Contents

INTRODUCTION........................................................................................................... 1
CHAPTER 1. ANTIBIOTICS: DEFINITION, CLASSIFICATION, MECHANISMS OF ACTION. .5
1.Definition.............................................................................................................. 5
1.2 Main classes of antibiotics................................................................................. 5
Chapter 2. Genetic and biochemical resistance to antibiotics in the main Grampositive and Gram-negative bacterial strains of clinical importance........................13
Resistance to antibiotics........................................................................................... 13
2.1 Natural resistance vs acquired resistance.......................................................23
2.2. Chromosomal versus plasmidial resistance....................................................28
2.3. Biochemical mechanisms of antibiotic resistance..........................................32
CHAPTER 3. PHENOTYPIC AND GENETIC ANALYSIS OF ANTIBIOTIC RESISTANCE IN
GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIAL STRAINS ISOLATED FROM THE
HOSPITAL ENVIRONMENT IN BUCHAREST, ROMANIA................................................37
3.1. Aim and objective........................................................................................... 38
3.2. Material and methods.................................................................................... 38
3.2.1. Isolation of microbial strains....................................................................42
3.2.2. Identification of microbial strains.............................................................53
3.2.3. Phenotypic analysis of antibiotic resistance patterns...............................56
1

3.2.4. Genotypic analysis of antibiotic resistance patterns................................67

3.3. Results and discussion................................................................................... 67
3.3.1. Resistance phenotypes in Gram negative bacterial strains isolated from
hospital infections1............................................................................................ 67
3.3.2. Resistance phenotypes in Gram positive bacterial strains isolated from
hospital infections Workors of antibiotics resistance in Gram positive
cocciisolated from hospital infections................................................................69
3.4. Conclusions.................................................................................................... 69
BIBLIOGRAPHY.......................................................................................................... 70

INTRODUCTION
2

The initial definition of antibiotics as chemical substances with small molecular weight
,produced by micro-organisms from byosinthesis processes,it was subsenquently completed
because antibiotics are chemical substances obtained from byosinthesis, semisynthesis or
chemical synthesis which in low concentration inhibits multiplication or kills micro-organisms.
Defining for the notion of antibiotics ,remains the capacity to be produced through byosinthesis
by micro-organisms.
The production of antibiotics has evolved as an inhibition ecological mechanism of
growth of other micro-organisms that compete for energy resources.The phenomenom it is called
antibiosis (describded by Pasteur ,1877) and it has a opposite functional meaning of
simbiosis.But producing microorganisms of antibiotics represent a very small proportion of the
total micro-organisms in the soil. The fact that antibiotics are synthesized at the end of grouwth
phase,it appears to not ward a real competitive advantage .Gratia and Dath (1924) studied the
micro-organisms in the soil and in acellular filter on cultivition of actinomicetes has
distunguished the inhibition effect of a substance that they called actinomycetes. Fleming
(1928) , in search of compounds with antibacterial potential,observed the inhibition growth
colonies of Staphylococcus aureus near fungical colonies of Penicilium notatum. Then he
showed that liquid enviroment of P. Notatum culture , diluted by 800 times , inhibated the
cultivation growth of Staphylococcus.
He also descovered a miracle medicine that he called it penicilin, but he didnt
isolated , for which one he was awarded with a Nobel prize.The qualities of that miracle
medicine were highlighted by Ernst Chain and Howard Florey (1939), who isolated and used it
in bacterial infections treatment in the Worl War 2. The industrial production has began in 1943.

The new medicine has been introduced in general clinical circuit in 1944 and it hasa huge impact
on the status of the human population. R. Dubos (1939) has isolated gramicidin and tirocidin
from Bacillus brevis ,active against gram positive bacteries.S.Waksman(1944-1945) isolated
streptomycin from Str. griseus ,a isolated micro-organism from soil , for that he was awarded
with a Nobel prize.
Streptomycin is active against some gram positive bacteries and against Mycobacterium
tuberculosis.He proposed the name of antibiotic,with meaning of chemical compound produced
by a micro-organism,which at low concentration,inhibits or kills other micro-organisms.
The antibiotics were essentials in fight with infectious diseases and have a essential
contribution at growth of life hope in XX century.After penicilins introduction in general
clinics(1944) , serious infections as (Streptococcus pharyngitis) became curable.Today the
humans addiction of antibiotics is total.Only in USA ,in 1998,for human use was used about
12,5 tons of antibiotics.If we add the one for dogs food and agriculture , it is estimated tha in the
last 50 years were produced and used about one million tons.
The antibiotics are produced by three groups of micro-organisms: actinomycetes ,
bacilli ,gram positive and microscopic filamentous funguses.Actinomycetes are the best at
producing antibiotics and other secundary metabolites with biological activity.The most
representative genre , Streptomyces, was isolated from a chickens digestive tract.
Cephalosporium was isolated from the sea,near a seawage outfall and Bacillus from a
tegumentary wound of a little girl (Tracy) and from there it was given the name of the bacitracin
antibiotic.

The estimations of antibiotic substances varies :some inventoried about 5 000 identified
antibiotics and others evaluates superior numbers about 10 000.Actinomycetes produce over 2/3
from the total of antibiotics and species g. Steptomyces produce 70-80% from secundary
metabolites.For the treatment of humans and animals infections is used a small number (about
100) ,produced by 5 symbolic genre of micro-organisms: Bacillus, Streptomyces,
Micromonospora, Penicillium, Cephalosporium.The others are toxic or they have adverse effects
on human body or they lack of selectivity.
The isolation of antibiotics producing microorganisms are very difficult , for example
from 134.700 isolated bacterial stems from about 5 000 diffent soil evidences , only one stem
presented practical interest.
During the descovery of the most antibiotics ( 1950-1960) were identified tetracycline ,
erythromycin and kanamycin , antifungics agents :candicyn and nystatin , as well as substances
with antineoplastic effect (adriamycin).The majority of the antibiotics are obtained in industrial
processes , microbiological way.Nowadays ,only the biggest segment of antibiotics molecule is
chemical synthetized so this obtain semisynthesis antibiotics or through bioconversion ways.In
some cases (as chloramphenicol),the all molecule is chemical synthetized because of the simple
molecular structure.
Conventionally in antibiotics category are included semisynthetis substances or the
artificially synthesized ones , but micro-organisms can synthesize total or partial.This mention is
necessary because from antibiotics category to be excluded compound with antibacterial effect
synthetized only chemical way(sulfanomides) or those produced in superior
organism(lysozyme).
5

Complex molecular structures of antibiotics combine derivatives of two or more groups

of metabolites: amino acids , carbohydrates , nucleid acid bases ,itermediates of lipids synthetis.
Acetyl-CoA and propionyl-CoA are forming long chains (as fatty acids synthesis) and results
poli- , ketones (RCO CH2CO CH2-),which condense to form rings of macrolides ,
polyenes , tetracyclines or segments of molecule from other antibiotics.
For half a century, antibiotics are the most important weapon in the fight against
dangerous infection-causing bacteria from the ear and the skin infection to the blood infection.
Infectious diseases causing thousands of deaths and much suffering in the past, such as
tuberculosis, are now under control with antibiotics.1
Despite their undeniable importance in modern medicine, these drugs are not always the best
therapeutic solution . There are two main types of germs that cause bacterial infections and
viruses. The most important points to note are:

- Antibiotics have no effect on diseases caused by viruses;

- Only diseases caused by bacteria can be treated with antibiotic;
At first administration of the antibiotic, the initial dose kills weak bacteria at which time
the general condition improves. The strongest bacteria survive, and if treatment is discontinued
before the period recommended by physicians, they multiply and become resistant to the
antibiotic (which can subsequently be transmitted to other family members and the community).
When a person is infected with a bacterium that has gained strength, it requires treatment
administration with stronger antibiotic substances.
1

Agerso et al., 2005

6

Besides the induction of antibiotic-resistant bacteria, abuse of these drugs lead to the appearance
of other side effects such as allergic reactions or serious diarrhea infections caused by
Clostridium difficile . In rare cases, these complications can even lead to death.2
According to American specialists, many infections spread among health care
professionals are caused by bacteria that have become resistant to many antibiotics. These
include:
- MRSA (a recent variety of Staphylococcus aureus bacterium );
- Enterococcus (resistant to vancomycin);
- K. pneumonia (resistance to cephalosporins);
- E. coli and Enterobacter spp. (Carbapenem resistant);
- P. aeruginosa (carbapenem resistance);

Agerso et al., 2005

7

CHAPTER 1. ANTIBIOTICS: DEFINITION,

CLASSIFICATION, MECHANISMS OF ACTION
1.Definition
Antibiotics (anti+bios = with adverse effects) = heterogeneous group of chemical substances
produced by microorganisms in the biosynthetic processes that kill or inhibit the growth of other
species of microorganisms.
Chemotherapeutic agents = chemical synthesis substances used to treat infections caused by
different microorganisms (bacteria, microscopic fungus, and protozoans) (P. Ehrlich -l904).
The present definition = chemical substances obtained by biosynthesis, semi-synthesis,or by
chemical synthesis, which, in low concentrations inhibits the multiplication or kills
microorganisms. = antimicrobial substances
> 500 identified substances having antibiotic properties
~ l00 used in therapy

1.2 Main classes of antibiotics

The large number of currently known antibiotics has questioned the classification of these
products. The following classifications has been proposed :3

SOCKETT. Et al., 2006

8

After the origin of producing microorganism :

antibiotics produced by bacteria

- Gramicidin
- Bacitracin
- Polymyxins

antibiotics produced by the actinomycete:

- Streptomycin
- Neomicima
- Kanamycin
- Nystatin

antibiotics produced by fungus

- penicillin
- grizeofulvin

2. After the chemical structure:

- antibiotics with aliphatic, aromatic structure

- heterocyclic

3. After biogenesis:
- antibiotics derived from amino acids
- from acetate units
- from carbohydrates
4. After pharmacological action:
- Antibacterial antibiotics
- Antituberculosis antibiotics
- Antiviral antibiotics
- Anticancer antibiotics
- Antifungal antibiotics

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The classification of antibiotic after structure

1. lactam: penicillins and cephalosporins

https://www.google.ro/url?
sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCIqM
pKmMx8cCFcbNFAod06YFGQ&url=http%3A%2F%2Fwww.psmicrographs.co.uk%2Fmouldfungus--penicillium-expansum-%2Fscience-image
%2F80200576&ei=mt_dVYrXFcabU9PNlsgB&psig=AFQjCNHVFX9XEQM3JUJ4KTDNu3PCj34vfg
&ust=1440690451933819

11

2. aminoglycosides gentamicin, kanamycin, streptomycin

3. Macrolides: erythromycin, clarithromycin
4. Tetracyclines: Tetracycline and Doxycycline
5. Chloramphenicol
6. sulfamides (are 23323k1021x synthetic)
bactericidal antibiotics: penicillins, cephalosporins, aminoglycosides.
bacteriostatic antibiotics: Erythromycin, Tetracycline, Chloramphenicol, sulfamides. broadspectrum antibiotics [1] broad-spectrum penicillin (Ampicillin, Amoxicillin), chloramphenicol,
tetracycline.
penicillins are antibiotics = drugs that destroy bacteria . There are several types:
Characteristics of some commonly used antibiotics
1. Beta-lactams (so called because of their chemical structure)
1.1. Peniciline
1.1.1. Narrow-spectrum penicillins
Benzylpenicillin (Penicillin G) is a natural penicillin. It is active against certain types of bacteria:
streptococcus ( that causes streptococcal angina, scarlet fever, erysipelas), pneumococcal (lobar
franca pneumonia ), meningococcal (meningitis), Treponema pallidum (syphilis), clostridia (gas
gangrene), the bacillus tetanus (tetanus) bacillus coal (anthrax).

12

It may not be administered orally because it is inactivated by stomach acid. It is usually

given intramuscularly, intravenously in severe cases. PenicilinaG can be potassium or sodium. A
patient with heart failure will prefer PenicilinaG potassic, because sodium attracts water and
overweight self circulation. A patient with kidney failure will prefer PenicilinaG sodium because
sodium is eliminated through the kidneys easier.
Phenoxymethylpenicillin (Penicillin V, Ospen) is resistant to gastric acid therefore is
administered orally. The absorption from the digestive tract is incomplete and performed blood
concentration of antibiotics is low, because of this drug is administered in case of low gravity
infections: For instance streptococcal angina. penicilinaG is administered in more severe
infections .
Benzathine benzylpenicillin (Moldamin) is a deposit penicillin. It injects deep
intramuscular where it performs a deposit of active substance (benzylpenicillin) which is
released slowly into the blood (for several weeks). The blood concentration is sufficient to
protect the body from a possible strep infection. It is used to prevent re-streptococcal infections
in persons with a history of poststreptococice disease (rheumatic fever)
Procainpeniclina (Efitard) is a combination of procaine and penicilinaG which is slowly
absorbed into the blood from the injection site. It is effective for 12 hours. It has increased risk of
allergic reactions due to the presence of procaine.
1.1.2. Staphylococcal penicillins: Oxacillin, Cloxacillin, Nafcillin
Synthetic penicillins can not be destroyed by penicillinase, so resistant germs are active on
narrow-spectrum penicillins. The only indication for these antibiotics are staphylococcal
infections (furunculosis, osteomyelitis, etc.)
13

1.1.3. Broad-spectrum penicillins: ampicillin, amoxicillin

Are active on the germs that are narrow-spectrum penicillins active and in addition to Gramnegative: Haemophilus influenza E.Colli, Salmonella, Shigella. Not active against staphylococci
that are inactivated by penicillinase.
Ampicillin can be taken internally or injected. It should be administered on an empty stomach,
because it decreases the absorption of food. It focuses much in the bile and urine, as indicated in
infections at these levels.
Amoxicillin has the advantage that it absorbs better digestive,the absorption is not affected by
food.
Amoxicillin may be associated with clavulanic acid which is an inhibitor of penicillinase,
resulting antibiotic called Augmentin (amoxiclav). Augmentin has a very broad spectrum of
action: it is active on all germs that are narrow-spectrum penicillins active, on staphylococci and
gram negative.

