancomycin or
teicoplanin) allowed selection of resistant isolates of S. aureus.
Glycopeptide- resistant mutants of S. aureus have been experimentally
selected by gradually increasing the levels of vancomycin present during
in-vitro growth (Rybak and Akins, 2001).
`Glycopeptides such as vancomycin are frequently the antibiotics of
choice for the treatment of infections caused by methicillin resistant
Staphylococcus aureus (MRSA) (Arthur et al., 1993). For the last 7 years
incidence of vancomycin intermediate S. aureus and vancomycin resistant
S. aureus (VISA and VRSA, respectively) has been increasing in various
parts of the world (Gtz, 2010).
Moreover, Cui et al. (2009) study indicated that vancomycin
resistance in S. aureus may occur under the selective pressure of prolonged
183
Discussion
vancomycin use and that the resistance to vancomycin in S. aureus is

reversible on removal of the drug.
The imipenem has proved to be an extremely useful antibacterial
agent because of its great - lactamase stability and high intrinsic activity
against a broad range of bacteria (Neu, 1999).
It was reported previously that, mechanism of resistance of
Staphylococci to vancomycin is still not clear (Fridkin, 2001). But transfer
of the vanA gene cluster from Enterococci to S. aureus has been
demonstrated in the laboratory by in vitro experiment of also on the skin of
mice. This finding has raised concern about the possibility of occurrence of
such mechanism of genetic transfer in clinical isolates of staphylococci,
which often co-colonize wound infection sites with enterococci.
Previously, there was no study showed the presence of any of van genes
clusters in clinical isolates of Staphylococci and so such mechanism could
not be proved (Smith et al., 2006).
Hiramatsu et al., (1997) reported that vancomycin-intermediate S.
aureus (VISA), first described in 1997, has continuously been a worldwide
problem in the treatment of methicillin-resistant S. aureus (MRSA) hospital
infection. VISA has a unique mechanism of resistance; the resistant cell
produces a thickened cell wall, whereby many vancomycin molecules are
trapped within the cell wall. The trapped molecules clog the peptidoglycan
meshwork and finally form a physical barrier towards further incoming
vancomycin molecules. VISA does not directly emerge from vancomycinsusceptible MRSA. It emerges from hetero-VISA that expresses
heterogeneous-type vancomycin resistance.
All S. aureus isolates that have acquired the vanA operon are
resistant to methicillin when grown in the absence of the glycopeptide. It
has been demonstrated that vancomycin (or teicoplanin) and oxacillin, at
184
Discussion
concentrations that are achievable in therapy, have a powerful synergistic

effect against VRSA both in vitro (Depardieu et al., 2006).
In the presence of representatives of both drug classes, such as
vancomycin and oxacillin, bacteria synthesize modified peptidoglycan
precursors ending in D-Ala-D-Lac which are not substrates for PBP2 in the
transpeptidation step. Because the TPase activity of PBP2 is inactivated by
oxacillin, synthesis of peptidoglycan cannot proceed, resulting in death of
the bacteria (Proft et al., 2000).
This study showed also that the activity of 12 antibiotics
(ciprofloxacin,
cefotaxime,
amoxycillin,
chloromphenicol, rifampicin, erythromycin, tetracyline, tobramycin,

cephardin, imipenem and cefepime) were determined against P. aeruginosa
and K. pneumoniae. It was found that, P. aeruginosa showed high
resistance to amoxycillin and rifampicin, while showed high susceptibility
to imipenem and tobramycin. Tested
P. aeruginosa showed high
intermediate resistance to cephardin.

Also the tested K. pneumoniae showed high resistant to many
antimicrobial agent as amoxycillin and rifampicin, while showed high
susceptibility to imipenem and ciprofloxacin. K. pneumoniae isolates
showed intermediate to cephardin.
The results are agreement with Giacommetti et al. (2000) they
reported that a very low susceptibility rate (10% sensitivity) was found for
ampicillin and amoxicillin against all Gram-positive and Gram-negative
isolated from surgical wound infections.
In addition, Zaid. (2001) reported that the resistance of 94 isolates of
P. aeruginosa and K.pneumoniae studied to 12 -lactam antibiotics and
found that all tested isolates were resistant to at least 7 -lactam antibiotics
(penicillin, ampicillin, amoxicillin, amoxicillin/ clavulanic acid, cepalexin,
cephardin, and cloxacillin).
185
Discussion
Moreover, Yamaguchi et al. (2002) reported that antibiotic

