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Characterization of methicillin-susceptible and -resistant staphylococci in the clinical setting: a multicentre study in Nigeria

Characterization of methicillin-susceptible and -resistant staphylococci in the clinical setting:... Background: The staphylococci are implicated in a variety of human infections; however, many clinical microbiology laboratories in Nigeria do not identify staphylococci (in particular coagulase negative staphylococci - CNS) to the species level. Moreover, data from multi-centre assessment on antibiotic resistance and epidemiology of the staphylococci are not available in Nigeria. This study investigated 91 non-duplicate staphylococcal isolates obtained from the microbiology laboratories of eight hospitals in Nigeria during the period January to April 2010. Methods: Identification and antibiotic susceptibility testing was performed using the VITEK 2 system, detection of resistance genes by PCR, and molecular characterization was determined by SCCmec typing, spa and multilocus sequence typing (MLST). Results: All the isolates were susceptible to mupirocin, tigecycline, vancomycin and linezolid, but 72.5% of CNS and 82.3% of Staphylococcus aureus were resistant to cotrimoxazole, while multiresistance was observed in 37 of the 40 CNS isolates. Untypeable SCCmec types (ccrC/Class A mec and ccr-negative/Class C2 mec gene complex) in two methicillin-resistant S. aureus (MRSA) were identified. Additionally, ccr-negative/Class A mec and ccr type 4/ Class C2 mec gene complex was detected in one isolate each of S. sciuri and S. haemolyticus, respectively. The S. aureus isolates were classified into 21 spa types including two new types (t8987, t9008) among the methicillin-susceptible S. aureus (MSSA) isolates. Two (CC8-SCCmecnon-typeable and CC88-SCCmec IV) and four (CC8-SCCmec III/IV/V; CC30-SCCmec II/III; CC88-SCCmec IV; and ST152-SCCmecnon-typeable) MRSA clones were identified in Maiduguri (North-East Nigeria) and South-West Nigeria, respectively. The proportion of Panton-Valentine leukocidin (PVL)-positive MSSA was high (44.4%) and 56.3% of these strains were associated with sequence type (ST) 152. Conclusions: The identification of multiresistant mecA positive S. haemolyticus and S. sciuri from clinical samples indicates that characterization of CNS is important in providing information on their diversity and importance in Nigeria. There is the need to develop new SCCmec classification methods for non-typeable methicillin-resistant staphylococci, and to curtail the spread and establishment of the S. aureus ST152 clone in Nigeria. The study presents the first report of a PVL-positive ST152-SCCmecnontypeable MRSA and SCCmec typing of methicillin-resistant CNS in Nigeria. Keywords: Coagulase negative staphylococci, Staphylococcus aureus, Multiresistance, mecA gene, Staphylococcus haemolyticus, Staphylococcus sciuri, Panton Valentine Leukocidin, SCCmec typing, ST152, Nigeria * Correspondence: bayo_shittu@yahoo.com Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria Division of Medical Microbiology, University of Cape Town, Cape Town, Republic of South Africa Full list of author information is available at the end of the article © 2012 Shittu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 2 of 10 http://www.biomedcentral.com/1471-2334/12/286 Background Town, South Africa using the VITEK 2 system (bioMér- The staphylococci are part of the normal cutaneous ieux, France) with the Gram-Positive Identification Card. ecosystem, but they are also opportunistic and invasive pathogens that cause a variety of human infections. Antibiotic susceptibility testing Staphylococcus aureus is the most important human Antibiotic susceptibility for penicillin G, oxacillin, teico- pathogen in this genus accounting for a high proportion planin, vancomycin, gentamicin, tetracycline, ciprofloxacin, of severe infections in hospitals and outpatient med- moxifloxacin, trimethoprim/sulfamethoxazole (cotrimox- ical care [1-3]. Although the clinical relevance of coagu- azole), fusidic acid, erythromycin, clindamycin, rifampi- lase negative staphylococci (CNS) is still controversial, cin, mupirocin, linezolid and tigecycline was determined patients at risk of CNS infections include neonates, by the broth microdilution method (VITEK 2 system, those with intravascular catheters, prosthetic devices, AST 603 cards, bioMérieux). S. aureus ATCC 29213 was postoperative sternal wound infections and immuno- the control strain and the VITEK 2 minimum inhibitory compromised hosts [4-7]. The remarkable ability of concentration (MIC) results were interpreted using S. aureus and CNS to acquire antibiotic resistance limits the Advanced Expert System of the VITEK 2. Multiresis- therapeutic options, and morbidity and mortality rates tance was defined as resistance to at least three classes of staphylococcal infections have increased the financial of antibiotics. burden on health care systems worldwide [8-14]. Although there are reports on the characterization of DNA extraction staphylococci in Nigeria [15-18], many of the studies Before DNA extraction, 5–8 colonies from an 18–24 h were limited to antibiotic susceptibility profiles. More- old culture on boiled blood agar was pretreated with over, one geographical region or health care institution lysostaphin (Sigma) (20 μg/ml for S. aureus;50μg/ml was investigated, and CNS were not identified to species for CNS) in 20 mM Tris-Cl, 2mM EDTA at 37°C for level. Only a few studies have investigated the molecular 30 min. The cells were harvested and DNA was isolated epidemiology and diversity of this group of organisms using a DNeasy tissue kit (Qiagen, Hilden, Germany). [19-23], however, they were not multi-centre surveys on S. aureus and CNS. This work describes a multi-centre PCR detection of the tuf gene study which was conducted in order to provide clinicians Phenotypic identification of the S. aureus isolates and infection control practitioners with information on was confirmed by detection of the tuf gene as described antibiotic resistance profiles, molecular characteristics of previously [24]. The PCR products were detected by coagulase positive and negative staphylococci, and to gel electrophoresis using 2% w/v agarose (Seakem, alert clinicians to the emergence of new clones in health Whittaker, USA) and run in 1X TAE buffer (pH 8.3) for care institutions (HCIs) in Nigeria. 1 h at 100 V. Thereafter, the gel was stained with eth- idium bromide, visualized under UV light and photo- graphed using a SynGene Bioimaging System. Methods Isolation and identification of staphylococcal isolatess PCR detection of the dfrA gene The study commenced with the request for non- The presence of the dfrA gene encoding trimethoprim duplicate staphylococcal isolates obtained from clinical resistance was investigated using the primer pairs: dfr- samples processed in the microbiology laboratories of AF: AAT AGA CGT AAC GTC GTA CT; dfr-AR: AAG eight health care institutions in Ile-Ife, Ogbomosho, Iwo, AAT GTA TGC GGT ATA GT and subsequent detec- Lagos (two centres) and Ibadan in South-West Nigeria; tion of a 289 bp product [25]. The PCR conditions were Jos in North-Central Nigeria, and Maiduguri in North- as follows: Initial denaturation at 95°C for 3min, fol- East Nigeria (Figure 1) during the period January to lowed by 30 cycles of amplification at 95°C for 30s, April, 2010. In this study, only staphylococcal isolates annealing at 55°C for 30s, extension at 72°C for 30s and were analyzed and human subjects, clinical samples or final extension at 72°C for 1min. human data were not investigated. All the hospitals, except the one located in Iwo, were tertiary health care SCCmec typing institutions, each of which were at least 500-bed facilities SCCmec typing was performed according to a previously providing medical care to about 500,000 to one million published protocol [26] using the control strains S. aur- people. Preliminary verification as staphylococci was eus COL (type I), N315 (type II), ANS46 (type III), based on culture characteristics on blood agar, catalase, MW2 (type IV), WIS (type V) and HDE288 (type VI). coagulase and DNase tests, while identification to spe- For non-typeable SCCmec elements, the investigation cies level was performed at the National Health Labora- of mec complexes and ccr allotypes as described by tory Service (NHLS), Groote Schuur Hospital, Cape Kondo et al. [27] was carried out on representative Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 3 of 10 http://www.biomedcentral.com/1471-2334/12/286 MAIDUGURI JOS OGBOMOSO IWO ILE-IFE IBADAN LAGOS Figure 1 Map indicating the location of the health care institutions in Nigeria. methicillin-resistant S. aureus (MRSA) and CNS isolates presented in Tables 1, 2 and 3. The clinical S. aureus iso- based on their spa-MLST profile and location of the lates were recovered from wounds, skin and soft tissue health care institutions, respectively. The profiles infections (SSI), osteomyelitis and burns (36.4%), geni- obtained were characterized and defined according to tourinary tract infection (GTI)/infertility (18.2%), septi- the current nomenclature used for MRSA [28]. caemia (15.9%), urinary tract infection (UTI) (6.8%), otitis media (6.8%), bronchitis (4.5%), and 11.4% were Detection of the PVL gene classified as other infections (Tables 1 and 