Penicillins have degenerative bactericidal effect (kills bacteria multiply). Prevents bacterial

wall synthesis.
1.2. Cephalosporins
Broad-spectrum antibiotics are indicated in serious infections that do not respond to other
antibiotics. Some are taken orally: cephalexin, cephalothin, cefuroxime (Zinnat)), others only by
injection (parenteral): Latamoxef, cefepime, cefpirome. If a person had a serious allergic reaction
to penicillin we recommend to avoid cephalosporin because of the risk of cross-allergy .
2. Aminglicozide: Streptomycin, gentamicin, kanamycin, neomycin.
14

These antibiotics are not used alone, because germs are quickly becoming resistant to them.
Always use together with other antibiotics.
Streptomycin is used to treat tuberculosis TB together with other chemotherapeutic agents.
Gentamicin and Kanamycin is associated with penicilinaG.
If taken digestive, these antibiotics are not absorbed. However sometimes it is administered
digestive to treat intestinal infections (enteritis, enterocolitis). For other infections (tuberculosis,
urinary tract infections, genital infections, bronchitis etc) aminoglycosides are administered by
injection. Neomycin is so toxic that is administered only digestive or external (skin or mucosa).

Erythromycin is an antibiotic that is given to patients allergic to penicillin. It is

bacteriostatic active, gram positive. It inhibits bacterial protein synthesis.

Co-trimoxazole (Biseptol) is a combination of two chemotherapy agents Sulfamethoxazole

and trimethoprim. It has bactericidal effect. It manages internal respiratory infections, digestive
and urinary.

Tetracycline is a broad spectrum antibiotic. It has bacteriostatic effect (stops the

multiplication of bacteria). It is indicated in periodontal infections (periodontal abscess). Do not

use in pregnant women or children younger than 12 years because it causes malformations of
teeth. If taken long, oral candidiasis occurs. Is NOT administered with milk or antacids that
decreases the absorption of tetracycline from the digestive tube.

Chloramphenicol is used in Salmonella infections. It has bacteriostatic effect. It can have

serious reactions: gray syndrome in infants and inhibiting hematopoiesis [2] in adults\

15

Antibiotic

Resistence mechanisms of bacteria

Betalactamine

Inactivating enzymes (beta-lactamases), (C, P)

(penicillins, cephalosporins,etc.)

Change PBP (C, P)

Reduction in cellular permeability (C)

Aminoglycosides

Enzymatic inactivation (aminoglycosides modifying enzymes)

(C, P)
The alteration of the ribosomal target protein (C)
Changing active transport systems through bacterial shells (C)

Quinolones

The affinity reduction of action target (changing DNA gyrase)

(C)
Reducing the permeability of the outer membrane (C)
Active efflux of antibiotics in bacterial cell (C, P)

Macrolides

Changing target share (C, P)

Active efflux of antibiotics in bacterial cell
ATB modifying enzymes bacteria acquisition (P)

Lincosamides

Changing target share (C, P)

ATB modifying enzymes bacteria acquisition (P)

Vancomycin

Changing target share

Tetracilcine

Active efflux of antibiotics in bacterial cell (P)

Changing ribosomal protein (P)

16

Chloramphenicol

Enzymatic inactivation (chloramphenicol acetyltransferase)

Reducing the bacterial wall permeability (C)

Sulphamides

Reducing the bacterial permeability (C, P)

Modification of enzymes (C, P)
Overproduction of APAB (C)

Trimethoprim

Reducing the bacterial permeability (C, P)

Nitrofurans

Reducing the bacterial permeability

Deficiency in nitrofuran reductase activity (C)

Nitroimidazoles

Reducing the bacterial permeability

Insufficient nitrofuran reductase activity

http://europroxima.com/products/antibiotics/nitrofurans/

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Chapter 2. Genetic and biochemical resistance to

antibiotics in the main Gram-positive and Gramnegative bacterial strains of clinical importance
Resistance to antibiotics
Antibiotic resistance is the naturally or acquired ability of a microorganism to resist the
effects of one or more antibiotics. Although this concept is also used relating to natural resistance
- intrinsic ability of bacteria to resist certain antibiotics - a greater scientific and clinical
importance is the acquired resistance.
Resistance to antibiotics can occur through natural selection or by mutation (in the mutagenic
effect of environmental factors or uncorrected errors in the DNA replication process). With
emerging antibiotic resistance, gene coding for this character may spread to other cells through
transfer of plasmids. A bacterium can use multiple antibiotic resistance gene, in this case being
called multi-resistant.6
Antibiotic resistance of a microorganism can also be artificially induced , by
transformation techniques (introduction of foreign genetic material into the cell and expression
of encoded characters). This method is useful in genetic engineering and biotechnology for the
selection of bacteria bearing a specific additional character, transmitted along with the gene of
resistance.
6

SOCKETT. Et al., 2006

18

The emergence of antibiotic resistance is a evolutionary phenomenon, caused by selective

pressure of environmental factors. Antibiotics are a selection factor and the bacteria that suffers a
benefical mutation (the emergence of resistance to antibiotic) will survive and will possibly send
these characters to descendants. The increasingly frequent use of antibiotics to treat infections
resulted in emergence of a growing number of bacterial strains resistant to a growing number of
antibiotics.
There are several mechanisms by which this phenomenon may occur:

Antibiotic inactivation or destruction - for example Penicillin inactivation by producing

enzyme (beta-lactamase) that break a beta-lactam bond in the molecule.
Changing a target (antibiotic binding site) so antibiotic molecule no longer can react with cellular
components - for example, ribosomes and enzymes involved in the synthesis of the bacterial wall
Change in the metabolic pathways in which antibiotics act - the case of bacteria resistant to
sulphonamides, which, instead of synthesizing folic acid starting from para-aminobenzoic acid,
they use preformant folic acid, like mammal cells.
inhibition of penetration of the antibiotic into the cell - for example the resistance mechanism of
E. coli to macrolide
Eliminating antibiotic (active efflux) - seen in some species of E. coli and S. aureus.

19

The natural result of the discovery of this phenomenon was the continuous search of new
antibiotics capable of inactivating microorganisms resistant to antibiotics already available.
However, it was found that after a certain time from the introduction of a new semi-synthetic
antibiotic, or a new class of antibiotic, the existance of resistant bacterial strains is, due to the
fact that antibiotic resistance is an evolutionary phenomenon. 8
Among the examples of continual evolution and adaptations of pathogenic bacteria to new
antibiotics, are increasingly frequent cases of multi-resistant tuberculosis and infections with
methicillin-resistant Staphylococcus aureus.

Conform http://www.britannica.com/science
8

Agerso et al., 2005

20

The concept of microbial resistance defines the ability of pathogenic germs to survive
and multiply in the presence of antibiotics. The germs are resistant or become "indifferent"
("tolerant") to antibiotics, thereby avoiding desired antibacterial effect through various ways ,
following multiple usual non-toxic doses . When exposed to an antibiotic, the bacteria is the
selected for resistance . This natural biological process leads to the survival of the resistant
strains (FEDES, 2000).
After HEINEMANN (1999), antimicrobials are approaching the end of their effectiveness.
Although evolution of resistance to these drugs was expected, mechanisms by which genes
conferring resistance will spread were not intuited . Therefore antimicrobial agents used in the
future must avoid effects caused by pathogens.

Types of antibiotic resistance:

Factors influencing bacterial antibiotic resistance. The term strength can be used in two ways, as
microbiological resistance and as clinical resistance.
Microbial resistance is an absolute concept and it must be distinguished from pseudo or false
strength, relative notion of practice clinical treatments.
Microbiological resistance
Absolute microbial resistance occurs when a microorganism is resistant in vitro, the antibiotic
used proving to be unable to stop the reproduction of them or when stopping the multiplication
is carried out only by very high concentrations of the antibiotics, which would require toxic
doses in vivo practically unusable in the patient. From a microbiological point of view, resistant
21

organisms are those that possess any type of mechanism of resistance or resistance genes. The
minimum inhibitory concentration (MIC) of an antibiotic gives quantitative information about
bacterial susceptibility. Usually an organism is considered susceptible when MIC is less than the
limit indicated by various laboratories authorized for such standards (SOCKETT. Et al., 2006).

Clinical resistance
Clinical resistance is more complex than microbiological resistance, since it is linked to
the likelihood of response to antimicrobial therapy. Prior to treatment, information concerning
the susceptibility of the bacteria involved in the infection are received from specialized
laboratories, although not fully reflects the antibacterial activity of antibiotics in clinical
conditions (Swartz, 2000).
Pseudo-resistance or false resistance
It represents the failure of antibiotic treatment due to characteristics of the host organism and of
the infection and especially incorrect use of antibiotics. Pseudo-resistance is a temporary
phenomenon, reversible, which shall be exercised only in vivo. In clinical terms, the
development of resistance is facilitated by pharmacokinetic characteristics of different classes of
antibiotics and an incorrect treatment (insufficient dose , too short duration of treatment or longterm use of antibiotics), as well as inefficient active concentration of the antibiotic at the place of
infection (hard sterilizable outbreaks,serous barriers, avascular tissue, inactivation of antibiotics,
unfavorable pH etc.). Another cause of treatment failure is relative resistance to
pathogens,sensitive in vitro, but became insensitive, usually temporarily, in vivo (persisters,
germs with reduced metabolism).
22

Moreover, other factors such as the immune status of the patient may affect the therapeutic
response. In most patients, antibiotics do not totally destroy pathogens, but assists the immune
system in an attempt to eliminate the infection. An enormous selection force is the large amount
of antibiotics used in agriculture as well as the therapy and prophylaxis of human and veterinary
medicine (HARDY, 2002; CROMWELL, 2002; DIBNER et al., 2005).
Microbial resistance to antibiotics can be natural and acquired: natural resistance is the
resistance of all members of a bacterial species to one or more antibiotics present in the
maximum dose tolerated by the treated body without risk, doses that can inhibit growth or
destroy other bacterial species . After ENGELKIRK Burton (2002), natural resistance is the
intrinsic property of the species thus total, genetically fixed. For instance, the resistance to
penicillin G of the strains of Salmonella;
Pseudomonas aeruginosa is naturally resistant to chloramphenicol; Proteus genus species
are resistant to tetracycline; anaerobes (Bacteroides, Clostridium, some streptococci) and Serratia
genus are resistant to aminoglycosides. Acquired resistance refers to the state arising from
naturally sensitive species after the entering of an antibiotic into therapy and consists in
decreasing or cancellation of antibiotic sensitivity.9
This is a phenotypic character correlated with an altered genetic material. In practice, the
bacteria is considered resistant when there is an subunitary ratio between medium serum levels
supported by the patient and minimum inhibitory concentrations of the antibiotic or antibacterial
(TODAR, 2002). Until now several acquired resistance types are known, and are classified by
various criteria:

Agerso et al., 2005

23

The moment infection is installed

The primary resistance, the state of resistance of the bacteria at the initiation of the infection.
Secondary resistance, state acquired by infected strain during treatment.
b. The number of antibiotics to which resistance is installed. Mono-resistance - microbial
resistance to a single antibiotic. Multi-resistance - microbial resistance to several antibiotics.
c. The rate of installing resistance to antibiotics. Streptomycin fast resistance type , consists of a
single stage (single mutant, interesting only one gene). Penicillin-type progressive resistance
constituted in several steps (successive mutations, interesting multiple processes)
d. The presence of antimicrobial factor. Inducible resistance - expressed only in the presence of
antibiotic resistance. Constitutive resistance - the ability of a gene to continuously express their
resistance independent of the presence or absence of the antibiotic.
e. Other types of resistance. Cross-resistance - the ability of some strains to manifest resistance
to some antibiotics related by chemical structure. Adaptive resistance - a condition which,
without interesting the genome is sometimes transmitted to successive generations and which
appears under the influence of subinhibitory doses of antibiotic, bacteria returning to its previous
state after several generations from the disappearance of inducing factor (ANGELESCU, 1998;
Swartz, 2000).

Prevention of antibiotic resistance

24

Antibiotic resistance is defined as a natural or acquired ability of a microorganism to survive the

effects of one or more antibiotics. This phenomenon is evolutionary caused by selective pressure
of environmental factors. Following frequent use of antibiotics in treating infections , a growing
number of bacterial strains resistant to more and more types of antibiotics, has appeared.10
Both patients and health professionals must collaborate to stagnate the phenomenon of antibiotic
resistance development. Experts recommend a series of measures by which the danger of
impossibility of treating serious infections does not become a harsh reality for mankind. Medical
experts recommend you
- carry out a specialist medical inspection to establish the exact nature of the infection (viral or
bacterial);
- Require access to antibiogram, so to make sure that your doctor has prescribed antibiotic in
removing bacteria or right contacted bacteria ;
- Follow antiobiotic treatment according to precise physician recommendations (dose, time,
etc.).
- Do not skip doses even if you feel better after only 1-2 days of treatment.
- Take only prescribed antibiotics personally do not share with anyone and do not use those
remaining from previous treatments (different types of antibiotics treat specific types of
infections
- the administration will postpone wrong substance healing and stimulate bacterial growth).