resistance analysis showed that all tested K.pneumoniae isolates were
highly resistant to ampicillin, piperacillin, carbenicillin, cephaloridine, and
cefotaxime; intermediately resistant to cefoxitin; moderately susceptible to
cephardin and ceftazidime; and susceptible to imipenem.
The present results are matched with Bagherwal and Shukla (1997)
who reported that Staphylococcus spp. and P. aeruginosa isolates were
resistant to amoxicillin, rifampicin, ampicillin, cloxacillin, and erythromycin
but sensitive to ciprofloxacin and pefloxacin.
The present study is in accordance with the study of Srinivasana et
al. (2007) who found that all tested P. aeruginosa isolates were resistant to
two or more antimicrobials used in veterinary medicine including
ampicillin, furadantine, cetazidime and cefotaxime. El-Aidy. (2008) found
that pencillin G and ampicillin showed low activity against all tested Grampositive and Gram-negative isolates.
In the present investigation, MICs and MBCs of vancomycin,
imipenem, ciprofloxacin and oxycillin antibiotics against the selected
isolates of S. aureus cultivated in liquid media were examined, MIC values
were determined visually. The results revealed that MICs of S. aureus no.
12 and 50 were 62.5 g/ml. On the other hand MICs of S. aureus no. 30
and 95 were 31.25 g/ml. The MBC of S. aureus no. 12, 30 and 95 were
62.5 g/ml., while S. aureus no. 50 was 125 g/ml.
Proft et al. (2000) found that the vancomycin MIC for the
vancomycin-resistant S. aureus (VRSA) isolate was 1024 g/ml, which is
1000 times the normal vancomycin MIC for S. aureus (i.e., 1 g/ml). Six
additional VRSA isolates for which the vancomycin MICs ranged from 16
to 512 g/ml. NCCLS guidelines define staphylococci for which the MIC
of vancomycin is 4 g/ml to be susceptible, while isolates for which the
186
Discussion
MIC is 8 to 32 g/ml are intermediate and those for which the MIC is 32
g/ml are resistant.
The present investigation recorded that the four VRSA isolates no.
12, 30, 50 and 95 had a resistant to vancomycin (MIC range, 16-70 mg/l)
This on line with Chang et al. (2003) who reported that the MIC for 335 of
358 isolates (93.57%) for vancomycin was 2 mg/l indicating that all were
sensitive to vancomycin. Sixteen isolates showed an MIC range between 48 mg/l, indicating vancomycin intermediate resistance. For the remaining
seven isolates, the MIC was in the range of 16-70 mg/l indicating that these
seven isolates were vancomycin resistant (VRSA). These were all MRSA
and resistant to majority of the other antibiotics tested. All these seven
isolates showed resistance to a minimum of six other antibiotics including
vancomycin and methicillin.
Furthermore, some studies have been reported the emergence of
VRSA in clinical isolates that. By definition, show in-vitro MICs of > 4 mg
/l. (Rybak and Akins 2001).
The results also indicated that MICs and MBCs of imipenem
antibiotic for S. aureus no. 12 and 95 were 62. 5 g/ml, also S. aureus no.
50 had the same MIC and MBC 15.625 g/ml, while MIC and MBC of S.
aureus no. 30 were 31.25 and 62.5g/ml, respectively. The results
demonstrated that MIC and MBC of ciprofloxacin for S. aureus no. 12 and
30 were 62. 5 and 125 g/ml, respectively. Also S. aureus no. 50 had the
same MIC and MBC 125 g/ml, while MIC and MBC of S. aureus no. 95
were 31.25 and 62.5g/ml, respectively.
The results demonstrated that MICs of oxycilline antibiotic were
125, 62.5, 31.25 and 15.625 g/ml, for S. aureus no. 12, 30, 50 and 95,
respectively. Also MBCs of oxycilline antibiotic were 250, 125, 62.5 and
15.625 g/ml, for S. aureus no. 12, 30, 50 and 95, respectively.
187
Discussion
The resistant to beta-lactam antibiotics may be expected related to

genes that coded for essential membrane proteins involved in the
biosynthesis of murein in peptidoglycane and protein components of the
cell wall (Udo and Grubb, 2001).
In vitro the activity of cefotaxime against 602 S. aureus isolates were
both methicillin and ciprofloxacin resistant (MIC > 15.6 mg/ml). For
ciprofloxacin susceptible, the MIC at which of organisms were inhibited
were 2 and 4 mg/ml), respectively (Low et al., 2002). On the other hand
Chang et al., (2003) reported that the MICs of oxacillin, vancomycin and
teicoplanin determined by agar dilution method.
Volatile oils had been largely employed for their properties already
observed in nature, i.e. for their antibacterial, antifungal, antivirus and
insecticidal activities. At present, approximately 3000 oils were known,
300
of
which
are
commercially
important
especially
for
the
pharmaceutical, agronomic, food, agriculture, sanitary, cosmetic and