2). Six species The presence of the PVL gene was determined by PCR accounted for the CNS isolates: S. haemolyticus (n=21), using primers luk-PV-1 and luk-PV-2 [29]. S. sciuri (n=9), S. saprophyticus (n=5), S. warneri (n=3), S. epidermidis (n=1) and S. hominis (n=1), which were Genotyping distributed across four HCIs located in Ogbomosho, Typing of S. aureus was based on sequencing of the Lagos (designated as Lagos2), Jos and Maiduguri hypervariable region of the protein A gene (spa). The (Table 3). Most of the S. haemolyticus isolates were from spa types were assigned using the Ridom StaphType the centres in Lagos2, Jos and Maiduguri, and 47.6% of (Ridom GmbH, Würzburg, Germany, version 1.5.21) the isolates were obtained from septicaemia, 14.3% from [30]. Multilocus sequence typing (MLST) was carried wound infections, 9.5% each from GTI, UTI, ocular- out according to the protocols described previously [31] related infections, and those categorised as other infec- on a representative isolate for the predominant and new tions (Table 3). spa types. PCR products were purified and sequenced at the Central Analytical Facility at the University of Stel- Antibiotic susceptibility of the staphylococcal isolates lenbosch, South Africa. Allelic profiles and sequence and detection of mecA and dfrA genes types (STs) were assigned using the MLST S. aureus All the staphylococcal isolates were susceptible to mupir- database (www.mlst.net). Isolates that were not typed by ocin, tigecycline, vancomycin and linezolid (Table 4). In MLST were assigned STs based on the Based Upon Re- addition to the antibiotics stated above, the methicillin- peat Pattern (BURP) via the Ridom StaphType software. susceptible S. aureus (MSSA) were susceptible to teico- planin, gentamicin, erythromycin, clindamycin, fusidic Results acid and rifampicin. About 94% of MSSA were suscep- Identification and source of the staphylococcal isolates tible to the fluoroquinolones and one isolate was suscep- Preliminary verification of isolates obtained from the tible to all the antibiotics tested. However, 80.6% of microbiology laboratories in eight HCIs in Nigeria iden- MSSA were resistant to cotrimoxazole and penicillin, but tified 91 staphylococcal isolates (51 S. aureus and all the cotrimoxazole-resistant MSSA were dfrA negative. 40 CNS) and the characteristics of each isolate are The predominant antibiotype was resistance to penicillin, Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 4 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 1 Characterization of MRSA from Nigeria by antibiotic resistance pattern, detection of genes and molecular typing Isolate Location Sample Or Antibiogram tuf mecA dfrA SCCmec PVL spa MLST Clonal No Clinical Diagnosis gene gene gene typing gene type (ST) Complex (CC) 50 Lagos1 Burns PEN, OXA, GEN, CIP, MOX, + + + III - t037 ST241 CC8 ERY, CLI*, TET, COT, RIF 2496 Maiduguri GTI PEN, OXA, GEN, CIP, ERY, + + - NT/UNT - t037** CLI, TET, COT 2589 Maiduguri GTI PEN, OXA, GEN, CIP, ERY, + + - NT - t037 CLI, TET, COT 60 Lagos1 ASOM PEN, OXA, TET, COT + + - NT/V - t064** ST8 CC8 69 Lagos1 Nasal swab/ PEN, OXA, GEN, TET, COT + + - NT - t064 Screening T39sB Iwo HIV/AIDS PEN, OXA, TET, COT + + - NT - t064 T6530 Ile-Ife Osteomyelitis PEN, OXA, GEN, CIP, TET, COT + + - IV - t064 TU32 Ibadan Wound Infection PEN, OXA, CIP, TET, COT + + - NT - t064 TU3 Ibadan Wound Infection PEN, OXA, CIP, TET, COT + + - NT/V - t064 T5056 Ile-Ife Bronchitis PEN, OXA, GEN, CIP, ERY, + + - III - t074 ST37 CC30 CLI*, TET, COT, FUS, RIF TD17 Ile-Ife Bronchitis PEN, OXA, ERY, CLI + + ND II - t007 ST39 TN45 Ile-Ife Wound Infection PEN, OXA, TET + + ND IV - t729 ST88 CC88 003B Maiduguri Septicaemia PEN, OXA, TET, COT + + + IV - t1603 008 Maiduguri Septicaemia PEN, OXA, COT + + + IV - t1603 T5843 Ile-Ife Osteomyelitis PEN, OXA, TET, COT + + - NT/UNT + t4690** ST152 singleton KEY: : Representative MRSA for SCCmec typing determined by Kondo et al. (2007). ¶Nasal samples of health care workers (HCWs) in one of the hospitals in Lagos (designated as Lagos1). Geographical region (Nigeria): South-West (Ile-Ife, Ibadan, Iwo and Lagos); North-East (Maiduguri); North Central (Jos). GTI: Genital Tract Infection; ASOM: Acute suppurative otitis media. Antibiotics - PEN: Penicillin; OXA: Oxacillin; GEN: Gentamicin; CIP: Ciprofloxacin; MOX: Moxifloxacin; ERY: Erythromycin; CLI: Clindamycin; TET: Tetracycline; FUS: Fusidic acid; RIF: Rifampicin; COT: Cotrimoxazole; *: Inducible resistance (clindamycin). UNT: Untypeable SCCmec types; NT: Non-typeable; ND: Not determined. Isolates with spa type t007, t074, t729 and t1603 were assigned STs based on the Based Upon Repeat Pattern (BURP) via the Ridom StaphType software. **: MLST on representative strains. tetracycline and cotrimoxazole, which was observed in recovered from clinical samples in four HCIs in Lagos2, 14 isolates (39%). Resistance to oxacillin was detected in Maiduguri, Jos and Ogbomosho (Table 3). In contrast, 15 S. aureus isolates and confirmed as MRSA by the de- resistance to fusidic acid was common in other species tection of the mecA gene. Of the 15 MRSA isolates, 13 of CNS, and they were susceptible to erythromycin, clin- were resistant to cotrimoxazole and tetracycline, while 6 damycin, teicoplanin and rifampicin (Table 4). The and 7 MRSA were resistant to gentamicin and ciproflox- S. sciuri isolates were resistant to oxacillin and obtained acin, respectively. Two of the 5 erythromycin-resistant primarily from various clinical samples in Ogbomosho MRSA expressed inducible resistance to clindamycin. (Table 3). On the whole, multiresistance was observed in Overall, 12 of the 15 MRSA were resistant to at least three 37 of the 40 CNS isolates, and the CNS isolates identi- classes of antibiotics (Table 1). The trimethoprim resist- fied as methicillin-resistant (VITEK) were mecA positive. ance gene, dfrA, was detected in only 3 MRSA isolates. All the S. haemolyticus isolates were resistant to peni- SCCmec typing cillin, oxacillin and cotrimoxazole, but only one isolate Fifteen MRSA were obtained from the HCIs in Ile-Ife exhibited resistance to fusidic acid. The proportion of (n=5); Maiduguri (n=4); Lagos1 (n=3); Ibadan (n=2) and S. haemolyticus isolates resistant to ciprofloxacin, genta- Iwo (n=1). Using the multiplex typing method [26], micin and tetracycline was 80.9%, 85.7% and 90.5%, SCCmec type IV was identified in two MRSA each from respectively (Table 4). Furthermore, 5 (50%) of the 10 Ile-Ife and Maiduguri, SCCmec type III in two MRSA, erythromycin-resistant S. haemolyticus isolates expressed one each from Ile-Ife and Lagos1, and SCCmec type II inducible resistance to clindamycin, and one isolate in one MRSA from Ile-Ife (Table 1). However, 8 of 15 showed intermediate resistance to teicoplanin (MIC: MRSA and 28 methicillin-resistant CNS (MRCNS) were 16μg/ml). Multiresistant S. haemolyticus isolates were non-typeable. Based on another PCR-based method [27], Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 5 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 2 Characterization of MSSA from Nigeria by antibiotic resistance pattern, detection of genes and molecular typing Isolate Location Sample Or Antibiotype tuf dfrA PVL spa MLST Clonal No Clinical Diagnosis gene gene gene type (ST) Complex (CC) 1229 Maiduguri GTI TET + ND - t311 ST5 CC5 652 Maiduguri Not available PEN, COT + - + t311 47 Lagos1 Leg Ulcer PEN, TET, CIP, MOX, COT + - - t064 CC8 54 Lagos1 SLE PEN, TET, COT, CIP + - - t064 49 Lagos1 Nasal swab/screening PEN, COT + - - t2331** ST8 10 Ogbomosho SSI PEN + ND + t084 CC15 62 Lagos1 UTI COT + - - t084 076 Maiduguri Not available PEN, TET, COT + - - t084 373 Maiduguri UTI COT, TET + - - t084** ST15 1253 Maiduguri SSI PEN, TET, COT + - - t084 2290 Maiduguri SSI PEN, TET, COT + - - t084 2696 Maiduguri Semen (Infertility) PEN, COT + - - t084 1038 Maiduguri SSI PEN, COT + - + t2216 2478 Maiduguri UTI PEN, TET, COT + - - t2216 6B Ogbomosho Septicaemia COT + - + t318 ST30 CC30 51 Lagos1 Nasal swab/screening PEN, COT + - - t318 1059 Maiduguri GTI COT + - - t318 2578 Maiduguri GTI PEN, TET, COT + - + t934 ST80 CC80 56 Lagos1 Otitis media PEN, COT + - - t159 ST121 CC121 67 Lagos1 Nasal swab/screening PEN, TET, COT + - + t2304 68 Lagos1 Nasal swab/screening PEN, TET, COT + - + t2304 006 Maiduguri Septicaemia PEN, TET, COT + - + t355** ST152 singleton 0017 Maiduguri Septicaemia PEN, TET, COT + - + t355 1097 Maiduguri SSI PEN, TET, COT + - + t355 2365 Maiduguri Semen (Infertility) PEN, COT + - + t355 259 Maiduguri Semen (Infertility) PEN, COT + - + t355 604 Maiduguri SSI PEN, TET, COT + - - t355 642 Maiduguri SSI PEN, TET + ND + t9008** 829 Maiduguri SSI PEN, TET + ND - t9008 J11 Jos Septicaemia PEN, TET, COT + - + t355 J13 Jos Septicaemia PEN, TET, COT + - + t355 61 Lagos1 SSI TET, COT + - + t8987** 64 Lagos1 Nasal swab/screening PEN + ND - t1510 ST508 CC45 55 Lagos1 CSOM PEN + ND - t8223 ND CC45 57 Lagos1 Haematoma Susceptible to all antibiotics + ND - t635 ND ND 70 Lagos1 Nasal swab/screening PEN, TET, COT + - - NT ND ND KEY: : Nasal samples of health care workers (HCWs) in one of the hospitals in Lagos (designated as Lagos1). GTI: Genital Tract Infection; SLE: systemic lupus erythematosus; SSI: Skin and soft tissue infection; UTI: Urinary Tract Infection; CSOM: Chronic suppurative otitis media. Antibiotics - PEN: Penicillin; TET: Tetracycline; CIP: Ciprofloxacin; MOX: Moxifloxacin; COT: Cotrimoxazole. ND: Not determined; NT: Non-typeable. Spa types (bold) are new types; Isolates with spa type t064, t159, t311, t318, t934, 1510, t2216 and t2304 were assigned STs based on the Based Upon Repeat Pattern (BURP) via the Ridom StaphType software. **: MLST on representative strains. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 6 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 3 Identification and Characterization of CNS by antibiotic resistance pattern, mecA gene detection and SCCmec typing Isolate Location Sample/Clinical VITEK 2 Antibiogram mecA SCCmec No Condition Identification gene Typing 2 Ogbomosho Wound Infection S. saprophyticus PEN, TET, COT, FUS - ND 4 Ogbomosho UTI S. saprophyticus PEN, TET, COT, FUS - ND 7 Ogbomosho Osteomyelitis S. saprophyticus PEN, TET, COT, FUS - ND 13B Ogbomosho UTI S. saprophyticus PEN, COT, FUS - ND 14 Ogbomosho Septicaemia S. sciuri PEN, OXA, GEN, CIP, FUS, MOX + NT/UNT 16 Ogbomosho SSI S. warneri PEN, TET, COT, FUS - ND 16B Ogbomosho Osteomyelitis S. warneri TET, COT, FUS - ND 18 Ogbomosho SSI S. saprophyticus PEN, TET, COT, FUS - ND 66 Lagos2 Wound Infection S. sciuri PEN, OXA, GEN, CIP, FUS + NT L2 Lagos2 Septicaemia S. haemolyticus PEN, OXA, TET, COT + NT/V L5 Lagos2 Eye swab S. haemolyticus PEN, OXA, CIP, MOX, TET, COT + NT L6 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, TET, RIF, COT + NT L7 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, MOX, ERY, TET, COT + NT L8 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, COT + NT L9 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, TET, COT + NT L10 Lagos2 Eye swab S. haemolyticus PEN, OXA, GEN, CIP, MOX, ERY, TET, COT + NT 1103 Maiduguri Wound Infection S. warneri PEN, OXA, TET + NT 1268 Maiduguri Wound Infection S. haemolyticus PEN, OXA, GEN, CIP, MOX, TET, COT + NT 024 Maiduguri Wound Infection S. haemolyticus PEN, OXA, GEN, TET, COT + NT/V 2491 Maiduguri UTI S. haemolyticus PEN, OXA, GEN, CIP, ERY, TET, COT, TEICOi + NT 2412 Maiduguri Wound Infection S. haemolyticus PEN, OXA, GEN, CIP, MOX, TET, COT + NT 2362 Maiduguri GTI S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, COT + NT/UNT 825 Maiduguri GTI S. haemolyticus PEN, OXA, GEN, CIP, TET, COT + NT 2502 Maiduguri UTI S. haemolyticus PEN, OXA, GEN, COT ND ND J2 Jos Septicaemia S. hominis PEN, OXA, TET ND ND J5 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, RIF, COT ND ND J6 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, RIF, COT ND ND J10 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, RIF, COT ND ND J14 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, TET, FUS, COT + NT/V J15 Jos Prostatis S. haemolyticus PEN, OXA, TET, COT + NT J16 Jos Otitis media S. haemolyticus PEN, OXA, GEN, CIP, COT + NT J17B Jos Osteomyelitis S. epidermidis PEN, OXA, COT + NT 151 Ogbomosho Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, MOX, TET, COT + NT 12 Ogbomosho Conjunctivitis S. sciuri PEN, OXA, GEN, CIP, FUS + NT 19B Ogbomosho SSI S. sciuri PEN, OXA, GEN, CIP, TET, FUS + NT 11 Ogbomosho Wound abscess S. sciuri PEN, OXA, GEN, CIP, FUS + NT 17 Ogbomosho Wound infection S. sciuri PEN, OXA, CIP, GEN, FUS + NT 11B Ogbomosho UTI S. sciuri PEN, OXA, GEN, CIP, MOX, FUS + NT 15 Ogbomosho UTI S. sciuri PEN, OXA, GEN, CIP, MOX, FUS + NT 6 Ogbomosho UTI S. sciuri PEN, OXA, GEN, CIP, FUS + NT KEY: : Representative MRCNS for SCCmec typing determined by Kondo et al. (2007); UTI: Urinary Tract Infection; SSI: Skin and soft tissue infection; GTI: Genital Tract Infection. Antibiotics - PEN: Penicillin; OXA: Oxacillin; GEN: Gentamicin; CIP: Ciprofloxacin; MOX: Moxifloxacin; ERY: Erythromycin; CLI: Clindamycin; TET: Tetracycline; FUS: Fusidic acid; RIF: Rifampicin; COT: Cotrimoxazole; TEICO: Teicoplanin. *: Inducible resistance (clindamycin); ND: Not determined; NT: Non-typeable; UNT: Untypeable SCCmec types. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 7 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 4 Antibiotic resistance profile of staphylococci from t2331, spa non-typeable) were identified among the Nigeria MSSA from nasal samples of health care workers Number (%) of isolates resistant among (HCWs) (Table 2). MSSA isolates with spa type t084 and t2331 were assigned ST15 and ST8 respectively, Antibiotics MSSA MRSA S. haemolyticus Others (CNS) while ST152 accounted for the new spa types (t8987, n=36 n=15 n=21 n=19 t9008) and t355. Nine and 11 of the 36 MSSA clustered Penicillin 29 (80.6) 15 (100) 21 (100) 18 (94.7) with ST15 and ST152 respectively (Table 2). Oxacillin 0 (0) 15 (100) 21 (100) 12 (63.2) Teicoplanin 0 (0) 0 (0) 1* (4.8) 0 (0) Vancomycin 0 (0) 0 (00 0 (0) 0 (0) Discussion Gentamicin 0 (0) 6 (40) 18 (85.7) 9 (47.4) The identification of bacterial isolates to species level is Tetracycline 21 (58.3) 13 (86.7) 19 (90.5) 9 (47.4) of great importance in the clinical microbiology labora- Ciprofloxacin 2 (5.6) 7 (46.7) 17 (80.9) 9 (47.4) tory to obtain information on the diversity and signifi- Moxifloxacin 1 (2.8) 1 (6.7) 6 (28.6) 3 (15.8) cance of each species in human infection. Moreover, Erythromycin 0 (0) 5 (33.3) 10 (47.6) 0 (0) species identification is a prerequisite for epidemio- logical studies. Many clinical microbiology laboratories Clindamycin 0 (0) 5 (33.3) 5 (23.8) 0 (0) in Nigeria do not identify staphylococci (in particular Fusidic acid 0 (0) 1 (6.7) 1 (4.8) 16 (84.2) CNS) to species level, which is important in understand- Tigecycline 0 (0) 0 (0) 0 (0) 0 (0) ing their variety and clinical relevance. Two species, Mupirocin 0 (0) 0 (0) 0 (0) 0 (0) S. haemolyticus and S. sciuri, accounted for the majority Linezolid 0 (0) 0 (0) 0 (0) 0 (0) of the 40 CNS isolates investigated in this study (Table 3). Rifampicin 0 (0) 2 (13.3) 4 (19) 0 (0) Most of the S. haemolyticus isolates were obtained from Cotrimoxazole 29 (80.6) 13 (86.7) 21 (100) 8 (42.1) blood culture samples in HCIs in Lagos2 and Jos, and *Intermediate resistance. S. sciuri from samples in Ogbomosho (South-West Nigeria). S. haemolyticus is regarded as the second most two of the four representative MRSA and three S. hae- frequently isolated staphylococci from blood cultures molyticus possessed the SCCmec V element (ccrC/Class [32] and one of the most clinically relevant CNS, par- C2 mec). However, untypeable SCCmec elements were ticularly in immunocompromised patients [33]. S. sciuri, detected in two MRSA (ccrC/Class A mec and ccr- principally found in animal species, and although not negative/Class C2 mec) and one isolate each of S. sciuri considered important from the clinical standpoint, has and S. haemolyticus (ccr-negative/Class A mec and ccr been associated with infections such as endocarditis type 4/Class C2 mec gene complex) (Tables 1 and 3). [34], urinary tract infection [35] and wound infections [19,36]. Multiresistance to antibiotics was detected in Detection of PVL gene 37 of the 40 CNS isolates, and all the S. haemolyticus One MRSA from Ile-Ife was PVL positive (Table 1). The isolates were resistant to at least three classes of antibio- gene was also detected in 16 MSSA, of which 10 were tics. Apart from the possible clinical importance because isolated from patients with SSI and septicaemia of its serious impact on the course of infection, multire- (Table 2). sistant CNS are a potential source of genes encoding resistance to antibiotics for other staphylococci patho- Molecular typing of MSSA and MRSA genic for man. The isolation of multiresistant mecA posi- A total of 21 spa types were identified and representative tive S. haemolyticus from blood samples (septicaemia) S. aureus strains assigned as t037, t064, t084, t355, and S. sciuri from various clinical materials (Table 3) in t2331, t4690, and new types t8987 and t9008 were some HCIs clearly indicate that they could be of clinical selected for MLST (Tables 1 and 2). In MRSA, seven dif- importance in Nigeria. A number of reports have indi- ferent spa types were identified and isolates with spa cated an increase in the resistance of staphylococci types t037, t064, and t4690 were assigned ST241, ST8 to cotrimoxazole in Nigeria [15,18,21,23,37]. In this and ST152, respectively. Overall, the clonal complexes study, 72.5% of CNS and 82.3% of S. aureus isolates for MRSA were distributed across CC8, CC30, CC88 were resistant to cotrimoxazole. The antibiotic has wide and ST152 (Table 2). In MSSA, fifteen different spa clinical application, inexpensive, orally administered and types, including two new types (t8987 and t9008) were available over-the-counter where they are sold with identified of which only t064 was identified also in or without prescription in Nigeria. This could possibly MRSA (Tables 1 and 2). The predominant spa types explain the high level of staphylococcal resistance were t355 (8 isolates), t084 (7 isolates) and t318 (3 iso- observed in this study. Most of the cotrimoxazole- lates) and a diversity of spa types (t318, t1510, t2304, resistant S. aureus were dfrA-negative; hence more Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 8 of 10 http://www.biomedcentral.com/1471-2334/12/286 studies are needed to understand the molecular mechan- The MSSA were assigned mainly to clonal complexes ism of resistance in Nigeria. CC5, CC8, CC15, CC30, CC45, CC80 and ST152 Methicillin resistance in staphylococci is due to the (Table 2). A high proportion of MSSA from Maiduguri expression of the mecA gene, which