10

Swartz, 2000
25

- Do not save antibiotics for possible future diseases and do not recommend them to others just
because they worked in your treatment.
- No doctor urges an antibiotic prescription if he does not deem it necessary. Remember always
that these drugs have side effects!
- Prevent infection by washing hands and correct immunization (vaccination).
For children with symptoms like fever, fatigue, muscle pain or irritated throat, do not quickly
conclude that they contacted bacteria . Most often, it is a common cold, against which antibiotics
have no effect. Carry out a pediatric inspection and help children fight viral infection by
- Prolonged bed rest;
- Additional hydration by water, fruit juices, soups and tea;
- Anti-inflammatory drugs based on ibuprofen;
- Nasal sprays with saline solution and cough syrup;
- Steam baths;
Prevention of antibiotic resistance depends largely on healthcare professionals, who are the only
ones authorized to issue prescriptions on which patients can purchase antibiotics. They must:11
- Prescribe antibiotics correctly (crop sampling, performing sensitivity testing, establishing the
required dosage and administration period, issuing the prescription after the patient has been
tested properly).

11

Ibidem

26

- To document on the dose, timing and indications on the prospectus of antibiotics sold in
pharmacies.
- constantly worrying about the high degree of risks in the preading of antibiotic resistant
bacteria in the medical unit in which they work.
- Attend and support all measures taken by hospital practices and authorities, to discourage
improper antibiotic prescriptions for patients.
- Respect all rules of hygiene and other infection control measures with each patient treated.
- Inform patients about the dangers they face when abusing antibiotics.12

13

12

Swartz, 2000

13

http://www.google.ro/imgres?
imgurl=http://evolution.berkeley.edu/evolibrary/images/interviews/resistance.gif&imgrefurl=
http://evolution.berkeley.edu/evolibrary/article/bergstrom_03&h=238&w=484&tbnid=2SYhL
vqfuxO4hM:&docid=N6590bDHHJrnIM&ei=VHdVe26L4HwUvidqPAB&tbm=isch&ved=0CB8QMygBMAFqFQoTCK3C1v2Nx8cCFQG4FAodA4KHg

27

2.1 Natural resistance vs acquired resistance 14

Efflux phenomena

Besides impermeability, there is another mechanism that explains accumulation in

bacteria; excretion or active efflux. The antibiotic enters in the bacteria, but before it could fix
on target, it is taken by membrane proteinsoutside and excreted outside the bacteria. This system
works with a cytoplasmic membrane protein that is the carrier (pump), a protein the outer
membrane that forms the excretion channel and a protein periplasmic responsible for the link
between the previous ones.15
Natural resistance

This system occur naturally in certain bacteria. Also in P. aeruginosa, such a system
largely explains the high natural resistance against a large number of antibiotic molecules. This
system exists in staphylococci against quinolones.
Gained resistance

First resistance efflux acquired by E. coli was reported in 1980. It was the tetracycline excretion.
Since then, this resistance mechanism has been revealed to:
14

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257838/

15

Swartz, 2000
28

- E coli, P. aeruginosa and Staphylococcus aureus against quinolones;

- staphylococci against macro-LIDE ;
- P. aeruginosa against beta lactams;
This may involve acquiring resistance genes as a result of introduction of a plasmid or, most
often, a mutation engaging existing chromosomal gene overexpression but little or no casted.
The lack of affinity

After cell penetration of the antibiotic, there is a stage of target recognition.This type of
resistance occurs at the remembered level. It's either a natural resistance with a weak affinity of
certain antibiotics for targets, or a resistance acquired with the modification of targets and the
affinity loss to antibiotics for these targets.
Natural resistance

Various molecules in the family does not have all the same affinity for the bacterial targets.
Aztreonam (monobactams) has weak affinity for the PLP of Gram-positive and rigorous
anaerobic bacteria , which explains their natural resistance against this antibiotic.16

Likewise, first-generation quinolones (nalidixic acid and pipemidic) have only weak affinity for
DNA gyrase Staphylococcus.
Acquired resistance
16

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257838/

29

This type of mechanism is responsible for a large number of acquired resistance. We quote
Staphylococcus resistance against beta-lactams. It is about the acquisition of genetic material
(mecA gene). This gene encodes the synthesis of a new PLP: PLP 2a or 2 'which possesses a
weak affinity for beta-lactams, whatever the molecule, also explaining cross-resistance against
the whole family.
Similarly, Streptococcus pneumoniae, sensitive to penicillin G is no longer sensitive (only in
40% of the cases in France). The acquisition of foreign genes by transformation from other
streptococci or pneumococcus is responsible for this resistance.
Another way of acquiring this type of resistance is met in bacteria: a chromosome mutation
touches the gene ruling the target . DNA gyrase gene mutations train modified enzyme
synthesizing possessing low affinity against quinolones molecules.17

Resistance by enzymatic modification

Quantitatively and qualitatively, this type of mechanism is certainly the most important. Many
classes of antibiotics and virtually all bacterial species are concerned. To be active, the antibiotic
must reach its target intact. If there is modification of the antibiotic by enzymes present in
17

Ibidem

30

bacteria, at any level, modified form of the antibiotics molecule is most often inactivate. These
enzymes occurr naturally or acquired at bacteria of clinical interest .

The natural resistance

The most important enzymes are enzymes directed clinically against beta-lactam betalactamases. These enzymes destroy beta-lactam ring, making the antibiotic inactive. Within this
family, the molecule choice is obviously based on the certain or supposed bacterium . The first
criterion is the presence of a natural resistance, more often the presence of beta-lactamases . For
this family, it is essential to have a good knowledge of wild phenotypes, so natural resistances.
Many species naturally produce these inactivating enzymes . This is the case of
Enterobacteriaceae, Pseudomonas and acinar-tobacter. Similarly, the Group II of
Enterobacteriaceae possess beta-lactamase that inhibits penicillin (amino, carboxy,
ureidopeniciline). Enterobacteriaceae from Groups III and IV, like Pseudomonas and
Acinetobacter produce another enzyme: a cephalosporinase explaining the natural resistance of
these bacteria. This type of resistance is not only beta-lactam reserved. In its natural state,
certain bacteria produce enzymes that inactivate aminozide.This is the case of Serratia marcescensus , that inactivates tobramycin, netilmicin and amikacin, as the Providencia stuartii that
inactivates gentamicin, tobramycin and netilmicin.18
Accquired resistance
Following the beta-lactams described, it is clear that it is important to detect MECA-resistant
organisms through the acquisition of enzymes. Geneticaly , beta-lactamases should distinguish
18

Ibidem

31

the plasmid origin from the chromosomal origin. In most cases, it is a genetic material
acquisition through plasmids. We are talking about plasmid resistance . Depending on their
spectrum of activity, we organize:in penicillinases, as staphylococci; in broad spectrum senior
penicillinases and beta-lactamases, as the enterobacteria.
If we talk about aminozide, there arevnumerous inactivating enzymes of plasmid origin .
Depending on their type, they are responsible for the inactivation of one or more molecules used
in clinical gentamicin, tobramycin, netilmicin or amikacin. They are frequent in
Enterobacteriaceae, Pseudomonas and, more frequently, in staphylococci.
Another mechanism of acquiring resistance is often seen in Gram negative bacteria. This is betalactams. This is no longer foreign gene, but mutations in genes that govern the synthesis of
natural cephalosporinase. This is no longer synthesized at low levels, but at a very high level,
responsible for inhibiting a greater number of molecules of beta-lactams.

Mechanisms to acquire antibiotic resistance. The interaction of the microbial resistance and antibacterial agents appears in a direct and indirect manner (RUBINSTEIN, 1999):
directly - by the development of resistance to the antibiotics used or to the antimicrobial agents
belonging to the same class: for example, betalactamase induction by both gram-positive and
gram-negative; as well as through the development of resistance to the components of the
different classes of antibiotics used to treat for example, loss of susceptibility to penicillin of
Streptococcus pneumoniae, together with a simultaneous loss of susceptibility to erythromycin
and tetracycline.

32

 indirectly - microbial resistance can develop by selection of resistant organisms when the
patient is treated with antibiotics, when the environment is contaminated with antibiotics
(intensive care unit) or the antibacterial agents used in agriculture or as animal growth promoters

2.2. Chromosomal versus plasmidial resistance

Genetic mechanisms

19

https://www.google.ro/url?
sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCNqh
n5WOx8cCFcVcFAodxksO3Q&url=http%3A%2F%2Flyonlive.com%2Fsupport
%2Fext1731.html&ei=ieHdVZq5DMW5UcaXuegN&psig=AFQjCNFZyG666dmesjr5qT2Qae2l0L
R5OQ&ust=1440690949701197

33

Antibiotic resistance in bacteria can be induced by mechanisms of genetic variation both

endogenous (mutations and translocations) and exogenous (chromosomal recombination,
plasmid transfer resistant, etc.). 20
The chromosomal resistance. This type of resistance occurs due to mutations in the bacterial
chromosome nucleotide sequences that determine the synthesis of proteins and other
macromolecules that differ to a great extent from original chemical entities that interfere with
antibiotic activity. Chromosome mutations can be spontaneous or induced by mutagen agents
(especially antibiotics) (Henderson, 1999; Kuhn et al., 2003), are genotyped, with vertical
transmission (performed without contact with the antibiotic), appear suddenly in one medicine
and are final. However, the number of resistant mutants will decrease after the end of exposure to
the antibiotic (Swartz, 2000).
In a population exposed to an antibiotic, development of chromosome resistance is usually a
gradual process, careful monitored, carried out through several consecutive mutations; However,
for some antibiotics a single mutation may determin resistance resulting in an intense increase of
MIC. The emergence of resistant mutants is much less frequent in vivo than in vitro, probably
because mutations leading to resistance are usually associated with other cellular changes that
may be disadvantageous for bacteria. For this reason, some scientists see the development of
resistance determined by mutations on chromosome as a small problem compared with
transferable resistance. However, in practice, these mutations have little significance due to the
fact that rarely occur (10%) (Jackson et al., 2004).

20

Ibidem

34

Transferable resistance.
Bacteria have extremely efficient genetic transfer systems capable of exchanging and
accumulating resistance genes. Some genes, including genes which encode resistance, can move
between the elements of chromosomal and extrachromosomal DNA of the bacteria.
Extrachromosomal mutations are more frequent (90%), so of great practical importance. Genes
can be transmitted between bacteria of the same species or different species or different genres of
bacteria (horizontal transfer), that have common habitat in the body. Interspecies transfer means
that, once transferable resistance gene have occurred, the bacteria carrying the gene will remain
a potential donor for other bacterial genes (Agerso et al., 2005).
The most important carrier for the transfer of resistance genes in bacteria are plasmids,
transposons and integrons (Swartz, 2000). As specified by McDermott (2002), in recent years a
number of resistance genes have been associated with the massive transfer of extrachromosomal
DNA elements called plasmids, which can be other mobile DNA elements such as transposons
and integrons.
It has been shown that these mobile elements transmit genetic determinants of antimicrobial
resistance mechanisms and they matter in the dissemination of resistance genes between different
bacteria. Plasmids are extrachromosomal DNA molecules, replicable, which may contain
resistance genes (MARBLE, 1999). Replication is independent of chromosomal DNA.21
They are important in the development of bacterial plasmids, since they affect the replication,
metabolism, bacteria fertility, as well as resistance to bacterial toxins (bacteriocins), antibiotics
and bacteriophages, thus ensuring a better chance for survival and propagation. However, the
21

Ibidem

35

general plasmids are not strictly necessary for the survival of the bacteria. They have been
identified in the majority of bacterial species, having the ability to be transferred (conjugative),
or co-transferred (non-conjugative) from a bacterium to another, thereby leading to a wide
dissemination of the plasmid-encoded characteristics within an ecosystem.
The genes encoded by the plasmid is inherently more mobile than chromosomal genes because
plasmids can be transferred within a bacterial species or between different species (Swartz,
2000). R-plasmids are plasmids containing the resistance gene (AOKI, 1993, KIM. Et al., 1996,
Diaz et al., 2006). The purchase of new resistance determinants may occur more rapidly than the
R-plasmid genetic mutation.
One R-plasmid may encode resistance simultaneously to more than 10 different antibiotics.
There have been found many different r-plasmids. A single bacterial cell may contain several
different plasmids and each plasmid may have more resistance genes. Plasmids isolates from
human and veterinary seem to be very similar, suggesting even their transmission from animals
to humans (WRAY C. et al., 1986) or from humans to animals (Sannes et al., 2004).
Dissemination of plasmids can occur through clonal distribution and intra- and inter-species
transfer, leading to a gradual increase in the proportion of microorganisms in a bacterial
community transporting one or more factors R.22
Transmission of plasmids from one bacteria to another is done in several ways: - conjugation
(recombinant) - by sexual pili; - Transducing phage - the plasmid is taken from a bacteriophage,
and transferred to a plasmid-free bacteria; - Transformation - the plasmid is taken directly from
another organism, after a bacteria destruction (KEHRENBERG, 2000; FURUSHITA, 2003).