perfume industries (Perry et al., 2003 and Silva et al., 2003).
Another experiment in our study was directed to study the effect of
different essential plant oils on the growth of multi resistant S. aureus no.
(12, 30, 50 and 95). All oils were tested for their antimicrobial activity
using the well agar diffusion method. The antimicrobial activity was
expressed as millimeter (mm.) diameter of inhibition zone. The essential
oils of plants used as antioxidant, laxatives, erosive gastritis, dermatology,
urology, sleep and nervous disorders, cardiac and vascular system, and cold
& coughs (Tkachenko et al., 1995).
The volatile oils of plants exhibited various reduction in the growth
according to its chemical composition. Many research groups screened
various plant extracts as secondary metabolites to detect biological
activities (Hassanein & El-Dokch., 6DODPD =DLG DQG
Abo-Ghalia et al., 2004).
188
Discussion
The results in our investigation revealed that, thyme oil and tea tree
oil had the highest antibacterial activity against the four S. aureus (12, 30,
50 and 95) with inhibition zones (8.2, 7.5, 8.0 and 9 mm), respectively,
while tea tree oil with inhibition zones (8.0, 8.3, 7.5 and 9 mm),
respectively followed by clove, lemon grass. Cinnamon oil had the lowest
antibacterial activity against the four S. aureus (12, 30, 50 and 95) with
inhibition zones (0, 1.2, 1.3 and 3.5 mm), respectively.
Our results are in agreement with Deans and Ritchie (1987) where
they found that lemon grass and cinnamon oil had no inhibitory effect
against tested isolates. These results were agreement with Zafra-Polo et al
(1989). They reported that the essential oil of Thymus leptophyllus showed
higher antimicrobial activity against S. aureus and other tested bacteria.
Also the essential oil of Thymus vulgaris (thyme oil) was very effective
against S. aureus, E. coli and Salmonella typhimurium.
The antimicrobial activity of different species of thymus was tested
against bacteria. The essential oil of Thymus broussonettii was most
efficient for killing the S. aureus, E. coli and inhibiting their growth
(Tantaoui et al., 1993).
In this relation, Ela et al. (1996) screened sixteen essential oils for
their antimicrobial activity against S. aureus, by using the agar diffusion
method and measuring their inhibition zone. They classified the tested oils
DFFRUGLQJ WR WKHLU DFWLYLW\ VWURQJO\ DFWLYH LQKLELWLRQ ]RQH ! PP
moderately active (inhibition zone > 6 to < 8 mm), and inactive (no
inhibition zone < 6 mm). Basil and parsley were inhibitory to S. aureus.
The antimicrobial activity of different species of thymus was tested
against bacteria the essential oil of Thymus vulgaris was most efficient for
killing E .coli, other bacteria & fungi and inhibiting their growth (ElGayyare et al., 2003).
189
Discussion
Also El-Aidy. (2008) reported that thyme and tea tree oil were the
most effective oil against all tested isolates E .coli, S. aureus, P.
aerugenosa and K. pneumonieae.
The volatile oils of plants exhibited various reduction in the growth
according to its chemical composition. Many research groups screened
various plant extracts as secondary metabolites to detect biological
activities (Salama & Zaid, 2000 and Abo-Ghalia et al., 2004). The results
in accordance with Rota et al. (2004) who reported that, the bactericidal
properties of thyme essential oil are supposed to be associated with high
levels of carvacrol and linalool. Kalemba and Kunicka (2003) reported
that, because of the great number of cell constituents, volatile oils seem to
have no specific cellular targets. As typical lipophiles, they pass through
the cell wall and cytoplasmic membrane, disrupt the structure of their
different layers of polysaccharides, fatty acids, phospholipids and
permeabilize them. The mode of action of antimicrobial agents also
depends on the type of microorganisms and is mainly related to their cell
wall structure and the outer membrane arrangement cytotoxicity appears to
include such membrane damage. In bacteria, the permeabilization of the
membranes is associated with loss of ions and reduction of membrane
potential, collapse of the proton pump and depletion of the ATP pool
(Turina et al., 2006).
Moreover the volatile oils can act as prooxidants affecting inner cell
membranes and organelles such as mitochondria. Depending on type and
concentration, they exhibit cytotoxic effects on living cells but are usually
non genotoxic (Bakkali et al. (2008).
It is worthy to mention here that the minimum inhibitory
concentration and minimum bactericidal concentration were determined for
thyme oil and tea tree oil. The results showed that the MIC and MBC value
for thyme oil against S. aureus12 were 62.5 ug/ml. Also the MIC value of
190
Discussion
thyme oil for S. aureus 30 was 15.62 ug/ml and MBC recorded 31.25
ug/ml, while MIC and MBC value for thyme oil against S. aureus 50 and S.
aureus 95 were 7.8 and 15.62 ug/ml, respectively. The MIC and MBC
value for tea tree oil against S. aureus 30 were 31.25ug/ml. Also the same
MIC and MBC for S. aureus (50) 125 ug/ml. The MIC of tea tree oil for S.
aureus (95) was 125 ug/ml and MBC recorded 250 ug/ml.
The present investigation was extended to include the study of
combination between (vancomycin + thyme oil) & (vancomycin + tea tree
oil) which gave a synergetic effect against each strain of S. aureus (12, 30,
50 and 95), respectively, where the inhibition zone of VAN disc saturated
with 30 l thyme oil or saturated with 30 l tea tree oil were more than
that of either VAN disc or thyme oil or tea tree oil each separately. This
experiment was carried out for the medical applications in the future
research. Rational for the use of antimicrobial combination was to decrease
emergence of resistance isolates; decreased dose-related toxicity of
antimicrobial using the other agent(s) (Mandell and Sande. 1980) and to
polymicrobial infection (it is one of the reasons for broad-spectrum
coverage).
Cappelletty and Rybak. (1996) reported that combination of
antimicrobial agents are considered to be synergetic if the effect of
combination is greater than the effect of either agent alone or greater than
the sum of the effect of individual agents. Antagonism result occurred if the
combination provides an effect worse than the effect of either agent alone
or worse than the sum the effect of individual agents.
This is in agreement with Mostafa. (2005) who reported that the
combination between oil and antibiotic was more active than each
individual alone. Also the synergistic effect from the association of
antibiotic with oil against resistant bacteria leads to new choices for the
treatment of infectious diseases. This effect enables the use of the
191
Discussion
respective antibiotic when it is no longer effective by itself during

therapeutic treatment (Adam et al., 1998).
El-Aidy (2008) found that combination between impenem antibiotic
and thyme oil gave highly synergetic effect against E. coli and S. aureus as
27 and 30 mm. respectively when used the combination mixture consisting
of 10 l of thyme oil and the equal potency of the antibiotic that used. Also
Abd El-Rahman (2008) studying the effect of combination between thyme
oil and beta-lactam antibiotics (which carried out by absorbance of 10 l of
thyme oil on amoxicillin and ampicillin disc 10 g each separately) against
three different isolates of P. aeruginosa and found that these combinations
gave highly synergetic effect against all tested isolates as 10, 10 & 9.5 mm.
and 9.4, 9 & 8.5 mm., respectively.
Abd El-Hamid. (2010) found that the combination between caraway
oil and ciprofloxacin gave synergetic effect on A. calcoaceticus. The
combination treatment of caraway oil and ciprofloxacin was carried out by
using different MICs percentages (0, 10, 15, 20, 25, 30, 35, 40, 45, and
50%) of caraway oil and ciprofloxacin. The growth of A. calcoaceticus
was completely inhibited when 50% MICs of caraway oil and ciprofloxacin
mixture was used.
Further study was concerned with investigating the potency of the
selected isolates to produce different degrading enzymes and certain
virulence factors. Crude cell free filtrates of 24h cultures of tested isolates
were used to determine activity of bacterial isolates on Blood agar, egg
yolk agar and casein agar plates using well diffusion agar method. Increase
in diameters of clear zones denoting enzymatic activities was directly
proportional with the increase in volume of filtrates by using agar diffusion
method (culture filtrates in wells using different volumes 10, 20 & 30 l).
Several methods for extracellular proteins detection from isolated
isolates had been developed through the years, including biological,
192
Discussion
immunological, chromatographic and molecular assays (Palma et al.

