International Educational Applied Scientific Research Journal

ISSN (Online): 2456-5040

Volume: 1 | Issue: 3 | December 2016

ANTIBIOTICS SUSCEPTIBILITY PATTERN OF COAGULASE

NEGATIVE STAPHYLOCOCCI ISOLATES FROM
ORTHOPAEDIC PATIENTS
Obajuluwa A.F1, Onaolapo J.A2, Olayinka B.O.2, Adeshina G.O.2
1
Department of Pharmaceutics and Pharmaceutical Microbiology, Kaduna State University, Kaduna, Nigeria.
2
Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria, Nigeria.

ABSTRACT
A total number of 74 coagulase negative Staphylococci were isolated from orthopaedic patients in Ahmadu Bello University
Teaching Hospital, Zaria, Nigeria. They were further characterized into various Staphylococci species using API STAPH
identification kit: Staph xylosus (31.1%), Staph lentus (10.8%), Staph hominis (10.8%), Staph cohnii cohnii (5.4%), Staph
epidermidis (4.1%) others were Staph cohnii ureal., Staph hyicus, Staph lugdunensis (2.7% each) Staph caprae , Staph capitis,
Staph haemolyticus, Staph scuiri, Staph chromogenes and Staph warneri (1.4% each). Microcossus spp was 8.2% while 13.5%
isolates were undetermined. Kirby Baurer disk method was used for the antibiotics susceptibility test, the result showed gentamicin
and ciprofloxacin to be most active (96.6%), followed by vancomycin (93.1) and pefloxacin (87.9). The isolates were resistant to
ampicillin (96.6), amoxicillin clavulanic acid (65.5%), clindamycin 41.4%).

The aim of this study is to classify the coagulase negative Staphylococci isolates into species and to determine their antibiotic
susceptibility

Key words: coagulase negative Staphylococci, antibiotics, susceptibility.

Introduction common forms of osteomyelitis attributable to

Staphylococci are members of the family Micrococcaceae.
They are Gram-positive, catalase-positive and occur singly
and in irregular grapelike clusters, a description from which
is the origin of their name. Staphylococci are broadly divided
into a group that produce coagulase (S. aureus) and those that
do not (Levinson, 2010). Most of the coagulase-negative
staphylococci associated with clinical disease are common
inhabitants of the skin and mucous membranes. S.
epidermidis is the most prevalent species, accounting for
approximately 60-70% of all coagulase-negative
Staphylococci on the skin. Coagulase-negative staphylococci
are frequently associated with nosocomial infections, 41% of
the time when bacteremia is present, and of these many are
line infections (Favre et al, 2005).
Host factors that predispose to coagulase-negative
staphylococci infections include immunosuppression and the
presence of a medical device. However, there are some
species of coagulase-negative staphylococci that have been
associated with particular infections other than
immunosuppression or a medical device, implanted foreign
bodies such as vascular access catheters, prosthetic joints
and other orthopaedic hard ware (Karsten et al, 2014). For
example, S. saprophyticus accounts for up to 10% of
uncomplicated urinary tract infections in young women
(Innes et al, 1994; Peters and Clissold, 1992). S. schleiferi, S.
lugdunensis and S. haemolyticus are associated with native
valve endocarditis (John and Harvin, 2007; Kanafani and
Telavancin, 2006). S. lugdunensis when isolated from an
otherwise sterile bodily site is almost always a pathogen
(Hellbacher et al, 2006; Viganego et al, 2007). The two most
coagulase-negative staphylococci infection are sternal
osteomyelitis, following cardiothoracic surgery, and
infection of bone surrounding a prosthetic joint
(Chandrasekar and Brown, 1994).
One of the characteristics of CoNS is their resistance to
multiple antimicrobial agents commonly used for the
treatment of staphylococcal infections. Since the 1970s, it has
been suggested that CoNS are of great importance as true
pathogens, and they have become a common cause of a wide
variety of infections (Dubois et al., 2010; Otto, 2009).
Materials and Methods
Isolation and Classification of Staphylococci strains
After obtaining the patients consent, all staphylococci used
were isolated from the wounds, beds, and skin of orthopaedic
patients in the orthopaedic ward of Ahmadu Bello University
Teaching Hospital Zaria, Nigeria. Using sterile swab sticks
114 samples were aseptically collected and purified.
Biochemical tests including coagulase test was used to isolate
the coagulase negative Staphylococci strains from the
coagulase positive. The CoNS isolates were further
characterized into species using API STAPH identification
kit (bioMerieux, Inc.,Durham, NC).
Antibiotics susceptibility test
Antibiotics susceptibility test of the characterized CoNS
strains were determined by the Kirby Bauer disk diffusion
method on Mueller-Hinton agar (Oxoid, Basingstoke)
according to the Clinical and Laboratory Standards Institute
(CLSI) (Clinical and Laboratory Standards Institute., 2013).