22

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257838/

36

The resistance plasmid is dependent on the genotype, having the transmision both horizontally
and vertically as possible from the one or more antibiotics (poly-resistant) (Johnson et al., 1994).
Transposons ("jumping genes") are short sequences of DNA that can move from plasmids,
between the plasmid and the bacterial chromosome or between a plasmid and a bacteriophage
(bacterial virus) (Henderson, 2000). In contrast to plasmids, transposons are not able to replicate
independently and must be maintained in a functional replicon (eg. Plasmid or chromosome).
Transposons at gram-negative bacteria are non-conjugative, while at gram-positive bacteria
Bacteriodes spp., may be either conjugative or non-conjugative. However, if a gram-negative
bacteria of the transposon DNA is part of a conjugative plasmid, horizontal transfer is possible.
Transposons, including those carrying resistance genes (KEHRENBERG et al., 1998; RIBERA
et al., 2003),are easily purchased by the plasmids and then incorporated into bacterial DNA.
Transposons are often more crowded on the same plasmid, causing the transfer of several
resistance determinants in a single conjugations. Also, plasmids from different backgrounds can
carry multiple sets of identical genes of resistance.
The intracellular transfer of the transposon from plasmids between bacterial chromosomes and
plasmids also interbacterian transfer of conjugative plasmids and transposons can lead to a rapid
development of resistance in bacterial populations. Expression of the resistance gene located on
transposons (eg. The production of enzymes) may require the presence of the antibiotic.
Moreover, the presence of antibiotic resistance will promote the transfer.
Antibiotics create an environment in which the resistance determinants possession is
advantageous and, in addition, will increase the rate of transfer of resistance genes. Integrons.
Integrons are natural components of gene expression. They are composed of two conserved
37

regions and a variable interposed region, containing cassettes of genes for resistance to
antibiotics (Hall and Collis 1995).
Gene cassettes are elements that include a single gene and a place ("Site") of recombination.
More than 40 cassettes have been identified, , and all but 5 of them contain the resistance gene
(Agerso et al., 2005). One of the conserved regions of integrons contains integrase gene, which is
responsible for specific insertion at the place ("Site") in the boxes (Swartz, 2000). Integrons can
be located in chromosomal DNA, but more often are located in plasmids (Diaz et al., 2006) or
transposons and are therefore mobile. For example, the chromosome resistance characteristic
pattern of Salmonella typhimurium DT104 is associated with the presence of integrons (Swartz,
2000).

Antibiotic resistance 1940-2000

38

2.3. Biochemical mechanisms of antibiotic resistance

Bacterial resistance to antibiotics, both the natural and acquired resistance, is achieved through a
variety of mechanisms (ANGELESCU, 1998):
1.Enzymatic inactivation of antibiotics, resistant plasmids and, more rarely, the
chromosomes, encodes enzymes of antibiotics inactivation . We know several categories of
inactivating enzymes: beta-lactam antibiotics, aminoglycoside antibiotics, chloramphenicol.
Betalactamazele. They are enzymes found in most bacterial species, which have the ability to
hydrolyze the beta-lactam ring of beta-lactam antibiotics, which leads to complete inactivation of
the antibiotic. Betalactamase may have chromosomal or plasmid-determinism. 23
The genes for the beta-lactamases are located on chromosome or on plasmid, and can be
translocated in the chromosome or in another plasmid by the transposon. Transfer within and
between species or genres successfully explains the spread of resistance mediated by these
ennzime. Betalactamazese classification can be based on molecular characteristics and / or
functional characteristics: Staphylococcus aureus beta-lactamase - there are four serologically
distinct forms that are closely related at the molecular level. Its production can be mediated
plasmidic or chromosomial.
They are particularly active in the penicillin. Chromosomal cephalosporinases of gram-negative
bacteria - virtually all gram-negative bacteria produce enzymes mediated chromosomyal. Most
of these preferential beta-lactamases hydrolyze cephalosporins. Some of these chromosomal

23

Hall and Collis 1995

39

beta-lactamases are able to hydrolyse betalactmine newer such as cefotaxime, cefuroxime,

moxolactam, aztreonam, imipenem.
Plasmidic-mediated betalactamase - are more common gram-negative bacteria and can be
divided in their turn into three main groups: broad spectrum penicillinases (TEM), which
hydrolyze penicillins and cephalosporins in similar proportions, the most widespread in
Enterobacteriaceae; oxacilinaze's (OXA), which rapidly hydrolyze oxacillin and cloxacillin;
carbenicilinaze's (CARB), which hydrolyze preferentially carbenicillin. Metallo-beta-lactamases
- perhaps the most formidable beta-lactamases known. Rapidly hydrolyze most beta-lactam
agents, including carbapenems.It is resistant to Beta-lactamase inhibitors.
These enzymes have been limited to a few strains originally isolated from B. cereus and
Bacteroides fragilis, as chromosomal enzymes, but were subsequently identified in Japan on
plasmids carried by Bacteriodes fragilis, Serratia marcescens, Klebsiella pneumoniae and
Pseudomonas aeruginosa.
Such strains appear to be confined to localized areas, but have potential for wide
dissemination. Enzymes amending aminoglycosides. Nucleotidiltransferase, acetyltransferase
genes encoded plasmid phosphotransferase or transposable elements that often gives them a high
level of resistance. Aminoglycosides are modified by these enzymes at the level of amino and
hydroxyl groups especially through mechanisms of phosphorylation, acetylation or adenilare,
and these changes lead to inactivation of antibiotics.
Inactivating enzymes of chloramphenicol. Resistance is likely a plasmid and is due to the
production of an enzyme by the bacterium, acetyltransferase, causing antibiotic acetylation,
resulting its inactivation.
40

2. Antibiotic resistance through bacteria changes a. modifying the permeability of bacterial

coatings
Structural alterations of bacterial coatings - outer membrane permeability reduction by
reducing the synthesis of porins so as decreasing the number of functional porins applicable
gram-negative bacteria which are in this manner impermeable to betalactamine and quinolones development of impermeable cell walls due to extremely narrow porins for example
Pseudomonas aeruginosa compared to multiple antibiotics - strengthening the barrier function of
the outer membrane proteins by producing additional intercalated protein in its complex
structure. thus producing E. coli tetracycline resistance - reducing bacterial wall permeability
through the loss of a outer membrane protein . resistance of bacteria compared to
chloramphenicol - reducing bacterial permeability deficient in intracellular penetration of
antibiotics is performed compared to sulfonamides, trimethoprim, nitrofurans, nitroimidazoles
modification of active antibiotics transport systems through bacterial coatings for example
Pseudomonas aeruginosa compared to aminoglycosides
b. alteration of headquarters - the target of inhibitory activity of the antibiotic - modification of
PBP (penicillin binding proteins), inserted proteins on the external membrane of the cell, as
action target: betalactamine - ribosomal protein modification target: aminoglycosides,
tetracyclines - macrolides, lincosamides, glycopeptides also determined the modifying of the
target of action - reducing the affinity of action for quinolones is caused by a structural change in
DNA gyrase. 24

24

Hall and Collis 1995

41

c. Active efflux of antibiotics in the bacterial cell - impedes the effective concentrations of
antibioticsin the cell : tetracycline, fluoroquinolones (Staphylococcus aureus), erythromycin
d. modifying enzymes: dihidropterat synthase (DHPS) for sulfonamides and dihydrofolate
reductase (DHFR) to trimethoprim, so that the chemotherapeutic agents can not achieve
competition with the enzymes involved in folate synthesis
e. lack of action of some enzymes: nitrofuran-reductase in the case of nitrofurans and nitroreductase in the case of nitroimidazoles
f. the acquisition by the bacteria of some enzymes - esterase, nucleotidiltransferases, enzymes of
plasmid origin, changes macrolides and lincosamides .
g. metabolite increased production of the antimicrobial agent is in competition - increasing
paraaminobenzoic acid synthesis (APAB) cancels competitive inhibitory action of sulfonamides.
Therefore, given the complexity of resistance to antibiotics and continued growth of this
phenomenon, constant surveillance is needed, involving medical- veterinary laboratories and the
empowered authorities worldwide.

42

CHAPTER 3. PHENOTYPIC AND GENETIC ANALYSIS

OF ANTIBIOTIC RESISTANCE IN GRAM-POSITIVE
AND GRAM-NEGATIVE BACTERIAL STRAINS
ISOLATED FROM THE HOSPITAL ENVIRONMENT IN
BUCHAREST, ROMANIA

Antibiotics-secundary metabolites

The biogenic substances taken by cell as a simple form are used by it in next essential
directions:

43

-to synthesize primary metabolites (amino acids ,purine and pyrimidine bases , enzymes ,
fatty acids),required by structural constituentss byosinthetis , the outcome being the growth of
cell.These compounds are synthesized in primary growth phase , named trofophase;
-for energy production in metabolism and metabolisms products (products of fermentation
alcoholic ,lactic,butyric,propyonic ,acidic);
-for production of secundary metabolites (antibiotics,alkaloids,ergotin , gibberellins).
Secundary metabolites are created only by some species of one genre , as family of
closely related compounds: at least 10 natural penicillins , 10 bacitracins , 25 actinomycines.The
different components proportion in mix depends of genetical factors , environmental factors and
is due to relative lack of specificity of enzymes involved in secundary metabolism.In contrast to
biosynthesis processes of primary metabolites are always catalyzed by high specificity enzymes:
the enzyme knows a single substratum and forming a single product.The specificity of enzymes
action which catalysis the primary metabolites synthesis is due to byosinthesis errors of
essentials celular components are generally lethal and the errors occurring in secundary
metabolism doesnt have significant consequences for main cell , because modified secundary
metabolism is keeping ,usually,biological activity.Secundary metabolites are synthesizing a
larger variety of ways than primary one.
Although they have very diversified chemical structures and it is synthesizes on various
ways , secundary metabolites are assembling from a limited number of intermediate metabolites.
Antibiotics are secundary metabolites whose synthesis starts later in growth phase ,at the
beginning of stationary phase.Autoaudiography experiences with marked amino acids
highlighted that during the time when mycelium is growing at high rate , amino acids are
44

incorporating in cellular proteins ,but doesnt synthesize actinomycin.After the micro-organism

closed the growth phase the rate of incorporation of amino acids in proteins is reduced
considerably.Waksman(1961) has intuited that the characteristic of certain micro-organisms to
synthesize antiobiotic is not correlated with any essential mechanism of nutrition and growth of
cell.In the main , synthesis of secundary metabolites from antibiotics category is underpressor
while the cells are in active multiplication phase and the fastest after that culture enter the
stationary phase.Comparative with primary metabolites , antibiotics have reduced specificity of
byosinthesis because the same organism synthesized many time a group of related molecules.

3.1. Aim and objective

One of the biggest scientific breakthroughs in the medical field, in the twentieth century,
was, undoubtedly, the discovery of antibiotics. This phenomenon became so soon a serious and
complex problem, caused by the development of antibiotic resistance at various pathogens.
Bacteria have special genetic capacity to develop resistance to antibiotics by using any source of
resistance genes and all forms of horizontal transmission of these genes. Genetic, biochemical
and physiological mechanisms, by which pathogens acquire antibiotic resistance, are special and
complex. (Julian Davies and Dorothy Davies, Origins and Evolution of Antibiotic Resistance,
Microbiol Mol Biol Rev. 2010 Sep; 74(3): 417433)
It is essential to understand both of these, mechanisms and causes, which lead to the
evolution of this phenomenon in order to adopt the best measures for combat and prevent.

45

The effects of bacterial antibiotic resistance are very serious; it greatly increases the
incidence of serious infectious diseases and increases mortality.
Various local and national organizations are following and assessing this serious problem.
They proposed several recommendations and resolutions, but in many areas of the world, this
phenomenon was not only stopped but evolved further.
From this point of view the situation in Romania has worsened in recent years, antibiotic
consumption has skyrocketed and simultaneously pathogen resistance to antibiotics has
increased.
"On comparative maps, in 2010, Romania appears in orange but for 2013 was to be
colored in red. There is no progress, "said Dr. Marc Sprenger, director of the European Centre for
Disease Prevention and Control (ECDC), in an interview for the magazine ,,Viata medicala.
Your minister should say that reducing the use of antibiotics it is important for Romania, "said
Sprenger.It is very important to invest in combating this phenomenon. But it all starts with
political will. "
Of all pathogens, bacteria are the most dangerous in terms of morbidity and mortality.
Recently there has been made a database containing more than 20,000 genes with potential
resistance for antibiotics, 257 antibiotics, 632 genomes, 933 species. (Liu B1, Pop M., ARDB-Antibiotic Resistance Genes Database., Nucleic Acids Res. 2009 Jan;37(Database issue):D443-7.
doi: 10.1093/nar/gkn656. Epub 2008 Oct 2.)
In recent years there have been extensive scientific and clinical studies that have set out
to discover the mode of action and resistance mechanisms developed by these pathogens. Also,
the pharmaceutical industry was involvement in carrying out this research. Mechanisms of action
of antibiotics or their biological function is not fully understood. The term, antibiotic" means the
46

class of organic molecules that inhibit and kill bacterial pathogens interacting with receptors
from these organisms. They determine specific cellular responses and biochemical mechanisms
of resistance in pathogens.
Most common nosocomial infections are caused by bacteria of the genus Acinetobacter
baumannii, Burkholderia cepacia, Campylobacter jejuni, Citrobacter freundii, Clostridium
difficile, Enterobacter spp., Enterococcus faecium, Enterococcus faecalis, Escherichia coli,
Haemophilus influenzae, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa,
Salmonella

spp.,

Serratia

spp.,

Staphylococcus

aureus,

Staphylococcus

epidermidis,

Stenotrophomonas maltophilia, and Streptococcus pneumoniae. (Julian Davies* and Dorothy

Davies, Origins and Evolution of Antibiotic Resistance, Microbiol Mol Biol Rev. 2010 Sep;
74(3): 417433)
The forms of pathogens that have developed multiple mutations have reached a high level
of resistance to antibiotics of different classes are now referred by the term ,,superbug". Some of
this are extremely virulent and even has an enhanced transmissibility.
Gram-negative bacteria most commonly, such Escherichia coli, Salmonella enterica and
Klebsiella pneumoniae cause a lot of diseases in humans and animals. Between the use of
antibiotics in treating these disorders and the development of antibiotic resistance there is a direct
and powerful correlation. This situation is more evident in classes of -lactam antibiotic and in
appropriate inactivating enzymes, -lactamases.
From 1970 until now, there has been identified over 1000 -lactamases enzymes that
confer resistance to -lactam antibiotics. (Fig. 1)

47

Fig. 1 Numbers of unique -lactamase enzymes identified since the introduction of the first lactam antibiotics. (Julian Davies* and Dorothy Davies, Origins and Evolution of Antibiotic
Resistance, Microbiol Mol Biol Rev. 2010 Sep; 74(3): 417433)
Hospital acquired infections have become a major threat worldwide. In Romania things
are becoming more serious. Officially, each year more than 8,500 Romanians were infected in a
hospital. The actual number of nosocomial infections is ten times higher, says Romanian doctors.
These infections are difficult to treat and cause high morbidity and mortality.
The increase in antibiotic resistant bacteria combined with a lack of new antibiotic
treatments, is now a serious risk to public health.
The analysis of antimicrobial resistance trends of gram-negative pathogens isolated from
hospital-acquired infections is important for the development of antimicrobial stewardship
programs.

