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International Educational Applied Scientific Research Journal

ISSN (Online): 2456-5040

Volume: 1 | Issue: 3 | December 2016


Obajuluwa A.F1, Onaolapo J.A2, Olayinka B.O.2, Adeshina G.O.2
Department of Pharmaceutics and Pharmaceutical Microbiology, Kaduna State University, Kaduna, Nigeria.
Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria, Nigeria.

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

Key words: coagulase negative Staphylococci, antibiotics, susceptibility.

Introduction common forms of osteomyelitis attributable to

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

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

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

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

infections. Data from the National Nosocomial Infections testing approved standard M100-S23. Clinical and
Surveillance (NNIS) system collected between 1992 and Laboratory Standards Institute, Wayne, PA.
1997 revealed that CoNS accounted for 36 percent of all
3. Diekema D.J, Pfaller M.A, Schmitz F.J, et al. (2001).
bloodstream isolates in intensive care units, making these
Survey of infections due to Staphylococcus species:
organisms the most common cause of nosocomial frequency of occurrence and antimicrobial susceptibility
bloodstream infections (Richards et al, 1999). A survey from of isolates collected in the United States, Canada, Latin
the Surveillance and Control of Pathogens of Epidemiologic America, Europe, and the Western Pacific region for the
Importance (SCOPE) database of nosocomial bloodstream SENTRY Antimicrobial Surveillance Program,
infections in United States hospitals between 1995 and 2002 1997-1999. Clin Infect Dis; 32 Suppl 2:S114.
also identified CoNS as the most common cause of
hospital-acquired bloodstream infections, accounting for 31 4. Dubois,D.,Leyssene,D.,Chacornac,J.P.,Kostrzewa,M.,Sc
percent of cases (Wisplinghoff, 2004). hmit,P.O.,Talon, R., Bonnet, R. & Delmas, J. (2010).
Identification of a variety of Staphylococcus species by
Among all the antibiotics used in this study gentamicin, an
matrix-assisted laser desorption ionization- time of flight
amyloglycoside and ciprofloxacin, a fluororquinolone were mass spectrometry. J Clin Microbiol 48 , 941945.
the most active followed by vancomycin a glycopeptide.
The aminoglycosides are bactericidal inhibitors of protein 5. Favre B, Hugonnet S, correa S, Sax H, Rohner P, Pittet D.
synthesis they have been widely used to treat staphylococcal (2005). Nosocomial bacteremia: clinical significance of a
infections, often in combination with other single blood culture positive for coagulase-negative
antistaphylococcal agents. High level of resistance was staphylococci. Infect Control Hosp
observed with ampicillin (96.6%) and other beta lactam epidemiol;26:697-702.
antibiotics, the production of beta lactamase enzymes by
6. Hellbacher C, Tornqvist E, Soderquist B. (2006).
most CoNS isolated in this study might be responsible for
Staphylococcus lugdunensis: clinical spectrum, antibiotic
this, these enzymes act by inactivating the beta lactam rings susceptibility, and phenotypic and genotypic patterns of
of beta lactam antibiotics. Olsen et al, 2006 reported that 39 isolates. Clin Microbiol Infect.;12(1):43-9.
Staphylococcal penicillinases confer resistance to penicillins,
ampicillin, amoxicillin, azlocillin, mezlocillin, 7. Innes A, Burden R.P, Finch R.G, Morgan A.G. (1994).
carbenicilli, piperacillin, and ticarcillin. Resistance to Treatment of resistant peritonitis in continuous
penicillin among the coagulase-negative staphylococci ambulatory peritoneal dialysis with intraperitoneal
(CoNS) approaches 90 to 95 percent and resistance to urokinase: a double-blind clinical trial. Nephrol Dial
semisynthetic penicillins has been observed in more than 80 Transplant;9(7):797-9.
percent of CoNS isolates (Diekema et al, 2001); these isolates
8. John J.F, Harvin A.(2007). History and evolution of
are often resistant to multiple classes of antibiotics in addition antibiotic resistance in coagulase negative staphylococci:
to beta-lactams. susceptibility profiles of new anti-staphylococcal agents.
Therapeutics Clin Risk Management. 2007
The multiple antibiotic resistance index observed in this
study is high, this is an indication that the samples were 9. Kanafani Z.A. (2006). Telavancin: a new
collected in an area where antibiotics are being misused this lipoglycopeptide with multiple mechanisms of action.
is of great concern including the level of multi-drug Expert Rev Anti Infect Ther.t;4(5):743-9. Review.
resistance observed in this study. These might lead to
treatment failure and extension of days of staying in the PETERS (2014) COAGULASE-NEGATIVE STAPHYLOCOCCI.
hospital, also in cases of fracture there may be delayed or CLIN. MICROBIOL. REV. ,27(4); 870-926
non-union of fractured bone. S. haemolyticus was reported to
be multi-drug resistant and that it may also be resistant to 11. Levinson, W. (2010). Review of Medical Microbiology
teicoplanin and vancomycin (Patterson, 2000; and Immunology (11th ed.). pp. 9499.
Miranda-Novales et al, 2006).
12. Miranda-Novales G, Leanos-Miranda B.E, Vilchis-Perez
Conclusion M, Solorzano-Santos F.(2006). In vitro activity effects of
Coagulase negative Staphylococci can be said to be generally combinations of cephalothin, dicloxacillin, imipenem,
vancomycin and amikacin against methicillin-resistant
resistant to beta lactam antibiotics while gentamicin and
Staphylococcus spp. strains. Ann Clin Microbiol
ciprofloxacin can be considered as drugs of choice in the Antimicrob. Oct 12;5:25.
treatment of Staphylococci infections
13. Olsen J.E, Christensen H, Aarestrup F.M. (2006).
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International Educational Applied Scientific Research Journal
ISSN (Online): 2456-5040
Volume: 1 | Issue: 3 | December 2016

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