mediates penicillin were grouped in ST15 or ST152 indicating that these binding protein 2a (PBP2a), a transpeptidase with low clones were successful in North East Nigeria. PVL- affinity for beta-lactams [7,38]. The mecA gene is carried encoding genes were detected in 16 MSSA (44.4%) iso- by a mobile genetic element (MGE) termed the staphylo- lates belonging to almost all clonal complexes identified coccal cassette chromosome (SCCmec), and though the in this study. Moreover, 56% of these strains were asso- mec origin remains unknown, it has been suggested that ciated with ST152 and 10 of 16 (62.5%) PVL positive mecA CNS may act as potential SCCmec donor account- MSSA isolates were associated with SSI and septicaemia. ing for the rise of new MRSA clones [39]. Eleven These observations confirm previous studies that PVL- SCCmec elements have been described to date: SCCmec positive MSSA ST152 are widespread in African coun- I-IV [40-42], and V-XI [43-48], but few reports exist tries [50-52] including Nigeria [22,23]. Furthermore, on the detection of mecA gene and characterization of MSSA isolates (ST121) from nasal samples of HCWs SCCmec types in Nigeria [21-23]. There was excellent were PVL positive. Our data suggests that the sequence correlation between results of antibiotic susceptibility types for pandemic PVL-positive MSSA identified in this testing (VITEK) and detection of the mecA gene for the study did not function as a reservoir for PVL-positive confirmation of methicillin-resistant staphylococci. MRSA supporting the argument of Schaumburg et al. However, the MRCNS were non-typeable using the [52] that the intimate inter-relation between PVL-positive multiplex PCR protocol [26]. Based on the method of MSSA and MRSA, which could lead to the emergence Kondo et al. [27], the SCCmec V element (ccrC/Class and spread of PVL-positive MRSA clones has not been C2 mec) was identified in representative S. haemolyticus established. However, the detection of an ST152-PVL- isolates, as well as untypeable SCCmec elements (ccr- positive MRSA in this study suggests that surveillance for negative/Class A mec and ccr type 4/Class C2 mec gene this clone is strongly advocated in Nigeria. complex) in one representative isolate each of S. sciuri and S. haemolyticus (Table 3). Recent studies have indi- Conclusions cated that SCCmec V is the predominant element among This is the first multi-centre study on the characterization methicillin-resistant S. haemolyticus isolates [33,49]. To of coagulase positive and negative staphylococci using the best of our knowledge, this is the first report on the phenotypic and molecular methods in Nigeria. However, characterization of SCCmec elements in CNS in Nigeria. there were a number of limitations in this study. The It is likely that the mec gene complex and ccr genes number of isolates from the HCIs was low and variable in CNS are diverse and distinct, and as indicated in pre- which did not allow for comparison of data among the vious reports [7,28], there is the need to develop new participating centres. Moreover, the diversity of the classification schemes for non-typeable SCCmec types staphylococci analysed in this study does not represent in MRCNS. their distribution patterns in health care institutions in Molecular typing of the MRSA isolates indicated that Nigeria. Lastly, we could not conduct full characterization two clones (CC8-SCCmecnon-typeable and CC88- of the mecA-positive CNS isolates, and only representa- SCCmec IV) existed in Maiduguri, North-East Nigeria, tive isolates were selected for SCCmec typing and MLST. and four clones in South-West Nigeria (CC8-SCCmecIII/ However, this study has highlighted the importance of IV/V; CC30-SCCmecII/III; CC88-SCCmecIV and ST152- species identification for CNS, their clinical relevance, SCCmecnon-typeable) (Table 1). Previous studies on the and the need for longitudinal multi-centre surveys to epidemiology of MRSA in Nigeria identified MRSA provide data on antibiotic resistance and epidemiology of clones t064-ST8-CC8 and t037-ST241-CC8 in South- the staphylococci in Nigeria. Characterization of multire- West and North-East Nigeria, respectively [22,23]. A sistant mecA positive S. haemolyticus and S. sciuri from community-associated clone, CC88-MRSA-IV, previously clinical samples is important in understanding their epi- reported in Ibadan, Nigeria [21] was identified from demiology within hospitals in Nigeria. The identification wound and blood samples in Maiduguri and Ile-Ife, how- of untypeable SCCmec types in the study will be further ever, we were unable to determine whether the isolates evaluated and characterized; however, there is the need were community-associated. Untypeable SCCmec types to develop new SCCmec classification schemes for non- (ccrC/Class A mec; ccr-negative/Class C2 mec) were typeable methicillin-resistant staphylococci. There is an detected in two representative MRSA isolates, of which urgent need to curtail the spread and establishment one was a PVL-positive isolate (t4690-ST152) from a of PVL-positive MSSA, in particular, the MSSA ST152 patient with osteomyelitis in Ile-Ife (South-West Nigeria). clone, in Nigeria. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 9 of 10 http://www.biomedcentral.com/1471-2334/12/286 Abbreviations 6. Deepa S, Kumari A, Venkatesha D: Increasing trends of methicillin SCCmec: Staphylococcal Cassette Chromosome mec; CC: Clonal Complex; resistant coagulase negative Staphylococcus in neonatal septicaemia – PVL: Panton Valentine Leukocidin; ST: Sequence type; CNS: Coagulase a study in a tertiary care hospital, Mysore, South India. OJHAS 2010, negative staphylococci; HCIs: Health Care Institutions; MIC: Minimum 9(4):1–3. Inhibitory Concentration; MLST: Multilocus sequence typing; ASOM: Acute 7. Zong Z, Peng C, Lu X: Diversity of SCCmec elements in methicillin- suppurative otitis media; CSOM: Chronic suppurative otitis media; resistant coagulase-negative staphylococci clinical isolates. PLoS One SLE: Systemic lupus erythematosus; GTI: Genitourinary tract infection; 2011, 6(5):e20191. UTI: Urinary tract infection; SSI: Skin and soft tissue infection; 8. Venkatesh MP, Placencia F, Weisman LE: Coagulase negative MSSA: Methicillin-susceptible Staphylococcus aureus; MRSA: Methicillin- staphylococcal infections in the neonate and child: an update. Semin resistant Staphylococcus aureus; MRCNS: Methicillin-resistant coagulase Pediatr Infect Dis 2006, 17(3):120–127. negative staphylococci; NT: Non-typeable; ND: Not determined; 9. Anderson-Berry A, Brinton B, Lyden E, Faix RG: Risk factors associated with UNT: Untypeable; Antibiotics: ; PEN: Penicillin; OXA: Oxacillin; development of persistent coagulase negative staphylococci bacteremia TEICO: Teicoplanin; TET: Tetracycline; GEN: Gentamicin; CIP: Ciprofloxacin; in the neonate and associated short-term and discharge morbidities. MOX: Moxifloxacin; ERY: Erythromycin; CLI: Clindamycin; FUS: Fusidic acid; Neonatology 2011, 99(1):23–31. RIF: Rifampicin; COT: Cotrimoxazole. 10. Bassetti M, Trecharichi EM, Mesini A, Spanu T, Giacobbe DR, Rossi M, Shenone E, Pascale GD, Molinari MP, Cauda R, Viscoli C, Tumbarello M: Risk factors and mortality of healthcare-associated and community-acquired Competing interests Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2011, doi:10.1111/ The authors declare that they have no competing interest. j.1469-0691.2011.03679. Epub ahead of print. 11. de Kraker ME, Davey PG, Grundmann H, on behalf of the BURDEN study Authors’ contributions group: Mortality and hospital stay associated with resistant AOS, KO, AR, ST, FO, KO and GE participated in the design of the study. OO Staphylococcus aureus and Escherichia coli bacteremia: estimating the and FA performed the preliminary identification of the isolates. AOS carried burden of antibiotic resistance in Europe. PLoS Med 2011, 8(10):e1001104. out the molecular characterization of the isolates. All authors read and 12. Jain A, Agarwal A, Verma RK, Awasthi S, Singh KP: Intravenous device approved the final manuscript. associated blood stream staphylococcal infection in paediatric patients. Indian J Med Res 2011, 134(2):193–199. Acknowledgments 13. Jean-Baptiste N, Benjamin DK Jr, Cohen-Wolkowiez M, Fowler VG Jr, We would like to thank the management of the hospitals for their support in Laughon M, Clark RH Smith PB: Coagulase negative staphylococcal the collection of the staphylococcal isolates. We also appreciate the infections in the neonatal intensive care unit. Infect Control Hosp assistance of Professor Mark Nicol, Dr. Eliya Madikane, Melissa Jansen van Epidemiol 2011, 32(7):679–686. Rensburg, Malefu Moleleki (Division of Medical Microbiology, University of 14. Marra AR, Camargo LF, Pignatari AC, Sukiennik T, Behar PR, Medeiros EA, Cape Town, South Africa), staff of the National Health Laboratory Service Ribeiro J, Girão E, Correa L, Guerra C, Brites C, Pereira CA, Carneiro I, Reis M, (NHLS, Groote Schuur Hospital, Cape Town, South Africa), Dr Franziska Layer de Souza MA, Tranchesi R, Barata CU, Edmond MB, Brazilian SCOPE Study (Staphylococcal Reference Centre, Robert Koch Institute, Wernigerode, Group: Nosocomial bloodstream infections in Brazilian hospitals: analysis Germany), Professor Teruyo Ito and Dr. Xiao Han of the Department of of 2,563 cases from a prospective nationwide surveillance study. J Clin Bacteriology, Juntendo University, Japan. We appreciate the comments and Microbiol 2011, 49(5):1866–1871. suggestions of Drs Edet Udo, Birgit Strommenger, Ulrich Nübel and Professor 15. Akinkunmi EO, Lamikanra A: Species distribution and antibiotic resistance Iruka Okeke in the preparation of the manuscript. 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Ito T, Katayama Y, Asada K, Mori N, Tsutsumimoto K, Tiensasitorn C, • No space constraints or color figure charges Hiramatsu K: Structural comparison of three types of staphylococcal • Immediate publication on acceptance cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother • Inclusion in PubMed, CAS, Scopus and Google Scholar 2001, 45(5):1323–1336. • Research which is freely available for redistribution 42. Ma XX, Ito T, Tiensasitorn C, Jamklang M, Chongtrakool P, Boyle-Vavra S, Daum RS, Hiramatsu K: Novel type of staphylococcal cassette Submit your manuscript at chromosome mec identified in community-acquired methicillin-resistant www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Infectious Diseases Springer Journals