48

Due to the alarming increase of cases of bacterial infections resistant to antibiotic

treatment it is necessary and urgent to adopt radical measures. The causes of antibiotic resistance
are the following, according to Dr. Lo Fo Wong, Senior Consultant Euro-WHO in RAM
(PromoSan - CRSP Bucureti, Ziua European a Informrii despre Antibiotice ZEIA-2013 4.
Comunicat de pres (proiect):
Essentially, RA (antibiotic resistance) is fed by the excess and abuse of antibiotics.
In two of three countries in the Euro-WHO, the antibiotics can be obtained at
pharmacies without a prescription. "
The public must be educated about the fact that steadfast antibiotics destroy bacteria but
dont affect the viruses
Thus, in 9/10 cases, the irritation of the esophagus it is caused by a virus, so it is
unnecessary administration of antibiotics because this promotes RA. "
Integrated systems must be strengthened to monitor the consumption of A and RA;
statistics nationally and internationally, must become transparent and thus mobilize government
policies to control consumption antibiotics and to reduce RA.
In the EU & Norway and Iceland, 5-12% of patients acquire an infection during
hospitalization in recent years. About 400 000 people were infected with a resistant bacterial
strain, and 25 000 lost their lives (2010 data 2011 resumed support package goddess 2012). "
This is not just a matter of local or regional, bacteria potentially fatal easily surpass
borders: the last two years, virulent strains of Escherichia coli, resistant to almost all available
antibiotics, were imported into Europe from countries where they are endemic.
In Romania there are only a few studies and also too little research and statistical data
necessary in programs and projects for combat and prevent this phenomenon. In this regard, the
49

present study aims to determine phenotypic resistance of gram-negative bacterial strains isolated
from hospital.

3.2.Materials and methods

a. Blood culture

Blood culture it is a method of qualitative identification of microorganisms reached the

blood in case of bacteremia and sepsis.
Although technical laboratory blood culture (BC) is a classical method, today it is
considered a viable method for the diagnosis and identification of pathogens in the blood. In its
conventional form this method has low sensitivity and required too long a time for
determination.. Currently it has developed new methods of this technique which has improved
sensitivity and clinical benefit for detecting pathogens in blood. This methods implemented
techniques for the detection of bacterial and fungal DNA. These new molecular methods can
help to reduce hospitalization and lower mortality in patients infected with antibiotic-resistant
bacteria.(O. Liesenfeld 1

,*

L. Lehman, 2 K.-P. Hunfeld, 3 and G. Kost 4, Molecular diagnosis of

sepsis: New aspects and recent developments, Eur J Microbiol Immunol (Bp). 2014 Mar; 4(1):
125.)
Conventional blood culture techniques are based on a blood culture broth enrichment,
followed by testing and pathogen identification using standard biochemical techniques. These
analyzes are important for determining appropriate antibiotic therapy to be administered for each
case. An inappropriate antimicrobial therapy represents a very high risk in patients who have
life-threatening or for that are in critical condition.
50

In recent years laboratory analytical methods have been developed greatly. It was
developed enriched and diversified growth media, it was developed automated stirring systems,
it was elaborated new software that allow rapid detection of bacterial growth using algorithms
that automatically track growth curves. Bacteraemia detection through blood cultures before
initiating therapy and antibacterial choice is very important for life of the patients.
Currently blood cultures are performed using fully automated appliances that detect
microbial growth by analyzing the carbon dioxide released in blood with a fluorescent or
colorometric sensor.

Also, changes in pressure in the bottle headspace is a measure of

consumption and production of gas. These parameters are measured and indicate the rate of
bacterial growth. This method, although it has made great progress still has the disadvantage that
may delay too long for making immediately a medical decision regarding treatment.
Bacteremia is the presence of bacteria in the blood. Blood dissemination (bacteremia) is
when bacteria come from a focus on septic or injured mucosa. Status that may have bacteremia
or clinical expression and may be accompanied by chills and fever to whom can add symptoms
such lesion that caused bacteremia. Sepsis is an aggravated bacteremia with clinical outcome and
is manifested by chills, fever, irregular, toxemia , hypotension , rash.
Pathogens such Staphylococcus aureus are involved in sepsis (blood reach the cutaneous
barrier when it affected mucosa or catheters or drainage). Also coagulase-negative Staphylococci
(CNS), Escherichia coli and other Enterobacteriaceae, Pseudomonas, Streptococcus, anaerobic
bacteria , yeasts can causes bacteremia or sepsis .
Blood is drawn aseptically and inoculated into blood culture bottle whose composition
favors the development of germs aerobic , anaerobic and microaerophilic . It is added

51

environmental enrichment factors which favor the development of Germs such: peptones, casein,
gelatin, hemin, vitamin B6, CO2.
In the culture also it is added an anticoagulant, because otherwise it is the risk being
captured microorganisms in fibrin clot and not to develop colonies. At the same time, some
anticoagulants may be toxic to certain pathogens. It uses sodium polyanetholsulfonat (SPS), a
non-toxic anticoagulant that favors bacterial proliferation by neutralizing human serum
bactericidal activity by inhibiting the action of antibiotics ( streptomycin , kanamycin,
gentamicin , Polymixina B). SPS 's role may inhibit some strains of Peptostreptococcus and
Neisseria effect neutralized by the presence of gelatin in the culture medium .
For blood culture analyzes it uses semiautomatic or automatic computerized systems .
Blood culture bottle it can examine and macroscopically for signs of bacterial growth. If the
liquid in the bottle shows turbidity there is present the Gram negative bacteria, Staphylococcus
ssp, Bacteroides ssp. If there occurs haemolysis there are involved Streptococcus ssp,
Staphylococcus ssp, Lyster ssp, Clostridium ssp, Bacillus ssp. If there appear gas, there is
aerobic and anaerobic Gram-negative bacilli. If occurs blood clots there are S. aureus.
If blood culture is positive (in blood were discovered pathogens that was multiplied in
culture medium), the expansion chamber will pass and will rise above its green sleeve. Then it
will be taken from the bottom of the expansion chamber a small amount of medium to be used
for smears, which will be examined microscopically after Gram staining. In relation to the
outcome readings are subculturing on solid and liquid media: blood agar, Columbia blood agar
chocolate, Levine, Sabouraud Chloramphenicol, thyoglycolate broth with reassuring. Plates were
incubated aerobically at 37 C in 5-7 % CO2 atmosphere for 48-72 hours. Examine colonies
isolated was identified with following tests:
52

Catalase test,

Identification kit Staphylococcus ,

Streptococcus identification kit discs Optochin

Growth factor test using X,V, and XV factor paper disks or strips - Identification of
Haemophilus species by their growth requirements,

Oxidase reaction, identification of yeasts by Candifast test.

These methods are described below:

a. Catalase test is based on the detection of catalase enzyme in the bacteria. This method it
is important to differentiate between Micrococcaceae strong catalase-positive and
Streptococcaceae catalase-negative. Although it is primarily used in the differentiation
between different bacterial genera, this method it is also valuable in the speciation of
certain Gram positive bacteria, such as Aerococcus urinae (positive) viridians from
Aerococcus (negative) and also in the differentiation of Gram negative such as
Campylobacter jejuni and Campylobacter coli (all positive) from the other
Campylobacter species. Also by this method it is possible to make a presumptive
differentiation among some Enterobacteriaceae. The catalase test is also valuable in
differentiating aerobic and obligate anaerobic bacteria, as anaerobes are generally known
to lack the enzyme. In this context, the catalase test is valuable in differentiating aero
tolerant strains of Clostridium, which are catalase negative, from Bacillus, which are
catalase positive.

53

b. The Staphylococcus aureus Detection Kit is a rapid method for the detection of
Staphylococcus aureus. This method is based on the polymerase chain reaction (PCR). A
DNA amplification assay using the polymerase chain reaction (PCR) was designed to
identify S. aureus. Twenty-eight staphylococcal and non-staphylococcal strains tested to
verify the specificity were of the assay, and only S. aureus strains gave a positive
reaction. It may be possible to provide immediate and accurate information for the
identification of S. aureus. (Katsutoshi Saruta, Sadayori Hoshina1and Katsuhiko Machida,
Genetic Identification of Staphylococcus aureus by Polymerase Chain Reaction Using
Single-Base-Pair Mismatch in 16S Ribosomal RNA Gene, Microbiology and
Immunology, Volume 39, Issue 11, pages 839844, November 1995). This method is
highly selective for S. aureus identification in a wide variety of samples. Also, it presents
minimal risk of contamination.
c. Hardy Diagnostics Optochin Differentiation Disks are recommended for use in the
presumptive identification of Streptococcus pneumoniae from other alpha-haemolytic
streptococci. Sensitivity to optochin (ethylhydrocupreine hydrochloride) is a wellestablished phenomenon for Streptococcus pneumoniae. A positive presumptive
identification of S. pneumoniae is made when a well-defined zone of inhibition results
around the impregnated disk. Other alpha-haemolytic streptococci do not display this
clear zone of inhibition when in the presence of optochin.
d. Growth factor test using X,V, and XV factor paper disks or strips - Identification of
Haemophilus species by their growth requirements. Members of the genus Haemophilus
are unable to grow on media which do not contain one or both of two growth factors.
These factors, termed X and V, have been identified as haemin and NAD (nicotinamide
54

adenine dinucleotide) respectively and both are present in blood. Heating blood releases
V factor and so heated blood (chocolate) agar plates are used for isolation of haemophili.
A medium containing neither of the growth factors must be used.
e. CANDIFAST kit enables identification of the following yeasts, Candida, Trichosporon,
Cryptococcus, Rhodotorula and Saccharomyces genus. The identification method is
based on the actidione sensitivity of these yeasts, on fermentation of 7 carbohydrates, and
on urea hydrolysis. The test also can determine the yeasts resistance to different
treatments in order to choose the appropriate treatment of superficial mycoses:
Amphotericin

B,

Nystatin,

flucytosine,

econazole,

ketoconazole,

miconazole,

fluconazole.
Samples were tested for sensitivity to antibiotics or antifungal agents. If that were isolated
several bacterial species in a single bottle or bottles of the same patient different, they are kept in
observation in the following situations: immunosuppressed patient, comatose or cater maintained
for long.
Depending on the period of growth of the bacteria they are divided into:

those that grow in the first 1-2 days as Streptococcus pneumoniae, Enterobacteriaceae;

the growing bacilli after 3 day non-fermentative Gram-negative and anaerobic bacteria;

4-5 days for grow - Haemophilus and Staphylococcus coagulase-negative;

6-7 days for Candida species;

9 days for Corynebacteria

b. Urine culture
55

A urine culture is a lab test to check for bacteria or other germs in a urine sample. Is

analysis that uncovers urinary tract infections. Urinary tract infections are caused by various
microorganisms inflammatory diseases reaching the urinary tract, where it multiplies and
causes as changes in normal kidneys and urinary tract. After location, infections may be low
when the urethra and bladder are affected and high when covered and ureters and kidneys.
Urinary infections can be asymptomatic or symptomatic (presence of pain and /or burning on
urination , pain in the lower abdomen, cloudy and smelly urine ) may be common in boys
during the first year of life, in women with sexual activity, in people diabetic with cancer ,
renal failure , with urethral climb and the elderly (especially in men, due to prostate
problems). To have a threshold bacterial urinary infection must be 105 CFU (colony forming
units ) for Gram negative bacilli /ml urine for 5x104UFC/ml like Staphylococcus and
Candida albicans 104 CFU/ml. Pathogenic bacteria most commonly found in the etiology of
urinary tract infections are Gram negative bacilli glucose fermentation (E. coli, Klebsiella,
Proteus, Enterobacter, Citrobacter , Morganella , Providencia ), Gram-negative bacilli
nefermentativi glucose (Pseudomonas, Acinetobacter), Gram positive cocci ( Enterococcus
spp , Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus hemolytic group
B). Corynebacterium urealyticum causes urinary tract infections in patients with prolonged
antibiotic treatment or urological surgery. Candida albicans determines metabolic
uretrocistite uncontrolled diabetics .
Secretions from the wound produced by damaged tissue and the composition of epidermal cell
death, leukocytes, bacteria. Visual analysis, chemical and bacteriological these secretions are
important to prevent wound infection or discover the healing time and track patient progress .
The main pathogen of wound infection is S. aureus, Pseudomonas aeruginosa in burns.
56

c. Nasal exudate

Is the microbiological examination of nasal secretions for determining upper respiratory

pathogens . Normal bacterial flora of the nose presents a small number of bacterial species are
dominated by Staphylococcus albus , Staphylococcus aureus and difteromorfi. Rarely it can be
identified hemolytic streptococci, enterococci, neiserii nonpathogenic, Acinetobacter and
Moraxella. The relationship between etiologic agents and various diseases of the nose is not
specific only in few cases. The most common inflammatory lesions encountered in the nose are
folliculitis and furunculosis of staphylococcal origin, eczema and impetigo streptococci
vestibular origin, commonly caused by streptococcal infection staphylococcal. In bacterial
rhinitis or viral rhinitis frequently hyperinfected are frequently isolated coagulase-positive
staphylococci Neisseria menigitidis. In purulent rhinitis often is found association with
Staphylococcus pneumonia and those with false membrane, association Staphylococcus,
Pneumococcus and Corynebacterium diphtheriae. Bolilecronice Ozen and rinoscleromul nose
and are usually caused by Klebsiella rhinoscleromatis. Sampling is done by one sterile swab
moistened with saline, different for each nostril, which rotates in nasal cavity and intoduce easy
into cavity to detach secretion. Bacteriological examination of nasal secretion involves
performing cultures, microscopic examination is relevant only in case of porting the diphtheria
bacillus. In this case collects two pads, one serving to conduct a smear, and the other to the
sowing of crops. Usually, nasal swab seeded on blood agar. Pharyngeal exudate is
bacteriological examination of secretions from the pharynx. Discharge sample is taken from the
pharyngeal cavity with a sterile swab will then be placed in a recipientce presents a solution that