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 International Educational Applied Scientific Research Journal
ISSN (Online): 2456-5040
Volume: 1 | Issue: 3 | December 2016

In this study, Cefoxitin 30g, Ceftriaxone 30g, Vancomycin
30g, Ampicillin 10g, Gentamicin 10g, Pefloxacin 5g,
Ciprofloxacin 5g, Amoxicillin-clavulanic acid 30g,
Erythromycin 15g and Clindamycin 2g (Oxoid Ltd.
Basingstoke, London) were used.
Standard Institute (CLSI) standard. The results are shown on
Table 2.
Table 2: Antibiotic susceptibility of characterized
coagulase negative Staphylococci

Test for -lactamase production (Nitrocefin test) No of CoNS isolates (%) n= 58

Enzyme extracts of the S. aureus isolates were prepared as Antibiotics Resistan Intermediat Sensitiv
described by Caddick [4] with modification. Microplate t e e
Nitrocefin assay was carried out as follows: 1mg lyophilized Vancomycin 4(6.9) - 54
Nitrocefin powder (Oxoid, UK) was reconstituted in 1.9ml of (93.1)
0.1M phosphate buffer, pH7 supplied by the manufacturer. Ampicillin 56 - 2 (3.4)
The reconstituted nitrocefin was further diluted 1 in 10 with (96.6)
phosphate buffer solution to give 50g/ml solution. The Ceftriaxone 33 10 (17.2) 15
disrupted cell preparations were used immediately by (56.9) (25.9)
dispensing 50L of preparation into separate wells of a 96 Ciprofloxacin 1 (1.7) 1 (1.7) 56
well plate. 50L of diluted nitrocefin solution was added into (96.6)
each of the wells and incubated at 37C for 10 minutes. In the Gentamicin 1 (1.7) 1 (1.7) 56
presence of -lactamase, the chromogenic nitrocefin (96.6)
substrate changes colour from yellow to pink/red. Pefloxacin 6 (10.3) 1 (1.7) 51
(87.9)
Results Erythromycin 21(36.2) 12 (20.7) 25
Isolation and classification Staphylococci strains (43.1)
From a total of 114 samples obtained from the hospital, Clindamycin 24 15 (25.9) 19
40(35.1%) Staphylococi isolates were confirmed to be (41.4) (32.8)
coagulase positive being Staphylococcus aureus while 74 Amoxicillin-clavulanat 38 - 20
(64.1%) were coagulase negative. The result of the API e (65.5) (34.5)
STAPH identification kit used to characterized the coagulase
negative Staphylococci is presented in Table 1, ten isolates Antibiotic resistance pattern
were undetermined. Total number of CoNS isolates with multiple antibiotics
resistance (MAR) index greater than 0.2 is 50/58 (86.21) as
Table 1: Characterization of coagulase negative shown in Table 3. Twenty one out of the 58 characterized
Staphylococci CoNS (36.2%) are multidrug resistant being resistant to three
or more classes of antibiotics.
S/N Staphylococci species No of isolates (%)
n=74 Table 3: Multiple antibiotics index of CoNS isolates
1. Staph xylosus 23(31.1)
2. Staph lentus 8 (10.8) MAR index No of CoNS isolates
3. Staph hominis 8 (10.8) 0 1
4. Staph cohnii cohnii 4 (5.4) 0.1 3
5. Staph epidermidis 3 (4.1) 0.2 4
6. Staph cohnii ureal. 2 (2.7) 0.3 16
7. Staph hyicus 2 (2.7) 0.4 18
8. Staph lugdunensis 2 (2.7) 0.5 0
9. Staph caprae 1 (1.4) 0.6 12
10. Staph capitis 1 (1.4) 0.7 2
11. Staph haemolyticus 1 (1.4) 0.8 1
12. Staph scuiri 1 (1.4) 0.9 0
13. Staph chromogenes 1 (1.4) 1.0 1
14. Staph warneri 1 (1.4)
15. Micrococcus spp 6 (8.1) Beta lactamase test
16. Undetermined 10 (13.5) The beta lactamase test showed 21/58 (36.2%) of the CoNS
isolates to be positive.
Antibiotics susceptibility
The diameter of the zones of inhibition of the isolates to Discussion:
various antibiotics was interpreted using the standard In this study CoNS were isolated from the wound, skin and
interpretative chart updated according to Clinical Laboratory beddings of orthopaedic patients, this is an indication that
CoNS are possible causative agents of nosociomial