Characterization of methicillin-susceptible and -resistant staphylococci in the clinical setting: a multicentre study in Nigeria

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Springer Journals
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Copyright © 2012 by Shittu et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Infectious Diseases; Parasitology; Medical Microbiology; Tropical Medicine; Internal Medicine
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1471-2334
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10.1186/1471-2334-12-286
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23121720
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Abstract

Background: The staphylococci are implicated in a variety of human infections; however, many clinical microbiology laboratories in Nigeria do not identify staphylococci (in particular coagulase negative staphylococci - CNS) to the species level. Moreover, data from multi-centre assessment on antibiotic resistance and epidemiology of the staphylococci are not available in Nigeria. This study investigated 91 non-duplicate staphylococcal isolates obtained from the microbiology laboratories of eight hospitals in Nigeria during the period January to April 2010. Methods: Identification and antibiotic susceptibility testing was performed using the VITEK 2 system, detection of resistance genes by PCR, and molecular characterization was determined by SCCmec typing, spa and multilocus sequence typing (MLST). Results: All the isolates were susceptible to mupirocin, tigecycline, vancomycin and linezolid, but 72.5% of CNS and 82.3% of Staphylococcus aureus were resistant to cotrimoxazole, while multiresistance was observed in 37 of the 40 CNS isolates. Untypeable SCCmec types (ccrC/Class A mec and ccr-negative/Class C2 mec gene complex) in two methicillin-resistant S. aureus (MRSA) were identified. Additionally, ccr-negative/Class A mec and ccr type 4/ Class C2 mec gene complex was detected in one isolate each of S. sciuri and S. haemolyticus, respectively. The S. aureus isolates were classified into 21 spa types including two new types (t8987, t9008) among the methicillin-susceptible S. aureus (MSSA) isolates. Two (CC8-SCCmecnon-typeable and CC88-SCCmec IV) and four (CC8-SCCmec III/IV/V; CC30-SCCmec II/III; CC88-SCCmec IV; and ST152-SCCmecnon-typeable) MRSA clones were identified in Maiduguri (North-East Nigeria) and South-West Nigeria, respectively. The proportion of Panton-Valentine leukocidin (PVL)-positive MSSA was high (44.4%) and 56.3% of these strains were associated with sequence type (ST) 152. Conclusions: The identification of multiresistant mecA positive S. haemolyticus and S. sciuri from clinical samples indicates that characterization of CNS is important in providing information on their diversity and importance in Nigeria. There is the need to develop new SCCmec classification methods for non-typeable methicillin-resistant staphylococci, and to curtail the spread and establishment of the S. aureus ST152 clone in Nigeria. The study presents the first report of a PVL-positive ST152-SCCmecnontypeable MRSA and SCCmec typing of methicillin-resistant CNS in Nigeria. Keywords: Coagulase negative staphylococci, Staphylococcus aureus, Multiresistance, mecA gene, Staphylococcus haemolyticus, Staphylococcus sciuri, Panton Valentine Leukocidin, SCCmec typing, ST152, Nigeria * Correspondence: bayo_shittu@yahoo.com Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria Division of Medical Microbiology, University of Cape Town, Cape Town, Republic of South Africa Full list of author information is available at the end of the article © 2012 Shittu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 2 of 10 http://www.biomedcentral.com/1471-2334/12/286 Background Town, South Africa using the VITEK 2 system (bioMér- The staphylococci are part of the normal cutaneous ieux, France) with the Gram-Positive Identification Card. ecosystem, but they are also opportunistic and invasive pathogens that cause a variety of human infections. Antibiotic susceptibility testing Staphylococcus aureus is the most important human Antibiotic susceptibility for penicillin G, oxacillin, teico- pathogen in this genus accounting for a high proportion planin, vancomycin, gentamicin, tetracycline, ciprofloxacin, of severe infections in hospitals and outpatient med- moxifloxacin, trimethoprim/sulfamethoxazole (cotrimox- ical care [1-3]. Although the clinical relevance of coagu- azole), fusidic acid, erythromycin, clindamycin, rifampi- lase negative staphylococci (CNS) is still controversial, cin, mupirocin, linezolid and tigecycline was determined patients at risk of CNS infections include neonates, by the broth microdilution method (VITEK 2 system, those with intravascular catheters, prosthetic devices, AST 603 cards, bioMérieux). S. aureus ATCC 29213 was postoperative sternal wound infections and immuno- the control strain and the VITEK 2 minimum inhibitory compromised hosts [4-7]. The remarkable ability of concentration (MIC) results were interpreted using S. aureus and CNS to acquire antibiotic resistance limits the Advanced Expert System of the VITEK 2. Multiresis- therapeutic options, and morbidity and mortality rates tance was defined as resistance to at least three classes of staphylococcal infections have increased the financial of antibiotics. burden on health care systems worldwide [8-14]. Although there are reports on the characterization of DNA extraction staphylococci in Nigeria [15-18], many of the studies Before DNA extraction, 5–8 colonies from an 18–24 h were limited to antibiotic susceptibility profiles. More- old culture on boiled blood agar was pretreated with over, one geographical region or health care institution lysostaphin (Sigma) (20 μg/ml for S. aureus;50μg/ml was investigated, and CNS were not identified to species for CNS) in 20 mM Tris-Cl, 2mM EDTA at 37°C for level. Only a few studies have investigated the molecular 30 min. The cells were harvested and DNA was isolated epidemiology and diversity of this group of organisms using a DNeasy tissue kit (Qiagen, Hilden, Germany). [19-23], however, they were not multi-centre surveys on S. aureus and CNS. This work describes a multi-centre PCR detection of the tuf gene study which was conducted in order to provide clinicians Phenotypic identification of the S. aureus isolates and infection control practitioners with information on was confirmed by detection of the tuf gene as described antibiotic resistance profiles, molecular characteristics of previously [24]. The PCR products were detected by coagulase positive and negative staphylococci, and to gel electrophoresis using 2% w/v agarose (Seakem, alert clinicians to the emergence of new clones in health Whittaker, USA) and run in 1X TAE buffer (pH 8.3) for care institutions (HCIs) in Nigeria. 1 h at 100 V. Thereafter, the gel was stained with eth- idium bromide, visualized under UV light and photo- graphed using a SynGene Bioimaging System. Methods Isolation and identification of staphylococcal isolatess PCR detection of the dfrA gene The study commenced with the request for non- The presence of the dfrA gene encoding trimethoprim duplicate staphylococcal isolates obtained from clinical resistance was investigated using the primer pairs: dfr- samples processed in the microbiology laboratories of AF: AAT AGA CGT AAC GTC GTA CT; dfr-AR: AAG eight health care institutions in Ile-Ife, Ogbomosho, Iwo, AAT GTA TGC GGT ATA GT and subsequent detec- Lagos (two centres) and Ibadan in South-West Nigeria; tion of a 289 bp product [25]. The PCR conditions were Jos in North-Central Nigeria, and Maiduguri in North- as follows: Initial denaturation at 95°C for 3min, fol- East Nigeria (Figure 1) during the period January to lowed by 30 cycles of amplification at 95°C for 30s, April, 2010. In this study, only staphylococcal isolates annealing at 55°C for 30s, extension at 72°C for 30s and were analyzed and human subjects, clinical samples or final extension at 72°C for 1min. human data were not investigated. All the hospitals, except the one located in Iwo, were tertiary health care SCCmec typing institutions, each of which were at least 500-bed facilities SCCmec typing was performed according to a previously providing medical care to about 500,000 to one million published