57

will stimulate the growth of microorganisms, and all the buffer that will inoculate the culture
medium.
d. Pathogenic bacterial strains. In this study we have used pathogenic bacterial strains E. coli
and K. pneumoniae. E. coli, Klebsiella pneumonia and

Proteus mirabilis and Serratia

marcescens and Aeromonas hydrophila and coagulase-negative Staphylocci and Enterococcus

faecium, Enterococcus faecalis, Streptococcus agalactiae and Streptococcus spp and Candida
albicans and non-C. albicans strains, provided at Microbiology Laboratory of the Institute of
Cardiovascular Diseases "( Institutul Clinic Fundeni", Bucharest, in 2013)
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that
normally inhabits the soil and surfaces in aqueous environments. Its adaptability and high
intrinsic antibiotic resistance enable it to survive in a wide range of other natural and artificial
settings, including surfaces in medical facilities. Serious P. aeruginosa infections are often
nosocomial, and nearly all are associated with compromised host defenses such as in
neutropenia, severe burns, or cystic fibrosis (CF; Table 1; Lyczak et al., 2000). Therapeutic
options are increasingly limited due to the continued emergence and spread of antimicrobial
resistant strains; as a result, P. aeruginosa infections demonstrate high morbidity and mortality.
In the United States, P. aeruginosa is among the most common hospital pathogens and is the
second most common pathogen isolated from patients with ventilator-associated pneumonia
(VAP; Hidron et al., 2008). Given the severity of P. Aeruginosa infections and the limited
antimicrobial arsenal with which to treat them, finding alternative prevention and a treatment
strategy is an urgent priority.
Table 1
Infections

Major risk factors

58

Soft tissue

Burns, open wounds, post-surgery

Urinary tract

Use of urinary catheter

Bacteraemia

Immunocompromised

Diabetic foot

Old age, COPD, cystic fibrosis,

mechanical ventilation

Otitis external (swimmers Tissue injury, water blockage

ear)

in ear canal

Keratitis (corneal infection Extended contact lens wear,

Otitis media folliculitis

contaminated contact lens solution

Otitis media folliculitis

(hot tub rash)

(hot tub rash)

The most prevalent bacterial infections were Acinetobacter spp. (26%), Pseudomonas spp.
(18%), and Klebsiella spp. (9%), which represented a 1% decrease in the Pseudomonas spp.
prevalence rate and a 4% and 11% increase in prevalence of Acinetobacter spp. And Klebsiella
spp.

e. The Pseudalert test detects the presence of Pseudomonas aeruginosa in bottled, pool, and
spa water samples. The test is based on a bacterial enzyme detection technology that signals the
presence of Pseudomonas aeruginosa through the hydrolysis of a substrate present in the
Pseudalert reagent. Pseudomonas aeruginosa cells rapidly grow and reproduce using the rich
supply of amino acids, vitamins, and other nutrients present in the Pseudalert reagent. Actively
growing strains of Pseudomonas aeruginosa have an enzyme that cleaves the substrate to
59

produce a blue fluorescence under UV light. Pseudalert detects Pseudomonas aeruginosa at 1 cfu
in either 100 mL or 250 mL samples within 24 hours for non-carbonated water samples and
within 26 hours for carbonated samples.
The susceptibility of 104 isolates of P. aeruginosa to 13 different antibiotics was
determined by agar disk diffusion method. The alkaline lysis method was used for plasmid
extraction. PFGE technique was optimized for DNA fingerprinting of isolates
Pseudomonas aeruginosa is a Gram-negative, aerobic, monoflagelat, asporogenous,
nonfermentativ opportunist that is found in soil, water, and plant surface, have minimal
nutritional requirements and can withstand low temperatures. It is able to use food as a wide
range of organic products that adapt and thrive in many ecological niches, even in fossil fuel.
The water increases as biofilm or free cells. Although aerobic environments may increase
without O2, NO3 or arginine only if there is medium. The cell has a size of 1-5 long and 0.5 to
1 in diameter (B. Iglewski, 1996).
Pseudomonas aeruginosa is commonly found in contaminated water, sewage system, the
fountains, the swimming pools. Pseudomonas groups to form biofilms tend to adhere to a wide
variety of substrates, making them the same resistance to environmental factors and antibiotics.
This bacterium is an opportunistic pathogen because it causes serious infections in
immunocompromised patients, causing nosocomial infections as found on most medical
equipment, toilets , showers , sinks, and even the potent disinfectant soaps. Clinical isolates
secrete certain pigments and piocianin (colonies that secrete the pigment acquires a greenish
blue tint) , pioverdin (green pigment is secreted especially in environments lacking Fe)
piorubin (purple pigment) and piomelanin (brown pigment).
60

P. aeruginosa can cause suppurative infections in patients with burns or surgery can
cause pneumonia, bacteremia , endocarditis , meningitis and brain abscess , otitis externa and
media keratitis , endoftalmii , osteomyelitis , urinary tract infection , enterocolitis , chronic
infections patient with cystic fibrosis .
In vitro Pseudomonas colonies are usually smooth, fried egg may look flattened edges
and higher middle or secretaries may look mucoid alginate due mainly to strains isolated from
tracheal secretions and urine. Colonies also producing a characteristic odor of linden flowers and
green apple . P. aeruginosa grow in environments with an optimum temperature of 37 C, but
may grow to reach normal temperature and 42 C
.

3.3. Results and Discussion

Pseudomonas aeruginosa
From a total of 197 strains of P. aeruginosa, isolated during the year 2012, 79 strains were
isolated tracheal secretions (representing a 40%), 47 (24%) of the wound secretions, 25 (13%) of
urocultures , 22 (11%) from blood cultures, 17 (9%) of the various secretions (bronchial
secretions of eschar prosthesis iliac vein catheter, dialysis catheter, necrosis), 5 sputum and nasal
exudate 3 of of 2% and 1 % of isolates (fig.1).

61

Fig. 1. Graphical representation of the sources of isolation of strains Pseudomonas

aeruginosa.

Section with the highest prevalence of infection with P. aeruginosa was polling Surgery
Cardiovascular (100 cases), followed by vascular surgery department (57 cases), Cardiology 1
(24 cases), Anaesthesia and Intensive Care (7 cases), Cardiology 2 (4 cases), Cardiology 4 (3
cases) and Cardiology 3 in two cases. The highest number of deaths was recorded in
cardiovascular surgery department (63 cases). (Fig. 2)

62

Fig. 2. Graphical representation of the prevalence of infections caused by Pseudomonas

aeruginosa in different wards of the hospital.

Most patients who suffered infections with P. aeruginosa were over the age of 65 years (109
cases), 71 of them were aged between 50 and 64 years, 15 were aged between 35 and 49 years, 2
were classified in the category 20-34 years; there was no patient younger than 20 years (fig. 3).
The average age of all patients was 66 years.

Fig. 3. Graphical representation of patients with infections caused by

Pseudomonas aeruginosa by age.

63

Of all patients who have infections caused by P. aeruginosa 151 (77%) belonged to the male
gender and 46 (23%) female gender. (Fig. 4)

Fig. 4. Graphical representation of patients of patients caused by Pseudomonas

aeruginosa by sexes

The mean duration of hospitalization days in patients whose tracheal secretions was
isolated P. aeruginosa was 62 days, and their average age was 65 years. Of these 62 patients
(78%) were male, and 17 (22%) of the female. 15 (20%) of the patients had more than 100 days
of hospitalization, 28 (35%) had between 50 and 100 days of hospitalization and 36 (45%) stayed
hospitalized less than 50 days. From Figure 5 it can be seen that the days of hospitalization were
the more as the age of the patients was mad. The majority of patients who were hospitalized for
less than 21 days were under the age of 60 years.

64

Fig. 5. Plotting the number of days of hospitalization correlated with age in patients
whose tracheal secretions was isolated Pseudomonas aeruginosa.

Whose average age of patients of wound secretions were sampled P. aeruginosa is 65

years and the average duration of their hospital stay is 16 days. 37 (79%) of the patients with
infections caused by P. aeruginosa wounds were male and 10 (21%) were female. Most patients
were under 20 days of hospitalization, eight people were hospitalized between 20 and 35 days,
but there were two people that were hospitalized 87, and 110 days. Most patients with wound
infections were aged between 40 and 65 years (48%), 7 (14%) patients were over 80 years (fig.
6)

65

Fig. 6. Plotting the number of days of hospitalization correlated with age in patients
whose wound secretions of Pseudomonas aeruginosa was isolated.

Patients whose secretions various (eschar bronchial prosthesis iliac venous catheter, dialysis
catheter, necrosis) was isolated bacillus Pseudomonas had an average of 63 days of
hospitalization days, most being hospitalized for a period longer than 40 days, and 4 of them in
the hospital for more than 100 days. The mean age of patients was 61 years, most were over 55.
(fig. 7)

Fig. 7. Plotting the number of days of hospitalization correlated with the age of patients whose
discharge various (eschar bronchial prosthesis iliac venous catheter, dialysis catheter, necrosis) were
isolated
66

The mean duration of hospitalization days in patients whose blood cultures were isolated P.
aeruginosa was 57 days, and their average age was 67 years. Of these 13 patients (59%) were
male and nine (41%) of the female. 10 (45%) of the patients had more than 80 days of
hospitalization, 7 (32%) had between 20 and 60 days of hospitalization and four (18%) stayed
hospitalized for less than 10 days. Patients who were hospitalized more than 80 days had over 65
years. (fig.8).

Fig. 8. Plotting the number of days of hospitalization correlated with age in patients
whose blood cultures was isolated Pseudomonas aeruginosa.

In patients with urinary tract infections caused by P. aeruginosa, the average days of
hospitalization was 54 days, 6 (24%) of them internal state between 90 and 130 days, 5 (20%)
67

between 60 and 70 days, 9 (36%) of 10 to 30 days, and 3 (12%) in 10 days. The median age was
65 years. In patients with urinary tract infections 20 (80%) were male and 5 were female. (fig. 9)

Fig. 9. Plotting the number of days of hospitalization correlated with age in patients
whose urine cultures was isolated Pseudomonas aeruginosa.

Among patients whose sputum P. aeruginosa was isolated 3 had an age of 75 years and 2 had an
age of 40 years. Their length of stay not exceeding 21 days, average days of hospitalization was
12 days.

68

Fig. 10. Plotting the number of days of hospitalization correlated with age in patients
whose sputum Pseudomonas aeruginosa was isolated.

The results showed widespread resistance (51.1 91.2 %) of the isolates to all the antibiotics,
except nitrofurantoin with resistance rate of 7.3 % (Figure 10). Among the fluoroquinolones,
ofloxacin showed the least resistance (51.1 %), followed by Ciprofloxacin (65.7 %) and
Norfloxacin (86.9 %). The result also showed that the E. coli isolates were multiply-resistant
with the highest percentage of multiresistant isolates skewed toward the highest number of
antibiotics. Up to 50 (36.5%) strains were resistant to 10 out of the 11 antibiotics employed. A
total number of 20 (14.6 %), 18 (14.14 %) and 13 (9.5 %) isolates were resistant to 7, 8 and 9
antibiotics respectively, while 8 (5.8 %) of the isolates were resistant to all the antibiotics
employed.

Conclusions
During 2013 were isolated 337 strains of S. aureus isolation sources are the following: 51 % of
wound secretions, nasal exudate 30%, 8% from blood cultures, 5% of tracheal secretions, 5% of
69

secretions (aneurysm, discharge valves, ganglion, abscess, mediastinal fluid, vascular prosthesis,
venous catheter, dialysis catheter, and necrosis) and 1% of sputum.
This study revealed the presence of ESBL producing Eschericiha coli in Cotonou. It
demonstrated also high resistance rate to antibiotics commonly used for infections treatment.
Continuous monitoring and judicious antibiotic usage are required.
Of the 197 strains of P. aeruginosa isolated during 2012, 40 % tracheal secretions , 24% of
wound secretions , 13% of urine cultures , 11% of blood cultures , 9% of secretions ( bronchial
secretions of eschar prosthesis iliac venous catheter , dialysis catheter , necrosis) , 2% and 1%
sputum nasal exudate .
The study showed a high prevalence of infection with P. aeruginosa in the Department of
cardiovascular surgery. Nosocomial infections with P. aeruginosa species etiologic agent caused
the highest mortality rate (51%).
P.aeruginosa has been reported to have an innate resistance to several antibiotics due to the
presence of lipopolysaccharides in the outer membrane, but persistent administration of
antimicrobial agents, has resulted in the emergence of multi-resistant strains of P. aeruginosa
(Van Eldere, 2003).

