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. common forms of osteomyelitis attributable to coagulase-negative staphylococci infection are sternal osteomyelitis, following cardiothoracic surgery, and infection of bone surrounding a prosthetic joint (Chandrasekar and Brown, 1994).
Introduction 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).
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).
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
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). 4
International Educational Applied Scientific Research Journal ISSN (Online): 2456-5040 Volume: 1 | Issue: 3 | December 2016 In this study, Cefoxitin 30μg, Ceftriaxone 30μg, Vancomycin 30μg, Ampicillin 10μg, Gentamicin 10μg, Pefloxacin 5μg, Ciprofloxacin 5μg, Amoxicillin-clavulanic acid 30μg, Erythromycin 15μg and Clindamycin 2μg (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 β-lactamase production (Nitrocefin test) Enzyme extracts of the S. aureus isolates were prepared as described by Caddick [4] with modification. Microplate Nitrocefin assay was carried out as follows: 1mg lyophilized Nitrocefin powder (Oxoid, UK) was reconstituted in 1.9ml of 0.1M phosphate buffer, pH7 supplied by the manufacturer. The reconstituted nitrocefin was further diluted 1 in 10 with phosphate buffer solution to give 50μg/ml solution. The disrupted cell preparations were used immediately by dispensing 50μL of preparation into separate wells of a 96 well plate. 50μL of diluted nitrocefin solution was added into each of the wells and incubated at 37ᵒC for 10 minutes. In the presence of β-lactamase, the chromogenic nitrocefin substrate changes colour from yellow to pink/red.
Antibiotics Vancomycin Ampicillin Ceftriaxone Ciprofloxacin Gentamicin Pefloxacin
Results Isolation and classification Staphylococci strains From a total of 114 samples obtained from the hospital, 40(35.1%) Staphylococi isolates were confirmed to be coagulase positive being Staphylococcus aureus while 74 (64.1%) were coagulase negative. The result of the API STAPH identification kit used to characterized the coagulase negative Staphylococci is presented in Table 1, ten isolates were undetermined. Table 1: Characterization Staphylococci S/N
of
Staphylococci species 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Staph xylosus Staph lentus Staph hominis Staph cohnii cohnii Staph epidermidis Staph cohnii ureal. Staph hyicus Staph lugdunensis Staph caprae Staph capitis Staph haemolyticus Staph scuiri Staph chromogenes Staph warneri Micrococcus spp Undetermined
coagulase
Erythromycin Clindamycin Amoxicillin-clavulanat e
No of CoNS isolates (%) n= 58 Resistan Intermediat Sensitiv t e e 4(6.9) 54 (93.1) 56 2 (3.4) (96.6) 33 10 (17.2) 15 (56.9) (25.9) 1 (1.7) 1 (1.7) 56 (96.6) 1 (1.7) 1 (1.7) 56 (96.6) 6 (10.3) 1 (1.7) 51 (87.9) 21(36.2) 12 (20.7) 25 (43.1) 24 15 (25.9) 19 (41.4) (32.8) 38 20 (65.5) (34.5)
Antibiotic resistance pattern Total number of CoNS isolates with multiple antibiotics resistance (MAR) index greater than 0.2 is 50/58 (86.21) as shown in Table 3. Twenty one out of the 58 characterized CoNS (36.2%) are multidrug resistant being resistant to three or more classes of antibiotics.
negative
No of isolates (%) n=74 23(31.1) 8 (10.8) 8 (10.8) 4 (5.4) 3 (4.1) 2 (2.7) 2 (2.7) 2 (2.7) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 6 (8.1) 10 (13.5)
Table 3: Multiple antibiotics index of CoNS isolates MAR index 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
No of CoNS isolates 1 3 4 16 18 0 12 2 1 0 1
Beta lactamase test 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 various antibiotics was interpreted using the standard interpretative chart updated according to Clinical Laboratory
Discussion: In this study CoNS were isolated from the wound, skin and beddings of orthopaedic patients, this is an indication that CoNS are possible causative agents of nosociomial 5
International Educational Applied Scientific Research Journal ISSN (Online): 2456-5040 Volume: 1 | Issue: 3 | December 2016 testing approved standard M100-S23. Clinical and Laboratory Standards Institute, Wayne, PA.
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).
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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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