Dinges et al6KULYHU-Lake et al., 1DNDQRet al., 2004).
The virulence factors of pathogenic bacteria are divided into several
groups on the basis of the mechanism of virulence and function. Of the
important ones are secretary proteins such as toxins and enzymes (Wu et
al., 2004).
S. aureus is notorious not only for its ability to rapidly acquire
antibiotic resistance, but also for the wide variety of factors it uses in its
defense against host immunity. Some of these virulence factors are well
known, while others have been uncovered following the sequencing of the
genome (Lim et al., 2004). Results in our study show that S. aureus12 was
the highest concerning hemolytic activity, S. aureus 50 was the highest in
lecithinase activity, S. aureus 30 in proteinase activity, S. aureus 95 was
the second in lecithinase and proteinase activities.
These results are in accordance with data reported in other studies of
Balaban and Rasooly. (2001) who reported that most of isolated S aureus
isolates were found to produce extracellular enzymes: lecithinase,
gelatinase and protease.
These results are on line with Lake et al. (2003) who observed that
nine isolates were hemolytic, with seven of them producing both alpha-and
beta-
hemolysis and two producing only beta-hemolysis, the other 5
isolates producing lecithinase.

Furthermore, Wooldridge. (2009) published that the extracellular
proteins of pathogenic bacteria are main contributors of pathogenesis and
are indisputably involved in bacterial virulence. These proteins have a
range of biological functions ranging from host cell toxicity to more subtle
alterations of the host cell for the benefit of the invader.
Moreover, Su and Wong (1997) found that pathogenic isolates of S.
aureus are capable of producing one or more extracellular proteins and
193
Discussion
toxins simultaneously. Classically, Staphylococcal enterotoxins SEs have

been divided into five major serological types (SEA, SEB, SEC, SED and
SEE) on the basis of their antigenic properties.
The capability of tested bacteria to produce its virulence factors after
70%) as chemical disinfectants was examined. The results revealed the
correlation between zone diameters due to virulence factors (lecithinase,
hemolysin and proteinase) produced by tested disinfectant treated isolates.
The results indicated that 6% H2O2was sufficient to depress lecithinase
activity, while 7% H2O2 was sufficient to depress hemolysin and proteinase
activity. Also the results demonstrated that 70 % ethanol was sufficient to
depress lecithinase activity, hemolysin and proteinase activity.
Very few previous studies have been conducted examining the
effects of subinhibitory antimicrobial agents on the production of
hemolysin. hemolysin production by the Gram-positive S. aureus isolates
was reduced in the presence of 7.0% of ethyle alcohol (Iwalokun et al.,
2003).
Another study, using two S. aureus isolates, found that the presence
of H2O2 had significantly altered protease production after 24 h, compared
to the controls (Edwards-Jones and Foster, 2002). However, the
production of protease appeared to be delayed in the presence of H2O2.
Their study also showed that for one of the isolates the proportions of
proteases varied greatly, with control cells producing largely protease
whereas cells grown with H2O2 produced only a very small amount of
protease.
In our study the investigation of the virulence factor production
(lecithinase, hemolysin and proteinase) by S. aureus (12, 30, 50 and 95)
under treatment of tea tree oil were done. S. aureus (12, 30, 50 and 95)
showed lower levels of lecithinase activity when cultured with 0.3% tea
194
Discussion
tree oil. For four S. aureus (12, 30, 50 and 95) isolates, significantly lower
levels of hemolysin and Proteinase were produced in the presence of 0.6 %
tea tree oil when compared to control cells. In general, these experiments
demonstrate significant reduction in levels of protease, hemolysin and
lethinase after growth for 24 h. in the presence of 0.3 and 0.6% tea tree oil.
The suppression of toxins
and enzymes is an important
part in the
treatment and management of S. aureus (Chang etal.,2003).

Athorough
and complete understandingofthe infections
interactionof
these
S.aureusproductsand componentsisnecessarytoapplythecorrecttreatm
ent andtopreventinfections (Edwards-Jones and Foster, 2002).
These results are in accordance with data reported in study of
Iwalokun et al. (2003) who stated that the exposure to tea tree oil may alter
cell permeability leading to the leakage of extracellular proteins. It is also
hypothetically possible that the presence of tea tree oil affects the
outermost part of the cell wall in such as way as to cause the extracellular
proteins to become less highly associated with the cell wall and therefore
more extracellular proteins is found free in the supernatant. Increased
expression of VFs after treatment with sub-inhibitory levels of
antimicrobial agents has been reported previously, although infrequently. S.
aureus isolates resistant to fluoroquinolones have increased expression of
fibronectin-binding
proteins
after
treatment
with
sub-inhibitory
ciprofloxacin (Bisognano et al., 1997).