70

4. Resistance patterns of Staphylococcus aureus from Bucharest

Wisam abdulameer najm, Mariana-Carmen Chifiriuc*, Irina Gheorghe, Otilia Banu,

Mihailescu Dan

4.1. Aim and objective

Antibiotic resistance is an increasingly serious global problem due to rapid spread of

infections and because of the current inability to effectively control this phenomenon. Pathogens
develop resistance to antibiotic both naturally and through human action. Excessive consumption
of antibiotic determines each year death of about 23,000 people that were infected with
antibiotic-resistant bacteria. Every year about 2 million people become infected with antibioticresistant bacteria. These infections can occur anywhere within a community, but the highest
frequency of occurrence is found in clinics and hospitals. Antibiotic resistance is such a serious
problem worldwide. Antibiotics are ineffective and even harmful to human body. The
mechanisms of resistance can be divided into two categories; (1) Chemical affecting of the
binding interaction between a target site and the antibiotic, and (2) inactivation of the antibiotic.
The mechanisms of inactivation of antibiotics are commonly associated with bacteria that
develop this capability by excessive use of antibiotics by humans. An example of this is
Staphylococcus aureus that are fighting with antibiotics by deactivating -lactam binding
71

protein. Staphylococcus aureus is a gram positive bacterium that is present in the human
microbiota and in community and hospital environments. The presence of S. aureus on the skin
(microbiota) may actually be beneficial to humans because the bacteria enable and expand
memory of T cells. But if the bacteria infect the body, it becomes extremely dangerous and in
some cases even fatal. In 2005, more than 477,000 people were hospitalized due to S. aureus
infections. The mortality rate was approximately 20-40%. The first treatment for staphylococcal
infection was penicillin and then methicillin was synthesized. The bacteria quickly developed
resistance to the antibiotics. Today, S. aureus is a growing problem both in hospitals and
communities because of its virulence and because of the ability to adapt to different
environments. The bacterium has developed resistance to antibiotics through mechanism
including horizontal gene transfer and altering of antibiotics.
The first found that antibiotic treatment for S. aureus was penicillin in 1940, which significantly
decreased mortality rates of infected patients. Two years later, the bacteria had become resistant
to treatment. More than 80% of the Staphylococcus strains have been shown to be resistant to the
beginning of 1960. In these strains, Bond and Dietz found, in 1948, penicillinase, which is a
specific type of -lactamase.
Beta-lactam antibiotics such as penicillin acts by inhibiting the bacterial cell enzyme, in
this case, it is -lactamase. Inhibition of this enzyme slows down the formation of the cell wall;
also the osmotic pressure decreases and finally kills the cell. -lactam antibiotics are capable of
inhibiting the enzyme with -lactam ring, which binds to the enzyme in the bacterial cell. A
mechanism by which bacteria become resistant to antibiotics is through the hydrolysis of lactam ring. A specific gene reorganized and did not allow the enzyme to bind to -lactam ring.
This gene is easily mobile and is transmitted horizontally.
72

After 21 years, in 1961, it was synthesized methicillin, another antibiotic to combat those
strains of Staphylococcus aureus resistant to penicillin. In less than one year were identified
methicillin-resistant strains of Staphylococcus aureus. Since 1970 this bacterium has become an
increasing problem both in hospitals and in the community. As penicillin, methicillin acts by
inhibiting cell wall synthesis by turning off the formation that forms the cell wall peptidoglycan.
The mechanism of development of resistance to antibiotics is similar to the one developed by the
bacteria to penicillin.
The current study aims to investigate this dangerous phenomenon in a hospital from
Bucharest, Romania and to identify patterns of resistance of Staphylococcus aureus. To combat
this phenomenon it must be watched carefully and understood very well. You cannot destroy an
enemy if you do not know him very well and does not identify all weapons that he has.
If the enemy constantly adapts to our weapons also we must be always on guard against
him and to adapt continuously. Just so we can prevent and limit the phenomenon by making as
many studies.

4.2. Materials and methods

A total number of S. aureus strains (n=20) were isolated from hospitalized patients with
various cardiovascular diseases from different clinical sources, mostly from wound secretions
during 2014 and 2015 from Cardiovascular Disease Institute Prof. C.C. Iliescu in Bucharest
(Romania). Other sources of isolation were: nasal exudates, respiratory secretions, blood cultures
venous, peritoneal fluid, pleural fluid, tracheal aspirates, pharyngeal exudates, only one strain
was isolated from a venous catheter. The strains identification was performed in the
73

Microbiology Laboratory of the Cardiovascular Diseases Institute Prof. C.C.

Iliescu with the use of the coagulase test, the Mannitol enriched medium
and the automated VITEK 2 system.
Staphylococcus aureus is known to produce coagulase, which can clot plasma into gel in
tube or agglutinate cocci in slide. This test is useful in differentiating S.aureus from other
coagulase-negative staphylococci. The bound coagulase is also known as clumping factor. It
cross-links the and chain of fibrinogen in plasma to form fibrin clot that deposits on the cell
wall. As a result, individual coccus sticks to each other and clumping is observed.
Enriched media are media that have been supplemented with highly nutritious materials
such as blood, serum or yeast extract for the purpose of cultivating fastidious organisms.
Mannitol salt agar is both a selective and differential media used for the isolation of
pathogenic Staphylococci from mixed cultures.
The VITEK 2 system has everything healthcare laboratories need for fast, accurate
microbial identification, and antibiotic susceptibility testing. The innovative VITEK 2
microbial identification system includes an expanded identification database, the most automated
platform available, rapid results, improved confidence, with minimal training time. The VITEK
2 system next-generation platform provides greater automation while increasing safety and
eliminating repetitive manual operations. The rapid response time means results can be provided
more quickly than with manual microbial identification techniques.
The susceptibility analysis of the strains was performed by diffusion method (Kirby
-Bauer), following the recommendations of CLSI editions 2013 and 2014. Determination of
bacterial resistance to antimicrobials is an important part of the management of infections in
patients. The disk diffusion method of Kirby and Bauer has been standardized and is a viable
74

alternative to broth dilution methods for laboratories without the resources to utilize the newer
automated methods for broth microdilution testing.
When a 6-mm filter paper disk impregnated with a known concentration of an
antimicrobial compound is placed on a Mueller-Hinton (MH) agar plate, immediately water is
absorbed into the disk from the agar. The antimicrobial begins to diffuse into the surrounding
agar. The rate of diffusion through the agar is not as rapid as the rate of extraction of the
antimicrobial out of the disk, therefore the concentration of antimicrobial is highest closest to the
disk and a logarithmic reduction in concentration occurs as the distance from the disk
increases (7). The rate of diffusion of the antimicrobial through the agar is dependent on the
diffusion and solubility properties of the drug in MH agar (2) and the molecular weight of the
antimicrobial compound. Larger molecules will diffuse at a slower rate than lower molecular
weight compounds. These factors, in combination, result in each antimicrobial having a unique
breakpoint zone size indicating susceptibility to that antimicrobial compound.
Genomic bacterial DNA was extracted using the alkaline extration method. One to five
colonies of bacterial cultures were suspended in 1.5 ml tubes containing 20 L solution of NaOH
(sodium hydroxide) and SDS (sodium dodecyl sulphate). For the permabilization of cell
membrane the tubes were heated on thermoblock at 95oC for 5 minutes. 180 L TE buffer (Tris
+ EDTA)1X was added in the tubes and the tubes were centrifuged at 13000 rpm for 3 minutes.
The DNA in the supernatant was kept and stored at -4 oC before analysis. All PCR reactions were
performed using Thermal Cycler machine Corbet. The amplification products of each PCR
reaction (multiplex / simplex) were visualized by electrophoresis on a 2% agarose gel, stained
with ethidium bromide (10 g / ml) and identified based on their typical dimensions by means of
specific molecular weight markers (100pb, I Lader Bench Top 100bp DNA, Promega, USA).
75

Genotypic-characterized SCCmec cassette types present in analysed strains was

performed using PCR methods (simplex and multiplex) in order to elucidate the structure of
these genetic elements and obtaining the relevant data from the epidemiological point of view.
Two reactions were performed using the multiplex PCR five and four pairs of specific primers
for the various sequences at the SCCmec cassette to detect simultaneously the necessary
constituents of the primer. Their classification and parameters used to conduct of reactions
followed the protocol developed by Miheirico et al. (2007). Sequences of primers used, their
specificity and amplification programs used are listed in Tables 1 and 2 and the
components used in these reactions are shown in Table 3.
Table 1. Nucleotide sequences of primers used, their specificity and the size of the amplicons
obtained (after Milheirico et al., 2007).
Primeri

The nucleotide sequence

Size

Primeri

amplicon

Specificity

(pb)

(mec cassette
type, region)

CIF2 F2

5-TTC GAG TTG CTG ATG AAG AAG G-3

CIF2 R2

5-ATT TAC CAC AAG GAC TAC CAG C-3

RIF5 F10

5- TTC TTA AGT ACA CGC TGA ATC G-3

RIF5 R13
ccrB2 F2

495

I, region J1

414

III, region J3

311

II and IV,

5- GTC ACA GTA ATT CCA TCA ATG C-3

5-AGT TTC TCA GAA TTC GAA CG-3

complex ccr
76

ccrB2 R2

5-CCG ATA TAG AAW GGG TTA GC-3

mecI P2

5-ATC AAG ACT TGC ATT CAG GC-3

mecI P3

5-GCG GTT TCA ATT CAC TTG TC-3

209

II and III,
mec
complexes

mecA P4

5-TCC AGA TTA CAA CTT CAC CAG G-3

mecA P7

5-CCA CTT CAT ATC TTG TAA CG-3

SCCmecV J1 5-TTC TCC ATT CTT GTT CAT CC-3

F

162

gene mecA

377

V, region J1

342

I, II, IV and

5-AGA GAC TAC TGA CTT AAG TGG-3

SCCmecV J1
R
dcs F2

5-CAT CCT ATG ATA GCT TGG TC-3

dcs R1

5-CTA AAT CAT AGC CAT GAC CG-3

kdp F1

5-AAT CAT CTG CCA TTG GTG ATG C-3

kdp R1

5-CGA ATG AAG TGA AAG AAA GTG G-3

SCC mec III

J1 F

5-CAT TTG TGA AAC ACA GTA CG-3

VI, region J3
284

II, region J1

243

III, region J1

5-GTT ATT GAG ACT CCT AAA GC-3

SCCmec III
J1 R

Table 2. PCR conditions used to amplify the SCCmec element (after Milheirico et al. 2007).
The amplification program
temperature

94C

94C

53C

72C

72C
77

Duration
Number of

4 min

30 sec

30 sec

1 min

4 min

30

cycles

Table 3. Reaction components used in the PCR reactions

Volume

Volume PCR

Volume ultra

primers

Master Mix*

pure water

10 l

6,5 l

Volume ADN

The reaction
volume

(10M)
0,3 l

0,5 l

20 l

The two reactions were performed using the multiplex PCR of four pairs of specific
primers in order to distinguish types and subtypes I V SCCmec cassettes. Sequences of primers
used and deployment parameters of reactions followed the protocol developed by Zhang et al.
(2005). However, a simplex PCR gene detection followed CCRC, SCCmec cassette recombines
complex. Sequences of primers used, their specificity and amplification programs used are listed
in Tables 4, 5 and 6 and the components used in these reactions are shown in Table 3.
Table 4. The nucleotide sequences of primers used, their specific nature and size of amplicons
produced (by Zhang et al. 2005)

78

Primers

The nucleotide sequence of

Type I-F

5-GCT TTA AAG AGT GTC GTT ACA GG-3

Type I-R

5-GTT CTC TCA TAG TAT GAC GTC C-3

Type II-F

5-CGT TGA AGA TGA TGA AGC G-3

Type II-R

5-CGA AAT CAA TGG TTA ATG GAC C-3

Type III-F

5-CCA TAT TGT GTA CGA TGC G-3

Type III-R

5-CCT TAG TTG TCG TAA CAG ATC G-3

Type IVa-F

5-GCC TTA TTC GAA GAA ACC G-3

Type IVa-R

5-CTA CTC TTC TGA AAA GCG TCG-3

Type IVb-F

5-TCT GGA ATT ACT TCA GCT GC-3

Type IVb-R

5-AAA CAA TAT TGC TCT CCC TC-3

Type IVc1-F

5-TCT ATT CAA TCG TTC TCG TAT T-3

Type IVc1-R

5-TCG TTG TCA TTT AAT TCT GAA CT-3

Type IVd1-F

5-AAT TCA CCC GTA CCT GAG AA-3

Type IVd1-R

5-AGA ATG TGG TTA TAA GAT AGC TA-3

Type V-F

5-GAA CAT TGT TAC TTA AAT GAG CG-3

Type V-R

5-TGA AAG TTG TAC CCT TGA CAC C-3

ccrC-F

5-CGT CTA TTA CAA GAT GTT AAG GAT

ccrC-R

Amplic

Specific

on size

primers (mec

(pb)

cassette type)

613

SCCmec I

398

SCCmec II

280

SCCmec III

776

SCCmec IVa

493

SCCmec IVb

200

SCCmec IVc

881

SCCmec IVd

325

SCCmec V

495

ccr Tip 5

AAT-3
79

5-CCT TTA TAG ACT GGA TTA TTC AAA

ATA T-3

Table 5. The conditions used for PCR amplification of the genetic elements according to the
types of SCCmec cassettes (by Zhang et al. 2005)
The amplification program
Temperature

94C

94C

65C

72C

94C

55C

72C

72C

Duration

45

45

1,5

45 sec

45

1,5

10 min

min

sec

sec

min

sec

min

Number of cycle
1

10

25

Table 6. PCR conditions used to amplify gene CCRC (by Zhang et al. 2005).
The amplification of program
Temperature

94C

94C

50C

72C

72C

Duration

5 min

1 min

1 min

2 min

10 min

Number of

30

cycles

*DreamTaqGreen(ThermoScientific,SUA),2x.