protocol [26] using the control strains S. aur- people. Preliminary verification as staphylococci was eus COL (type I), N315 (type II), ANS46 (type III), based on culture characteristics on blood agar, catalase, MW2 (type IV), WIS (type V) and HDE288 (type VI). coagulase and DNase tests, while identification to spe- For non-typeable SCCmec elements, the investigation cies level was performed at the National Health Labora- of mec complexes and ccr allotypes as described by tory Service (NHLS), Groote Schuur Hospital, Cape Kondo et al. [27] was carried out on representative Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 3 of 10 http://www.biomedcentral.com/1471-2334/12/286 MAIDUGURI JOS OGBOMOSO IWO ILE-IFE IBADAN LAGOS Figure 1 Map indicating the location of the health care institutions in Nigeria. methicillin-resistant S. aureus (MRSA) and CNS isolates presented in Tables 1, 2 and 3. The clinical S. aureus iso- based on their spa-MLST profile and location of the lates were recovered from wounds, skin and soft tissue health care institutions, respectively. The profiles infections (SSI), osteomyelitis and burns (36.4%), geni- obtained were characterized and defined according to tourinary tract infection (GTI)/infertility (18.2%), septi- the current nomenclature used for MRSA [28]. caemia (15.9%), urinary tract infection (UTI) (6.8%), otitis media (6.8%), bronchitis (4.5%), and 11.4% were Detection of the PVL gene classified as other infections (Tables 1 and 2). Six species The presence of the PVL gene was determined by PCR accounted for the CNS isolates: S. haemolyticus (n=21), using primers luk-PV-1 and luk-PV-2 [29]. S. sciuri (n=9), S. saprophyticus (n=5), S. warneri (n=3), S. epidermidis (n=1) and S. hominis (n=1), which were Genotyping distributed across four HCIs located in Ogbomosho, Typing of S. aureus was based on sequencing of the Lagos (designated as Lagos2), Jos and Maiduguri hypervariable region of the protein A gene (spa). The (Table 3). Most of the S. haemolyticus isolates were from spa types were assigned using the Ridom StaphType the centres in Lagos2, Jos and Maiduguri, and 47.6% of (Ridom GmbH, Würzburg, Germany, version 1.5.21) the isolates were obtained from septicaemia, 14.3% from [30]. Multilocus sequence typing (MLST) was carried wound infections, 9.5% each from GTI, UTI, ocular- out according to the protocols described previously [31] related infections, and those categorised as other infec- on a representative isolate for the predominant and new tions (Table 3). spa types. PCR products were purified and sequenced at the Central Analytical Facility at the University of Stel- Antibiotic susceptibility of the staphylococcal isolates lenbosch, South Africa. Allelic profiles and sequence and detection of mecA and dfrA genes types (STs) were assigned using the MLST S. aureus All the staphylococcal isolates were susceptible to mupir- database (www.mlst.net). Isolates that were not typed by ocin, tigecycline, vancomycin and linezolid (Table 4). In MLST were assigned STs based on the Based Upon Re- addition to the antibiotics stated above, the methicillin- peat Pattern (BURP) via the Ridom StaphType software. susceptible S. aureus (MSSA) were susceptible to teico- planin, gentamicin, erythromycin, clindamycin, fusidic Results acid and rifampicin. About 94% of MSSA were suscep- Identification and source of the staphylococcal isolates tible to the fluoroquinolones and one isolate was suscep- Preliminary verification of isolates obtained from the tible to all the antibiotics tested. However, 80.6% of microbiology laboratories in eight HCIs in Nigeria iden- MSSA were resistant to cotrimoxazole and penicillin, but tified 91 staphylococcal isolates (51 S. aureus and all the cotrimoxazole-resistant MSSA were dfrA negative. 40 CNS) and the characteristics of each isolate are The predominant antibiotype was resistance to penicillin, Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 4 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 1 Characterization of MRSA from Nigeria by antibiotic resistance pattern, detection of genes and molecular typing Isolate Location Sample Or Antibiogram tuf mecA dfrA SCCmec PVL spa MLST Clonal No Clinical Diagnosis gene gene gene typing gene type (ST) Complex (CC) 50 Lagos1 Burns PEN, OXA, GEN, CIP, MOX, + + + III - t037 ST241 CC8 ERY, CLI*, TET, COT, RIF 2496 Maiduguri GTI PEN, OXA, GEN, CIP, ERY, + + - NT/UNT - t037** CLI, TET, COT 2589 Maiduguri GTI PEN, OXA, GEN, CIP, ERY, + + - NT - t037 CLI, TET, COT 60 Lagos1 ASOM PEN, OXA, TET, COT + + - NT/V - t064** ST8 CC8 69 Lagos1 Nasal swab/ PEN, OXA, GEN, TET, COT + + - NT - t064 Screening T39sB Iwo HIV/AIDS PEN, OXA, TET, COT + + - NT - t064 T6530 Ile-Ife Osteomyelitis PEN, OXA, GEN, CIP, TET, COT + + - IV - t064 TU32 Ibadan Wound Infection PEN, OXA, CIP, TET, COT + + - NT - t064 TU3 Ibadan Wound Infection PEN, OXA, CIP, TET, COT + + - NT/V - t064 T5056 Ile-Ife Bronchitis PEN, OXA, GEN, CIP, ERY, + + - III - t074 ST37 CC30 CLI*, TET, COT, FUS, RIF TD17 Ile-Ife Bronchitis PEN, OXA, ERY, CLI + + ND II - t007 ST39 TN45 Ile-Ife Wound Infection PEN, OXA, TET + + ND IV - t729 ST88 CC88 003B Maiduguri Septicaemia PEN, OXA, TET, COT + + + IV - t1603 008 Maiduguri Septicaemia PEN, OXA, COT + + + IV - t1603 T5843 Ile-Ife Osteomyelitis PEN, OXA, TET, COT + + - NT/UNT + t4690** ST152 singleton KEY: : Representative MRSA for SCCmec typing determined by Kondo et al. (2007). ¶Nasal samples of health care workers (HCWs) in one of the hospitals in Lagos (designated as Lagos1). Geographical region (Nigeria): South-West (Ile-Ife, Ibadan, Iwo and Lagos); North-East (Maiduguri); North Central (Jos). GTI: Genital Tract Infection; ASOM: Acute suppurative otitis media. Antibiotics - PEN: Penicillin; OXA: Oxacillin; GEN: Gentamicin; CIP: Ciprofloxacin; MOX: Moxifloxacin; ERY: Erythromycin; CLI: Clindamycin; TET: Tetracycline; FUS: Fusidic acid; RIF: Rifampicin; COT: Cotrimoxazole; *: Inducible resistance (clindamycin). UNT: Untypeable SCCmec types; NT: Non-typeable; ND: Not determined. Isolates with spa type t007, t074, t729 and t1603 were assigned STs based on the Based Upon Repeat Pattern (BURP) via the Ridom StaphType software. **: MLST on representative strains. tetracycline and cotrimoxazole, which was observed in recovered from clinical samples in four HCIs in Lagos2, 14 isolates (39%). Resistance to oxacillin was detected in Maiduguri, Jos and Ogbomosho (Table 3). In contrast, 15 S. aureus isolates and confirmed as MRSA by the de- resistance to fusidic acid was common in other species tection of the mecA gene. Of the 15 MRSA isolates, 13 of CNS, and they were susceptible to erythromycin, clin- were resistant to cotrimoxazole and tetracycline, while 6 damycin, teicoplanin and rifampicin (Table 4). The and 7 MRSA were resistant to gentamicin and ciproflox- S. sciuri isolates were resistant to oxacillin and obtained acin, respectively. Two of the 5 erythromycin-resistant primarily from various clinical samples in Ogbomosho MRSA expressed inducible resistance to clindamycin. (Table 3). On the whole, multiresistance was observed in Overall, 12 of the 15 MRSA were resistant to at least three 37 of the 40 CNS isolates, and the CNS isolates identi- classes of antibiotics (Table 1). The trimethoprim resist- fied as methicillin-resistant (VITEK) were mecA positive. ance gene, dfrA, was detected in only 3 MRSA isolates. All the S. haemolyticus isolates were resistant to peni- SCCmec typing cillin, oxacillin and cotrimoxazole, but only one isolate Fifteen MRSA were obtained from the HCIs in Ile-Ife exhibited resistance to fusidic acid. The proportion of (n=5); Maiduguri (n=4); Lagos1 (n=3); Ibadan (n=2) and S. haemolyticus isolates resistant to ciprofloxacin, genta- Iwo (n=1). Using the multiplex typing method [26], micin and tetracycline was 80.9%, 85.7% and 90.5%, SCCmec type IV was identified in two MRSA each from respectively (Table 4). Furthermore, 5 (50%) of the 10 Ile-Ife and Maiduguri, SCCmec type III in two MRSA, erythromycin-resistant S. haemolyticus isolates expressed one each from Ile-Ife and Lagos1, and SCCmec type II inducible resistance to clindamycin, and one isolate in one MRSA from Ile-Ife (Table 1). However, 8 of 15 showed intermediate resistance to teicoplanin (MIC: MRSA and 28 methicillin-resistant CNS (MRCNS) were 16μg/ml). Multiresistant S. haemolyticus isolates were non-typeable. Based on another PCR-based method [27], Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 5 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 2 Characterization of MSSA from Nigeria by antibiotic resistance pattern, detection of genes and molecular typing Isolate Location Sample Or Antibiotype tuf dfrA PVL spa MLST Clonal No Clinical Diagnosis gene gene gene type (ST) Complex (CC) 1229 Maiduguri GTI TET + ND - t311 ST5 CC5 652 Maiduguri Not available PEN, COT + - + t311 47 Lagos1 Leg Ulcer PEN, TET, CIP, MOX, COT + - - t064 CC8 54 Lagos1 SLE PEN, TET, COT, CIP + - - t064 49 Lagos1 Nasal swab/screening PEN, COT + - - t2331** ST8 10 Ogbomosho SSI PEN + ND + t084 CC15 62 Lagos1 UTI COT + - - t084 076 Maiduguri Not available PEN, TET, COT + - - t084 373 Maiduguri UTI COT, TET + - - t084** ST15 1253 Maiduguri SSI PEN, TET, COT + - - t084 2290 Maiduguri SSI PEN, TET, COT + - - t084 2696 Maiduguri Semen (Infertility) PEN, COT + - - t084 1038 Maiduguri SSI PEN, COT + - + t2216 2478 Maiduguri UTI PEN, TET, COT + - - t2216 6B Ogbomosho Septicaemia COT + - + t318 ST30 CC30 51 Lagos1 Nasal swab/screening PEN, COT + - - t318 1059 Maiduguri GTI COT + - - t318 2578 Maiduguri GTI PEN, TET, COT + - + t934 ST80 CC80 56 Lagos1 Otitis media PEN, COT + - - t159 ST121 CC121 67 Lagos1 Nasal swab/screening PEN, TET, COT + - + t2304 68 Lagos1 Nasal swab/screening PEN, TET, COT + - + t2304 006 Maiduguri Septicaemia PEN, TET, COT + - + t355** ST152 singleton 0017 Maiduguri Septicaemia PEN, TET, COT + - + t355 1097 Maiduguri SSI PEN, TET, COT + - + t355 2365 Maiduguri Semen (Infertility) PEN, COT + - + t355 259 Maiduguri Semen (Infertility) PEN, COT + - + t355 604 Maiduguri SSI PEN, TET, COT + - - t355 642 Maiduguri SSI PEN, TET + ND + t9008** 829 Maiduguri SSI PEN, TET + ND - t9008 J11 Jos Septicaemia PEN, TET, COT + - + t355 J13 Jos Septicaemia PEN, TET, COT + - + t355 61 Lagos1 SSI TET, COT + - + t8987** 64 Lagos1 Nasal swab/screening PEN + ND - t1510 ST508 CC45 55 Lagos1 CSOM PEN + ND - t8223 ND CC45 57 Lagos1 Haematoma Susceptible to all antibiotics + ND - t635 ND ND 70 Lagos1 Nasal swab/screening PEN, TET, COT + - - NT ND ND KEY: : Nasal samples of health care workers (HCWs) in one of the hospitals in Lagos (designated as Lagos1). GTI: Genital Tract Infection; SLE: systemic lupus erythematosus; SSI: Skin and soft tissue infection; UTI: Urinary Tract Infection; CSOM: Chronic suppurative otitis media. Antibiotics - PEN: Penicillin; TET: Tetracycline; CIP: Ciprofloxacin; MOX: Moxifloxacin; COT: Cotrimoxazole. ND: Not determined; NT: Non-typeable. Spa types (bold) are new types; Isolates with spa type t064, t159, t311, t318, t934, 1510, t2216 and t2304 were assigned STs based on the Based Upon Repeat Pattern (BURP) via the Ridom StaphType software. **: MLST on representative strains. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 6 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 3 Identification and Characterization of CNS by antibiotic resistance pattern, mecA gene detection and SCCmec typing Isolate Location Sample/Clinical VITEK 2 Antibiogram mecA SCCmec No Condition Identification gene Typing 2 Ogbomosho Wound Infection S. saprophyticus PEN, TET, COT, FUS - ND 4 Ogbomosho UTI S. saprophyticus PEN, TET, COT, FUS - ND 7 Ogbomosho Osteomyelitis S. saprophyticus PEN, TET, COT, FUS - ND 13B Ogbomosho UTI S. saprophyticus PEN, COT, FUS - ND 14 Ogbomosho Septicaemia S. sciuri PEN, OXA, GEN, CIP, FUS, MOX + NT/UNT 16 Ogbomosho SSI S. warneri PEN, TET, COT, FUS - ND 16B Ogbomosho Osteomyelitis S. warneri TET, COT, FUS - ND 18 Ogbomosho SSI S. saprophyticus PEN, TET, COT, FUS - ND 66 Lagos2 Wound Infection S. sciuri PEN, OXA, GEN, CIP, FUS + NT L2 Lagos2 Septicaemia S. haemolyticus PEN, OXA, TET, COT + NT/V L5 Lagos2 Eye swab S. haemolyticus PEN, OXA, CIP, MOX, TET, COT + NT L6 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, TET, RIF, COT + NT L7 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, MOX, ERY, TET, COT + NT L8 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, COT + NT L9 Lagos2 Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, TET, COT + NT L10 Lagos2 Eye swab S. haemolyticus PEN, OXA, GEN, CIP, MOX, ERY, TET, COT + NT 1103 Maiduguri Wound Infection S. warneri PEN, OXA, TET + NT 1268 Maiduguri Wound Infection S. haemolyticus PEN, OXA, GEN, CIP, MOX, TET, COT + NT 024 Maiduguri Wound Infection S. haemolyticus PEN, OXA, GEN, TET, COT + NT/V 2491 Maiduguri UTI S. haemolyticus PEN, OXA, GEN, CIP, ERY, TET, COT, TEICOi + NT 2412 Maiduguri Wound Infection S. haemolyticus PEN, OXA, GEN, CIP, MOX, TET, COT + NT 2362 Maiduguri GTI S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, COT + NT/UNT 825 Maiduguri GTI S. haemolyticus PEN, OXA, GEN, CIP, TET, COT + NT 2502 Maiduguri UTI S. haemolyticus PEN, OXA, GEN, COT ND ND J2 Jos Septicaemia S. hominis PEN, OXA, TET ND ND J5 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, RIF, COT ND ND J6 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, RIF, COT ND ND J10 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, CLI*, TET, RIF, COT ND ND J14 Jos Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, ERY, TET, FUS, COT + NT/V J15 Jos Prostatis S. haemolyticus PEN, OXA, TET, COT + NT J16 Jos Otitis media S. haemolyticus PEN, OXA, GEN, CIP, COT + NT J17B Jos Osteomyelitis S. epidermidis PEN, OXA, COT + NT 151 Ogbomosho Septicaemia S. haemolyticus PEN, OXA, GEN, CIP, MOX, TET, COT + NT 12 Ogbomosho Conjunctivitis S. sciuri PEN, OXA, GEN, CIP, FUS + NT 19B Ogbomosho SSI S. sciuri PEN, OXA, GEN, CIP, TET, FUS + NT 11 Ogbomosho Wound abscess S. sciuri PEN, OXA, GEN, CIP, FUS + NT 17 Ogbomosho Wound infection S. sciuri PEN, OXA, CIP, GEN, FUS + NT 11B Ogbomosho UTI S. sciuri PEN, OXA, GEN, CIP, MOX, FUS + NT 15 Ogbomosho UTI S. sciuri PEN, OXA, GEN, CIP, MOX, FUS + NT 6 Ogbomosho UTI S. sciuri PEN, OXA, GEN, CIP, FUS + NT KEY: : Representative MRCNS for SCCmec typing determined by Kondo et al. (2007); UTI: Urinary Tract Infection; SSI: Skin and soft tissue infection; GTI: Genital Tract Infection. Antibiotics - PEN: Penicillin; OXA: Oxacillin; GEN: Gentamicin; CIP: Ciprofloxacin; MOX: Moxifloxacin; ERY: Erythromycin; CLI: Clindamycin; TET: Tetracycline; FUS: Fusidic acid; RIF: Rifampicin; COT: Cotrimoxazole; TEICO: Teicoplanin. *: Inducible resistance (clindamycin); ND: Not determined; NT: Non-typeable; UNT: Untypeable SCCmec types. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 7 of 10 http://www.biomedcentral.com/1471-2334/12/286 Table 4 Antibiotic resistance profile of staphylococci from t2331, spa non-typeable) were identified among the Nigeria MSSA from nasal samples of health care workers Number (%) of isolates resistant among (HCWs) (Table 2). MSSA isolates with spa type t084 and t2331 were assigned ST15 and ST8 respectively, Antibiotics MSSA MRSA S. haemolyticus Others (CNS) while ST152 accounted for the new spa types (t8987, n=36 n=15 n=21 n=19 t9008) and t355. Nine and 11 of the 36 MSSA clustered Penicillin 29 (80.6) 15 (100) 21 (100) 18 (94.7) with ST15 and ST152 respectively (Table 2). Oxacillin 0 (0) 15 (100) 21 (100) 12 (63.2) Teicoplanin 0 (0) 0 (0) 1* (4.8) 0 (0) Vancomycin 0 (0) 0 (00 0 (0) 0 (0) Discussion Gentamicin 0 (0) 6 (40) 18 (85.7) 9 (47.4) The identification of bacterial isolates to species level is Tetracycline 21 (58.3) 13 (86.7) 19 (90.5) 9 (47.4) of great importance in the clinical microbiology labora- Ciprofloxacin 2 (5.6) 7 (46.7) 17 (80.9) 9 (47.4) tory to obtain information on the diversity and signifi- Moxifloxacin 1 (2.8) 1 (6.7) 6 (28.6) 3 (15.8) cance of each species in human infection. Moreover, Erythromycin 0 (0) 5 (33.3) 10 (47.6) 0 (0) species identification is a prerequisite for epidemio- logical studies. Many clinical microbiology laboratories Clindamycin 0 (0) 5 (33.3) 5 (23.8) 0 (0) in Nigeria do not identify staphylococci (in particular Fusidic acid 0 (0) 1 (6.7) 1 (4.8) 16 (84.2) CNS) to species level, which is important in understand- Tigecycline 0 (0) 0 (0) 0 (0) 0 (0) ing their variety and clinical relevance. Two species, Mupirocin 0 (0) 0 (0) 0 (0) 0 (0) S. haemolyticus and S. sciuri, accounted for the majority Linezolid 0 (0) 0 (0) 0 (0) 0 (0) of the 40 CNS isolates investigated in this study (Table 3). Rifampicin 0 (0) 2 (13.3) 4 (19) 0 (0) Most of the S. haemolyticus isolates were obtained from Cotrimoxazole 29 (80.6) 13 (86.7) 21 (100) 8 (42.1) blood culture samples in HCIs in Lagos2 and Jos, and *Intermediate resistance. S. sciuri from samples in Ogbomosho (South-West Nigeria). S. haemolyticus is regarded as the second most two of the four representative MRSA and three S. hae- frequently isolated staphylococci from blood cultures molyticus possessed the SCCmec V element (ccrC/Class [32] and one of the most clinically relevant CNS, par- C2 mec). However, untypeable SCCmec elements were ticularly in immunocompromised patients [33]. S. sciuri, detected in two MRSA (ccrC/Class A mec and ccr- principally found in animal species, and although not negative/Class C2 mec) and one isolate each of S. sciuri considered important from the clinical standpoint, has and S. haemolyticus (ccr-negative/Class A mec and ccr been associated with infections such as endocarditis type 4/Class C2 mec gene complex) (Tables 1 and 3). [34], urinary tract infection [35] and wound infections [19,36]. Multiresistance to antibiotics was detected in Detection of PVL gene 37 of the 40 CNS isolates, and all the S. haemolyticus One MRSA from Ile-Ife was PVL positive (Table 1). The isolates were resistant to at least three classes of antibio- gene was also detected in 16 MSSA, of which 10 were tics. Apart from the possible clinical importance because isolated from patients with SSI and septicaemia of its serious impact on the course of infection, multire- (Table 2). sistant CNS are a potential source of genes encoding resistance to antibiotics for other staphylococci patho- Molecular typing of MSSA and MRSA genic for man. The isolation of multiresistant mecA posi- A total of 21 spa types were identified and representative tive S. haemolyticus from blood samples (septicaemia) S. aureus strains assigned as t037, t064, t084, t355, and S. sciuri from various clinical materials (Table 3) in t2331, t4690, and new types t8987 and t9008 were some HCIs clearly indicate that they could be of clinical selected for MLST (Tables 1 and 2). In MRSA, seven dif- importance in Nigeria. A number of reports