Very few previous studies have been conducted examining the
effects of subinhibitory antimicrobial agents on the production of protease.
Protease production by the Gram-positive bacterium S. aureus isolates was
reduced in the presence of 0.75 and 1.0 g/ml Ocimum gratissimum
essential oil (Iwalokun et al., 2003).
195
Discussion
Another study, using four S. aureus isolates, found that the presence
of tea tree oil had significantly altered lethinase production after 24 h,
compared to the controls (Edwards-Jones and Foster, 2002).
It has been observed previously that the suppression of extracellular
protein synthesis occurs only with antibiotics that inhibit protein synthesis
and that antibiotics with other modes of action may in fact have a
stimulatory effect. Although tea tree oil is not an antibiotic, tea tree oil acts
by inhibiting the synthesis of the extracellular enzyme protease (Herbert et
al., 2001).
Our results are not matched with Kernodle et al. (1995) who had
several possible explanations for the apparent increases in protein synthesis
levels after growth in the presence of sub-inhibitory tea tree oil. Another
example is increased -toxin production by S. aureus after treatment with
subinhibitory levels of the -lactam antibiotics nafcillin and methicillin
(Doss et al.2KOVHQet al., 1998).
Moreover, Herbert et al. (2001) reported that the mechanisms
behind the apparent decrease in hemolysin seen in the present study may be
elucidated by the gene encoding hemolysin is lower expressed or
transcribed in the presence of tea tree oil.
Two studies have investigated this very possibility, both assessing
the effectiveness of tea tree oil products in the eradication of MRSA
carriage (Caelli et al.'U\GHQet al., 2004). Data from the first study
showed that tea tree oil formulated into nasal ointment and skin wash
compared favourably to mupirocin nasal ointment and Triclosan skin wash
for the decolonisation of MRSA carriage (Caelli et al., 2000). Similarly,
the second study indicated similar clearance rates for both the tea tree oil
regimen and standard treatment. These clinical outcomes may have
occurred by any number of mechanisms. Firstly, the topically applied tea
tree oil may have had a direct killing effect against the target organisms.
196
Discussion
However, it has been postulated that antimicrobial agents are often present
at only sub-inhibitory levels, and cannot therefore produce killing effects.
This means that sub-inhibitory levels of tea tree oil may somehow
responsible for eradicating S. aureus carriage or infection. Sub-inhibitory
tea tree oil may have any number of effects against the infecting organism,
host cells, and any interactions between the two. Since data from this study
suggest that extracellular VFs such as coagulase, protease and toxins are
not reduced in the presence of tea tree oil, a reduction in the levels of these
proteins is not likely to be the mechanisms by which tea tree oil is acting.
One possibility is that the tea tree oil alters cells of S. aureus in such a way
as to render them susceptible to the effects of the host immune system. This
mechanism and others require investigation (Dryden et al., 2004).
Several genomic targets have been effectively used for the
identification of Staphylococcus species, including the 16S rRNA gene, the
tRNA gene intergenic spacer, the internal transcribed spacer, the heat-shock
protein 60 (HSP60) gen, the chaperonin 60 gene, the femA gene, the sodA
gene, the gap gene and the nuc gene. Recently, an enterobacterial repetitive
intergenic consensus PCR and BOX-PCR were also used in the
identification of S. epidermidis isolates (Walker, 1998). Some antibiotic
resistance genes were found on non conjugative plasmids, these genes may
also move from one plasmid to another. The unstable nature of plasmids
that can spread even to multiple species of bacteria and may be lost or
acquired spontaneously, make plasmid fingerprinting, which was the first
molecular typing method to be used for epidemiological purposes often
poorly reproducible (Stefani & Agodi, 2008).
It is worthy to mention here that our study showed that the four
isolates no. (12, 30, 50& 95) which were isolated from wounds &blood and
selected as multi-resistant to most tested antibiotics were preliminary
identified as S. aureus. PCR amplification of 16s rRNA gene of the four
selected isolates using specific primers produced amplicons of size 1500bp
197
Discussion
for all isolates. PCR products confirmed the identity of the four isolates as
S. aureus. The partial nucleotide sequences of 16S rRNA gene of the four
tested isolates named S. aureus 12 (ZWFR 12) S. aureus 30 (ZWFR 30), S.
aureus 50 (ZWFR 50) and S. aureus 95 (ZWFR 95) were then submitted to
the DDBJ/EMBL/Gen Bank with the accession number(s) 12 [AB674512],
30 [AB674513], 50 [AB674514] and 95 [AB674515], respectively.
Also molecular studies represented in detection of mec A and van A
gene
by
using
different
TheuseofPCRforthedetectionofthemec
typinghasbeendescribedpreviouslyby
Agene
Oliveira
condition.
and
van
andcolleague
(2002),and usedasareferencemethodforSCCmectyping (Zhang etal.,