All PCR reactions were performed using Thermal Cycler machine Corbet. The amplification
products of each PCR reaction (multiplex / simplex) were visualized by electrophoresis on a 2%
80

agarose gel, stained with ethidium bormure (10 g / ml) and identified based on their typical
dimensions by means of specific molecular weight markers (100pb, I Lader Bench Top 100bp
DNA, Promega, USA).
4.3. Results and discussion
Given the increased number of genetic polymorphisms detected in the SCCmec cassette
is necessary to know the description and classification of them in order to understand the
molecular epidemiology of S. aureus resistant to methicillin (MRSA), a powerful pathogen
prevalent in both nosocomial and community. Given the increased number of genetic
polymorphisms detected in the SCCmec cassette is necessary to know the description and
classification of them in order to understand the molecular epidemiology of S. aureus resistant to
methicillin (MRSA), a powerful pathogen prevalent in both nosocomial and community.
Table 4. The results obtained in these experiments.

Bacter

SCCmec cassetetes genes

me

mec

CI

ccr

strains B2

cI

F2

S.a.2

S.a 3

S.a.4

S.a.5

S.a.6

S.a.7

S.a.8

ial

ccr

SCCme TypeI
cVJ1

Va

TypeI

TypeI

Typ

TypeI

Vb

Vc

eII

Vd

+
+

+
81

S.a.9

S.a.10

S.a.11

S.a.12
S.a.13

S.a.14
S.a.15
S.a.16
S.a.17
S.a.18
S.a.19
S.a.20

The molecular analysis through PCR arrays showed that 8 strains revealed the ccrB2 gene; only
1 strain expressed the mecI gene (Tab4, Fig 1). In the case of the ccr genes the genotypic analysis
pointed out those 9 strains presented the ccrC gene (Tab4, Fig 2). Regarding the types of
SCCmec cassettes 9 strains expressed the Type Iva (Tab. 4, Fig 3).

ccrB2-311
mecl209 bp
82

Figure 1. Gel electrophoresis with primers for corresponding elements of SCCmec cassettes:
ccrB2, mecI, mecA, CIF2, RIF5. The figure shows that MRSA isolates 2, 3, 4, 5, 6, 7, 8, 9
express the ccrB gene and only strain no.2 revealed mecI gene. Well 1(top and bottom) marker
gm: 100pb

ccrC-495 bp

Figure 2. Gel electrophoresis with primers for corresponding ccrC gene. Well 1(top and
bottom) marker gm: 100pb. (Promega , USA). Wells 2-20: S.aureus strain analysed . Wella: 2, 4,
5, 6, 8, 9, 14, 18, 19: positive.

Type IVa-776 bp

83

Figure 3. Gel electrophoresis with primers for corresponding Type Iva, Type IVb, zzType IVc,
and Type II genes. Well 1 (top and bottom) marker gm : 100pb.(Promega, USA).Wells 3-22:
S.aureus strain analysed. The wells: 5, 7, 8, 9, 10, 11, 13, 17, 19: positive for Type Iva
element(776 bp)

84

3.2.4. Genotypic analysis of antibiotic resistance patterns

3.3. Results and discussion

3.3.1. Resistance phenotypes in Gram negative bacterial strains isolated from hospital
infections1
Description of the device:
Identifyin the bacteria with the automatized system VITEK2
Compact VITEK2 is a automatized system with high performances (MacLowry and
colab.) which identifying and testing the bacteria sensitivity at antibiotics using available cards
of system VITEK2.
Procedure: transfer asepsis 3,0ml sterile saline solution (aqueous 0.45%-0.50% NaCl,pH 4.5-7.0)
into a transparent plastic tube (polystyrene 12 x75 mm).Select isolated colonies from primary
board or from testing subculture that grows on appropriate culture in conditions of recomended
85

incubation.Prepare a bacterial homogeneous suspension with a equivalent density of 0.5-0.63

standard McFarland using VITEK2 DENSICHECK.The suspension tube is placed in
cassette.Oped the foil of the VITEK2 card and put into cassette.The transfer of inoculum in
card it makes in the filling chamber of compact VITEK2 and afterwards it transfers in
reader/incubator room.After sealing , cards are loaded automatically in incubation carousel , and
the incubation of VITEK2 card is automatic in incorporated incubator of system.The
temperature of incubation is 35.5 +1C.The reading of card is automatically make.
Reading method:
-turbidimetrycally :for testing bacteria sensitivity at antibiotics.
-colormetrycally:for identifying of bacterial species.
Reading number:
-turbidimetry:48 points of reading (3x16)/automatic reading/well;
-colorimetry:48 points of reading (3x16)/automatic reading/well;
The changes of bacterian metabolisma and growth rate during the incubation lead changes of
transmittance values.Each cell is read every 15 minutes with a optical reader at a wavelenght of
430 or 568 nm.For each reading the measurements are performed automatically for each well in
16 different points 3 time (in total 48 points).The reading in dynamic allow getting a curve of
growth /well.
Reading performance:
For testing bacterias sensitivity at antibiotics:
86

-wavelenght.....660nm
-measuring accuracy.......2%
-bacterial detection level....5x107-5x108UFC/ml(0,2 McFarland until 2 McFarland).

The identification of a micro-organismusing system VITEK2 uses a methodology

based on comparing the species features with data about micro-organism and analysed
reactions.The interpretation of results allow getting the biochemical profile for all tests by
comparing with introduced results in database for each taxon which can be indentifying by card.
At bacterial species gram negative card contain 47 biochemical tests allowing the recognition of
158 species including negative control.The final results are completed over the period of 2-10
hours.
The sensitivity card at antibiotics for VITEK2 systems represent a automatic based
way on method C.M.I , each card test contain 64 microcells.The distribution of negative
control(culture environment) it is compulsory in all the cards , the others cells containing
different amounts of antibiotics in culture environment , tested in different concentrations. The
VITEK2 system is monitoring the growth of each cell of card in a period of time (18
hours).During the incubation , C.M.I values are determined for each antibiotics from card.

87

3.3.2. Resistance phenotypes in Gram positive bacterial strains isolated from hospital
infections Workors of antibiotics resistance in Gram positive cocciisolated from
hospital infections
Results and Discussion

S. aureus strains were isolated from hospitalized patients with various cardiovascular
diseases from different clinical sources, mostly from wound secretions. Other sources of
isolation were: nasal exudates, respiratory secretions, blood cultures venous, peritoneal fluid,
pleural fluid, tracheal aspirates, pharyngeal exudates, only one strain was isolated from a venous
catheter.
Using PCR (multiplex and simplex) sought evidence of gene-specified regions present in
the SCCmec elements to classify and characterize them. Given the increased number of genetic
polymorphisms detected in the SCCmec cassette is necessary to know the description and
classification of them in order to understand the molecular epidemiology of Staphylococcus
aureus resistant to methicillin (MRSA), a powerful pathogen prevalent in both nosocomial and
community.
Results of PCR experiments are shown in Figures 14. Table 13 summarizes the results
obtained in these experiments.

88

Fig. 14.Vizualizarea in gel electrophoresis of amplification products corresponding elements of

SCCmec cassettes using primers: SCCmecV J1, J1 SCCmecIII, KDP, DCS, S. aureus strains
analyzed. Gode 1: MW marker .: 100pb (Promega, USA). Wells 2 10: S. aureus strains analyzed.
The test of results Strain 2 positive gene and the rest tulpinilo SCCmecIII J1 except strain
no. 10 (ATCC) were negative by achieved using the PCR primer pairs specific for the different
parts of the SCCmec cassette, so the coding regions, non-coding regions as well as the allowed
typing to 19 strains of S. aureus, the remainder being non-tipabile USING methods.
Thus, a total of 7 strains containing type III SCCmec cassette, as confirmed by the
presence of the mecA gene, and match the specific sequences in the regions J3 and J1 (evidenced
by RIF5 primer pairs, respectively J1 SCCmec III). The results obtained using the method
proposed by Zhang et al. (2005), confirms membership in this category SCCmec cassette. It is
89

noted, however, the presence of gene CCRC (specific SCCmec V) in all 8 strains and the
presence of dcs region (characteristic SCCmec I, II, IV and VI) which is explained by the
extraordinary mobility of these genetic elements and susceptibility loss / acquisition of
components that may be present at any given time, certain strains of bacteria. It also noted the
presence of a strain containing both ccrB2 gene (specific SCCmec II and IV) and gene CCRC
box indicating possible presence SCCHg, with SCCmec III integrated in tandem at the bacterial
chromosome.
He noted the presence of four strains, although containing elements required classification as
SCCmec type III gene have no match.
A total of 11 strains could be classified as SCCmec type IVa cassette based on the presence
of the mecA gene, and locus ccrB2 dcs. The results obtained using the method proposed by
Zhang et al. (2005), confirms membership in this category SCCmec cassette and a strain
containing an element could be SCCmec type IVa.
Although strains exhibiting SCCmec cassette type IV are associated with community
infections, their prevalence is observed, which corresponds to reports of authors noted the
presence in 87% of S. aureus strains isolated over a period of two years, cassettes SCCmec type
IV (Valsesia et al., 2010). Szkely et al. (2012) reported also ntr.un study on the molecular
epidemiology of S. aureus in a clinical hospital in Romania, significant percentage of strains
containing SCCmec cassette type IV or III SCCmec cassette type V are reported only
sporadically. Typically, the carrier tape community strains SCCmec type IV and V are most
likely to - lactam antibiotics except that, unlike nosocomial strains carrying the SCCmec
elements of larger size (Gordon and Lowy, 2008).
Some groups indicates that most clones MRSA from MSSA strains that have acquired SCCmec
90

IV cassette, this being due to probably its small size, which facilitates the transfer of this genetic
element (Deurenberg et al., 2007).
A total of three strains tested, containing the mecA gene, according to the phenotypic
classification of the category of MRSA, but the methods used did not allow typing of SCCmec
cassettes. The use of pairs of specific primers for the elements of some other tape the fitting
could be set correctly. Thus described: a strain containing the mecA gene and match a strain
positive for the mecA gene and the CCRC, and a strain showing only the mecA gene.
Nine isolates studied showed no mecA gene, although other specific cassettes showed
SCCmec locus dcs (SCCmec cassettes characteristic I, II, IV, VI, Region J3), match, CCRC or
elements present in the J3 region of SCCmec III. The absence of the mecA gene from MRSA
strains with phenotype should be investigated, with the possibility of expressing the isolated gene
coding for a protein mecALGA251 alternative PBP2a, Romania although not reported, to date
such strains. Spa typing could also provide the necessary data on the origin of these strains.
The absence of a phenotype associated MRSA mecA gene could signify strength
independent of the expression of this gene, its possible causes can be represented by:
hypersecretion of - lactamase synthesis of meticilinaze acquisition of genes responsible for the
synthesis of different PBPs PBP2a structural as well and the presence of type tulipinilor SCV
(Small Colony Variants), whose structural and functional defects (defects of transport systems,
low metabolic rate, reduced synthesis of the cell wall) could facilitate - lactam antibiotic
tolerance (Brakstad and Mland, 1997; Plata et al., 2009).
However, keeping long stems WHO culture media type and their possible subcultivation
could lead to loss of the mecA gene, similar cases where cryopreservation strains being reported
Griethysen et al. (2005). 4 strains showed no SCCmec gene complex.
91

3.4. Conclusions

In the context of the emergence of a significant number of multi-resistant staphylococcal strains

to antibiotics, the MRSA strains dominating [(In Romania, in 2010, between 25 and 50% of S.
aureus strains isolated from blood cultures and CSF showed MRSA phenotype (ECDC
Surveillance report, 2012)], understanding mecansimelor responsible for resistance is essential.
emergence of MRSA strains associated with community infection makes it even more important
to understand the genetic basis of phenotypic MRSA and horizontal transmission mechanisms,
intra-and inter-species genetic elements responsible for these characteristics.
Staphylococcal chromosomal SCCmec cassettes are carriers of genetic elements determining the
Resistance so - lactam antibiotics and to other classes of antimicrobial substances. Genetic
polymorphisms associated with these structures (Ito, T., Y. Katayama, and K. Hiramatsu. 1999),
their variability and their acquisition mecansimele are particularly important to understand the
structure and functionality, their correct classification in order to establish accurate data on the
molecular epidemiology of MRSA strains.
Bacterial strains analyzed is characterized by structural heterogeneity of SCCmec cassettes, most
can, however, be included in boxes SCCmec type III and IV, the latter category, although
characteristic of staphylococcal strains of Community origin are becoming more frequently
reported in the context clinical. The strains considered non-tipabile by the methods used in the
present study, further investigation is required to establish proper ownership SCCmec cassettes
92

contained. Analysed strains is characterized by structural heterogeneity of SCCmec cassettes,

most can,however, be included in boxes SCCmec type III and IV, the latter category, although
characteristic of staphylococcal strains of Community origin are becoming more
frequentlyreported in the context clinical

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