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 International Educational Applied Scientific Research Journal
ISSN (Online): 2456-5040
Volume: 1 | Issue: 3 | December 2016
infections. Data from the National Nosocomial Infections
Surveillance (NNIS) system collected between 1992 and
1997 revealed that CoNS accounted for 36 percent of all
bloodstream isolates in intensive care units, making these
organisms the most common cause of nosocomial
bloodstream infections (Richards et al, 1999). A survey from
the Surveillance and Control of Pathogens of Epidemiologic
Importance (SCOPE) database of nosocomial bloodstream
infections in United States hospitals between 1995 and 2002
also identified CoNS as the most common cause of
hospital-acquired bloodstream infections, accounting for 31
percent of cases (Wisplinghoff, 2004).
Among all the antibiotics used in this study gentamicin, an
amyloglycoside and ciprofloxacin, a fluororquinolone were
the most active followed by vancomycin a glycopeptide.
The aminoglycosides are bactericidal inhibitors of protein
synthesis they have been widely used to treat staphylococcal
infections, often in combination with other
antistaphylococcal agents. High level of resistance was
observed with ampicillin (96.6%) and other beta lactam
antibiotics, the production of beta lactamase enzymes by
most CoNS isolated in this study might be responsible for
this, these enzymes act by inactivating the beta lactam rings
of beta lactam antibiotics. Olsen et al, 2006 reported that
Staphylococcal penicillinases confer resistance to penicillins,
ampicillin, amoxicillin, azlocillin, mezlocillin,
carbenicilli, piperacillin, and ticarcillin. Resistance to
penicillin among the coagulase-negative staphylococci
(CoNS) approaches 90 to 95 percent and resistance to
semisynthetic penicillins has been observed in more than 80
percent of CoNS isolates (Diekema et al, 2001); these isolates
are often resistant to multiple classes of antibiotics in addition
to beta-lactams.
The multiple antibiotic resistance index observed in this
study is high, this is an indication that the samples were
collected in an area where antibiotics are being misused this
is of great concern including the level of multi-drug
resistance observed in this study. These might lead to
treatment failure and extension of days of staying in the
hospital, also in cases of fracture there may be delayed or
non-union of fractured bone. S. haemolyticus was reported to
be multi-drug resistant and that it may also be resistant to
teicoplanin and vancomycin (Patterson, 2000;
Miranda-Novales et al, 2006).
Conclusion
Coagulase negative Staphylococci can be said to be generally
resistant to beta lactam antibiotics while gentamicin and
ciprofloxacin can be considered as drugs of choice in the
treatment of Staphylococci infections
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