2005).Inthecaseof
MRSA,PCR
basedassaysdetectthemecAgeneresponsible for mediating methicillinr

esistance instaphylococci(Fluitetal.,2001).
In agreement with our study, Biswajit et al. (2008) who used PCR
amplification for detection van A: The primer synthesis and the PCR
amplification of the vanA were done at Bioserve Biotechnologies,
Hyderabad, using Gene Amp (Applied Biosystems, USA) thermocycler.
The primers and the PCR conditions were as described: Forward primer
ATGAATAGAATAAAAGTTGC
Reverse
primer
TCACCCCTTTAACGCTAATA. The amplification conditions were initial

denaturation at 98C for 10 sec, annealing at 5C for 1 min, polymerization
at 72C foUPLQDQGILQDOH[WHQVLRQDW&IRUPLQ
In our finding, four selected isolates found mec A negative by PCR.
This in concordance with Pierard et al. (2004) who found that ten MRSA
isolates did not carry mecA gene. In contrary Tiwari and Sen. (2006)
found all MRSA were mec A positive, and all MSSA were mec A negative.
On the other hand after using different condition for PCR, only
primer of van A give positive for S. aureus 12 and S. aureus 50, although
198
Discussion
that the four isolates were VRSA May be another gene for vancomycin
resistant may van C or van R or other.
Actually, thickening of the cell wall of vancomcin- resistant
staphylococci has been found to be associated with complex reorganization of cell wall metabolism with extra cell wall material showing
reduced peptidoglycan cross-linking of D-Ala-D-Ala termini of side chains
(Walsh and Howe, 2002).
These studies on the role of cell wall thickening may provided us
with another explantation for the absence of van a gene in two out of four
vancomycin resistant isolates in the current study. This may mean that cell
wall thickness may be the effective mechanism for vancomcin- resistant
isolates lacking van A gene. Biswajit et al. (2008) reported that in view of
low level vancomycin resistance and the absence of van genes or any
altration in D-Ala-D-Ala residues of peptidoglycan , it would be reasonable
to consider the cell wall thickning as the major contributor to vancomycin
resistance staphylococci clinical isolates. However, in the current study the
level of vancomycin resistance was not low and the presence of other van
gene was not excluded.
This came in concordance with study of DeNiederhusern et al.
(2011) who reported that from 12 VRSA isolate contained vanA gene, six
isolates were negative for vanA by PCR whereas all the isolates had not
expressed mecA.
Our results on line with Assadullah et al. (2003) who reported that
sixty five percent of the VRSA isolates were Van-negative with multiplex
PCR assay.
Moreover, Reynolds. (1990) stated that multistep genetic alteration
is required for MRSA to achieve the level of vancomycin resistance of
VISA. In the progression of vancomycin resistance, isolates with
199
Discussion
heterogeneous vancomycin resistance, designated hetero-VISA, are

observed.
Furthermore Biswajit et al. (2008) reported that the glycopeptide
vancomycin was considered to be the best alternative for the treatment of
multi drug resistant MRSA. However, there are increasing numbers of
reports indicating the emergence of VRSA isolates exhibiting two different
resistance mechanisms. Initially VISA noted in Japan in 1996 and
subsequently in United States in 1997, was believed to be due to the
thickened cell wall, where many vancomycin molecules were trapped
within the cell wall. The trapped molecules clog the peptidoglycan
meshwork and finally form a physical barrier towards further incoming
vancomycin molecules. The second, noted in United States in 2002, S.
aureus was identical to the mechanism seen in vancomycin-resistant
Enterococcus. Vancomycin resistant E. faecium harbours the vanA operon,
which contains five genes, van S, -R, -H, -A andX. But Tiwari and Sen.
(2006) reported VRSA which is van gen-negative. Subsequent isolation of
VISA and VRSA isolates from other countries including France, United
Kingdom, Germany, India and Belgium15 has confirmed that the
emergence of these isolates is a global issue (Pierard et al., 2004)
These finding does not correlate with Jansen et al. (2007) who
observed that the genetic mechanism of vancomycin resistance in VRSA is
not well understood. Several genes have been proposed as being involved
in certain clinical VRSA isolates. The experimental transfer of the vanA
gene cluster from E. faecalis to S. aureus has raised fears about the
occurrence of such genetic transfer in clinical isolates of methicillin
resistant S. aureus. In this study, all the seven VRSA isolates contained
mecA, but only six contained vanA, also reported a van gene-negative
VRSA.
200
Discussion
The study depended on the same criteria for detection of the ten
vancomycin resistant isolates. However, other workers reported that the
most resistant isolates bear the vanA gene cluster and these are usually
found to be in the range 32-64 mg/l (Heggers et al., 2009). This may
explain why not all vancomycin resistant isolates by conventional methods
were PCR positive for vanA gene in the present study. All our VRSA
isolates showed MIC for vancomycin of 32 mg/l., which vanA gene or not
(Heggers et al., 2009). So this PCR method is confirmatory for the VRSA
isolate if give positive result (confirm nuc gene for S. aureus and vanA
gene for vancomycin resistance), but if positive for nuc gene and negative
for vanA gene, it cannot exclude the possibility to be VRSA. It should be
regarded that there are van resistance genes other than van A (which was
concerned in the current study), these genes include vanB, vanC, vanD and
vanE and vanG (Depardieu et al., 2006).
However, more studies are still needed to explore the definite
mechanisms by which these isolates acquire resistance to vancomycin
which may open the door to overcome this problem.
201
202
Summary
Summary
Multidrug-resistant microbial infections are becoming increasingly
common and difficult to treat. In the current study we aimed to study the
prevalence of antibiotic resistant bacteria specially MRSA and VRSA
strains in burn units at different Hospitals, study their antibiotic
susceptibility profile and suggest antibiotic oil combination therapy to
control and prevent the transmission of MRSA and VRSA strains to aid
burned patients in recovering from their injuries. In the future, these
alternatives may prove useful in treating not only burn infections but other
antibiotic-resistant infections as well.
Results obtained in this study can be summarized as follows:1.
The present work recovered seventy burned patients, 40 from Sidnawy

Hospital, Zagazig University, Egypt and 30 patients from Fahd king
2.
One hundred cultures were collected from seventy burned patients;

seventy six cultures were positive, while twenty four showed negative
results. The incidence of total bacterial isolates from Egypt and Saudi
Arabian in swab, blood, urine, and sputum culture showed that Grampositive bacteria were the most predominant (59. 50%) than Gramnegative (40.50%).
3.
One hundred and twenty one isolates classified into five bacterial
groups and were further on identified by studying morphological and
biochemical characteristics using conventional methods according to
the key described in Bergey's manual. Fifty seven isolates were
included in group 1, while 12 isolates in group 2, On the other hand
there were 14 isolates in group 3, while 36 Gram-negative isolates in
group 4, two isolates no. (8 and 65) were group 5. Isolated bacteria
related to groups I, 2, 3, 4 & 5 were preliminary identified as S.
203
Summary
aureus, S. epidermidis, P. aeruginosa, K. pneumoniae and

Micrococcus, respectively.
4.
The attention was then focused on the susceptibility of these 56

isolates of Staphylococcus to ciprofloxacin, cefotaxime, amoxycillin,
unasyne, chloromphenicol, rifampicin, erythromycin, tetracyline,
oxycilline, cephardin, vancomycin, ampicillin, imipenem, methicillin
and cefepime. Results revealed that imepenim and vancomycin were
the most effective antibiotics against Staphylococcus group. Also the
activities of 12 antibiotics (ciprofloxacin, cefotaxime, amoxycillin,
unasyne, chloromphenicol, rifampicin, erythromycin, tetracyline,
tobramycin, cephardin, imipenem and cefepime) were determined
against P. aeruginosa and K. pneumoniae. Imipinem showed highest
activity against K. pneumoniae P. aeruginosa.
5.
Depending on antibiotic susceptibility pattern four strains were

selected as multi-resistant isolates. Strains encoded S. aureus no. (12,
30, 50 and 95) were selected for further investigation. The four
mentioned strains were methicilin and vancomycin resistant, thus
suspected as MRSA and VRSA strains.
6.
The minimum inhibitory concentration (MIC) and minimum

bactericidal concentration (MBC) for four antibiotics (vancomycin,
imipenem, ciprofloxacin and oxycillin) as the most effective antibiotic
were tested against the selected multi-resistant isolates S. aureus no.
(12, 30, 50 and 95).
a-
Results revealed that MICs of S. aureus no. 12 and 50 were 62.5

g/ml showed highest resistance to vancomycin antibiotic. The
MBC of S. aureus no. 12, 30 and 95 were 62.5 g/ml., while S.
aureus no. 50 was 125 g/ml.
b-
In the same manner, MICs and MBCs values of imipenem against

resistant strains of
S. aureus no. 12 and 95 were 62. 5 g/ml,

204
Summary
also S. aureus no. 50 had the same MICs and MBCs 15.625
g/ml, while MICs and MBCs of S. aureus no. 30 were 31.25 and
62.5g/ml, respectively.
c-
Also the results indicated that MICs and MBCs of ciprofloxacin

for S. aureus no. 12 and 30 were 62. 5 and 125 g/ml,
respectively. S. aureus no. 50 had the same MICs and MBCs 125
g/ml, while MICs and MBCs of S. aureus no. 95 were 31.25 and
62.5g/ml, respectively.
d-
On the other hand MICs of oxycilline were 125, 62.5, 31.25 and
15.625 g/ml, for S.aureus no. 12, 30, 50 and 95, respectively.
Also MBCs of oxycilline antibiotic were 250, 125, 62.5 and
15.625 g/ml, for S.aureus no. 12, 30, 50 and 95, respectively.
7-
Ethanol (50, 60 and 70%) and (5, 6 and 7 %) of hydrogen peroxide

were tested against the selected multi resistant S. aureus (12, 30, 50
and 95) to detect the required exposure time needed when ethanol and
H2O2 were diluted. It was found that increasing in exposure time was
accompanied with a clear increase in inhibitory effect against the
a-
The dilution of ethanol to 50% will be ineffective for inhibition of

the tested isolates even exposure period was 30 minutes, while
duration at 60% ethanol for 20 minutes was satisfied to inhibit all
tested bacteria. On the other hand at the highest concentration
70%, the lowest exposure time was at 15 minutes. It could be
concluded that the best concentration of ethanol to be used as
GLVLQIHFWDQWZDVIRUPLQXWHVLWZDVVDWLVILHGWRLQKLELWDOO
associated bacteria.
b-
The dilution of hydrogen peroxide to 6% for inhibition 20

minutes all S. aureus did not show any growth, while duration at
7% ethanol for 15 minutes was satisfied to inhibit all tested
205
Summary
bacteria. On the other hand at the highest concentration 8%, the

lowest exposure time was at 10 minutes, so we could concluded
that the best concentration of hydrogen peroxide to be used as
disinfectant was 8 % for 10 minutes it was satisfied to inhibit all
8-
The antimicrobial susceptibility pattern of 9 different essential oils

against multi-resistant four S. aureus no. (12, 30, 50 and 95) with
accession numbers S. aureus 12 [AB674512], 30 [AB674513], 50
[AB674514] & 95 [AB674515], was determined. It was observed that
thyme and tea tree oil had the highest antibacterial activity against S.
aureus no. (12, 30, 50 and 95) with inhibition zones (8.2, 7.5, 8.0 and
9 mm), respectively, while tea tree oil with inhibition zones (8.0, 8.3,
7.5 and 9 mm.), respectively followed by clove, lemon grass.
Cinnamon oil had the lowest antibacterial activity against the four S.
aureus (12, 30, 50 and 95) with inhibition zones (0, 1.2, 1.3 and 3.5
mm.), respectively.
9-
The MIC and MBC of thyme and tea tree oil were determined to
obtain the lowest concentration that inhibits a visible growth in liquid
media and the lowest concentration at which no growth occurs on
solid media. The MIC and MBC value for thyme oil against S.
aureus12 were 62.5 ug/ml. Also the MIC value of thyme oil for S.
aureus 30 was 15.62 ug/ml and MBC recorded 31.25 ug/ml, while
MIC and MBC value for thyme oil against S. aureus 50 and S. aureus
95 were 7.8 and 15.62 ug/ml, respectively. The results reveal that the
MIC and MBC value for tea tree oil against S. aureus 30 were
31.25ug/ml. Also the same MIC and MBC for S. aureus (50) 125
ug/ml. The MIC of tea tree oil for S. aureus (95) was 125 ug/ml and
MBC recorded 250 ug/ml.
206
Summary
10- The influence of combination between thyme oil and vancomycin

antibiotic or tea tree oil and vancomycin antibiotic on viability of S.
aureus no. 12, 30, 50, 95 indicated that VAN disc (30g) saturated
with thyme oil (30 l) gave a synergetic effect against both multiresistant VRSA strains. The inhibition zones increased to be 15 &
18mm for both strains, respectively.
11- The selected bacterial strains were screened for their capability of
producing different virulence factors. Four examined S. aureus
produced the tested virulence factors including hemolysins causing hemolysis, licithinase degrading phospholipids into insoluble lipids in
the form of white ppt. and caseinase degrading casein protein as
extracellular degrading enzymes by using agar well diffusion assay.
12- The capability of tested bacteria to produce its virulence factors after
70%) as chemical disinfectants was examined. The results revealed the
correlation between zone diameters due to virulence factors
(Lecithinase, Hemolysin and Proteinase) produced by tested
disinfectant treated isolates. The results revealed that 7% H2O2 was
sufficient to depress lecithinase activity, while 8% H2O2was sufficient
to depress hemolysin and protease activity. Also the results
demonstrated that 70 % ethanol was sufficient to depress lecithinase,
hemolysin and protease activity.
13- The effects of tea tree oil on the production of VFs by S. aureus (12,
30, 50 and 95) had been investigated. The production of the lecithinase
by S. aureus (12, 30, 50 and 95) showed lower levels when cultured
with 0.3% tea tree oil. Four S. aureus (12, 30, 50 and 95) strains,
significantly lower levels of hemolysin and protease were produced in
the presence of 0.6 % tea tree oil when compared to control cells. In
general, these experiments demonstrate significant reduction in levels
207
Summary
of protease, hemolysin and lethinase after growth for 24 h. in the

presence of 0.3 and 0.6% tea tree oil.
14- Amplification of 16S rDNA gene of selected multi-resistant strains S.
aureus (12, 30, 50 and 95) revealed the occurrence of amplicons with
molecular size 1500bp for both strains. Thus four strains were
confirmed as S. aureus and were deposited in gene bank under
accession numbers 12 [AB674512], 30 [AB674513], 50 [AB674514]
& 95 [AB674515], respectively.
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other two strains. The suspected PCR fragments as van A genes were cut
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AIM OF THE WORK

2- Types of infection in burned patients

3- Pathogenic bacteria isolated from burned patients

4- The antibiotics activity against pathogenic bacteria

5- Resistant of bacteria to antimicrobial agent

6- Antimicrobial resistant in P. Aeruginosa

7- Antimicrobial resistant in Enterobacteriacae

8- Bactericidal action of different antimicrobial agent

9- Essential oils as antibacterial agents

10- Molecular studies

MATERIAL AND METHODS

2- Materials and equipment for samples collection and processing

3- Isolation and purification of pathogenic bacteria isolated from

5- Antibiotic susceptibility test

6- Determination of MICs and MBCs of different antibiotic against

8- Antimicrobial activity of some essential oils against multi resistant

12- Molecular identification and characterization assays of van A & 101

2- Factors affecting mortality

3- Distribution of collected isolates

4. Morphological and biochemical tests for identification of all 125

6. Antibiotic susceptibility test

7. Determination of minimum inhibitory concentration (MIC) and 145

Adult respiratory distress syndrome

Antibiotic sensitivity testing

Community associated- MRSA

Fractional Inhibitory Concentration

Methicillin resistant S. aureus

National Nosocomial Infections Surveillance

Polymerase chain reaction

Surgical site infections

Toxic shock syndrome

Urinary tract infection

Vancomycin resistant Enterococcus.

Deoxy ribonucleic acid

Erythromycin ribosome methylation ( macrolide),

Glycopeptide intermediate Staphylococcus aureus

Kilo base pairs

Minimal bactericidal concentration

Minimum inhibitory concentration

Methicillin-Resistant Staphylococcus aureus

Antimicrobial resistance can increase complications and costs

Respiratory complications rank as the major cause of death in burn

those without bacteremia. This is in contrast to Gram-positive bacteremia,

emerging threat of widespread vancomycin resistance poses a serious

for a number of applications including skin problems such as burns,

Aim of the Work

Aim of the Work

Isolation and purification of pathogenic bacteria from burned patients.

Identification of isolated bacteria according to morphological

Studying the susceptibility and resistance of isolated bacteria to

Determination of bactericidal action of ethanol and H2O2 as

Studying the susceptibility of multiresistant isolates to certain essential

Determination of the minimum inhibitory concentration and minimum

Studying the influences of combination between thyme or tea tree oil

Screening for the virulence factors and degrading enzymes produced

Studying the effect of bactericidal action of different antimicrobial

Aim of the Work

a. Confirmed identification of multiresistant isolates by 16S rDNA and

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