have indi- ferent spa types were identified and isolates with spa cated an increase in the resistance of staphylococci types t037, t064, and t4690 were assigned ST241, ST8 to cotrimoxazole in Nigeria [15,18,21,23,37]. In this and ST152, respectively. Overall, the clonal complexes study, 72.5% of CNS and 82.3% of S. aureus isolates for MRSA were distributed across CC8, CC30, CC88 were resistant to cotrimoxazole. The antibiotic has wide and ST152 (Table 2). In MSSA, fifteen different spa clinical application, inexpensive, orally administered and types, including two new types (t8987 and t9008) were available over-the-counter where they are sold with identified of which only t064 was identified also in or without prescription in Nigeria. This could possibly MRSA (Tables 1 and 2). The predominant spa types explain the high level of staphylococcal resistance were t355 (8 isolates), t084 (7 isolates) and t318 (3 iso- observed in this study. Most of the cotrimoxazole- lates) and a diversity of spa types (t318, t1510, t2304, resistant S. aureus were dfrA-negative; hence more Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 8 of 10 http://www.biomedcentral.com/1471-2334/12/286 studies are needed to understand the molecular mechan- The MSSA were assigned mainly to clonal complexes ism of resistance in Nigeria. CC5, CC8, CC15, CC30, CC45, CC80 and ST152 Methicillin resistance in staphylococci is due to the (Table 2). A high proportion of MSSA from Maiduguri expression of the mecA gene, which mediates penicillin were grouped in ST15 or ST152 indicating that these binding protein 2a (PBP2a), a transpeptidase with low clones were successful in North East Nigeria. PVL- affinity for beta-lactams [7,38]. The mecA gene is carried encoding genes were detected in 16 MSSA (44.4%) iso- by a mobile genetic element (MGE) termed the staphylo- lates belonging to almost all clonal complexes identified coccal cassette chromosome (SCCmec), and though the in this study. Moreover, 56% of these strains were asso- mec origin remains unknown, it has been suggested that ciated with ST152 and 10 of 16 (62.5%) PVL positive mecA CNS may act as potential SCCmec donor account- MSSA isolates were associated with SSI and septicaemia. ing for the rise of new MRSA clones [39]. Eleven These observations confirm previous studies that PVL- SCCmec elements have been described to date: SCCmec positive MSSA ST152 are widespread in African coun- I-IV [40-42], and V-XI [43-48], but few reports exist tries [50-52] including Nigeria [22,23]. Furthermore, on the detection of mecA gene and characterization of MSSA isolates (ST121) from nasal samples of HCWs SCCmec types in Nigeria [21-23]. There was excellent were PVL positive. Our data suggests that the sequence correlation between results of antibiotic susceptibility types for pandemic PVL-positive MSSA identified in this testing (VITEK) and detection of the mecA gene for the study did not function as a reservoir for PVL-positive confirmation of methicillin-resistant staphylococci. MRSA supporting the argument of Schaumburg et al. However, the MRCNS were non-typeable using the [52] that the intimate inter-relation between PVL-positive multiplex PCR protocol [26]. Based on the method of MSSA and MRSA, which could lead to the emergence Kondo et al. [27], the SCCmec V element (ccrC/Class and spread of PVL-positive MRSA clones has not been C2 mec) was identified in representative S. haemolyticus established. However, the detection of an ST152-PVL- isolates, as well as untypeable SCCmec elements (ccr- positive MRSA in this study suggests that surveillance for negative/Class A mec and ccr type 4/Class C2 mec gene this clone is strongly advocated in Nigeria. complex) in one representative isolate each of S. sciuri and S. haemolyticus (Table 3). Recent studies have indi- Conclusions cated that SCCmec V is the predominant element among This is the first multi-centre study on the characterization methicillin-resistant S. haemolyticus isolates [33,49]. To of coagulase positive and negative staphylococci using the best of our knowledge, this is the first report on the phenotypic and molecular methods in Nigeria. However, characterization of SCCmec elements in CNS in Nigeria. there were a number of limitations in this study. The It is likely that the mec gene complex and ccr genes number of isolates from the HCIs was low and variable in CNS are diverse and distinct, and as indicated in pre- which did not allow for comparison of data among the vious reports [7,28], there is the need to develop new participating centres. Moreover, the diversity of the classification schemes for non-typeable SCCmec types staphylococci analysed in this study does not represent in MRCNS. their distribution patterns in health care institutions in Molecular typing of the MRSA isolates indicated that Nigeria. Lastly, we could not conduct full characterization two clones (CC8-SCCmecnon-typeable and CC88- of the mecA-positive CNS isolates, and only representa- SCCmec IV) existed in Maiduguri, North-East Nigeria, tive isolates were selected for SCCmec typing and MLST. and four clones in South-West Nigeria (CC8-SCCmecIII/ However, this study has highlighted the importance of IV/V; CC30-SCCmecII/III; CC88-SCCmecIV and ST152- species identification for CNS, their clinical relevance, SCCmecnon-typeable) (Table 1). Previous studies on the and the need for longitudinal multi-centre surveys to epidemiology of MRSA in Nigeria identified MRSA provide data on antibiotic resistance and epidemiology of clones t064-ST8-CC8 and t037-ST241-CC8 in South- the staphylococci in Nigeria. Characterization of multire- West and North-East Nigeria, respectively [22,23]. A sistant mecA positive S. haemolyticus and S. sciuri from community-associated clone, CC88-MRSA-IV, previously clinical samples is important in understanding their epi- reported in Ibadan, Nigeria [21] was identified from demiology within hospitals in Nigeria. The identification wound and blood samples in Maiduguri and Ile-Ife, how- of untypeable SCCmec types in the study will be further ever, we were unable to determine whether the isolates evaluated and characterized; however, there is the need were community-associated. Untypeable SCCmec types to develop new SCCmec classification schemes for non- (ccrC/Class A mec; ccr-negative/Class C2 mec) were typeable methicillin-resistant staphylococci. There is an detected in two representative MRSA isolates, of which urgent need to curtail the spread and establishment one was a PVL-positive isolate (t4690-ST152) from a of PVL-positive MSSA, in particular, the MSSA ST152 patient with osteomyelitis in Ile-Ife (South-West Nigeria). clone, in Nigeria. Shittu et al. BMC Infectious Diseases 2012, 12:286 Page 9 of 10 http://www.biomedcentral.com/1471-2334/12/286 Abbreviations 6. Deepa S, Kumari A, Venkatesha D: Increasing trends of methicillin SCCmec: Staphylococcal Cassette Chromosome mec; CC: Clonal Complex; resistant coagulase negative Staphylococcus in neonatal septicaemia – PVL: Panton Valentine Leukocidin; ST: Sequence type; CNS: Coagulase a study in a tertiary care hospital, Mysore, South India. OJHAS 2010, negative staphylococci; HCIs: Health Care Institutions; MIC: Minimum 9(4):1–3. Inhibitory Concentration; MLST: Multilocus sequence typing; ASOM: Acute 7. Zong Z, Peng C, Lu X: Diversity of SCCmec elements in methicillin- suppurative otitis media; CSOM: Chronic suppurative otitis media; resistant coagulase-negative staphylococci clinical isolates. PLoS One SLE: Systemic lupus erythematosus; GTI: Genitourinary tract infection; 2011, 6(5):e20191. UTI: Urinary tract infection; SSI: Skin and soft tissue infection; 8. Venkatesh MP, Placencia F, Weisman LE: Coagulase negative MSSA: Methicillin-susceptible Staphylococcus aureus; MRSA: Methicillin- staphylococcal infections in the neonate and child: an update. Semin resistant Staphylococcus aureus; MRCNS: Methicillin-resistant coagulase Pediatr Infect Dis 2006, 17(3):120–127. negative staphylococci; NT: Non-typeable; ND: Not determined; 9. Anderson-Berry A, Brinton B, Lyden E, Faix RG: Risk factors associated with UNT: Untypeable; Antibiotics: ; PEN: Penicillin; OXA: Oxacillin; development of persistent coagulase negative staphylococci bacteremia TEICO: Teicoplanin; TET: Tetracycline; GEN: Gentamicin; CIP: Ciprofloxacin; in the neonate and associated short-term and discharge morbidities. MOX: Moxifloxacin; ERY: Erythromycin; CLI: Clindamycin; FUS: Fusidic acid; Neonatology 2011, 99(1):23–31. RIF: Rifampicin; COT: Cotrimoxazole. 10. Bassetti M, Trecharichi EM, Mesini A, Spanu T, Giacobbe DR, Rossi M, Shenone E, Pascale GD, Molinari MP, Cauda R, Viscoli C, Tumbarello M: Risk factors and mortality of healthcare-associated and community-acquired Competing interests Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2011, doi:10.1111/ The authors declare that they have no competing interest. j.1469-0691.2011.03679. Epub ahead of print. 11. de Kraker ME, Davey PG, Grundmann H, on behalf of the BURDEN study Authors’ contributions group: Mortality and hospital stay associated with resistant AOS, KO, AR, ST, FO, KO and GE participated in the design of the study. OO Staphylococcus aureus and Escherichia coli bacteremia: estimating the and FA performed the preliminary identification of the isolates. AOS carried burden of antibiotic resistance in Europe. PLoS Med 2011, 8(10):e1001104. out the molecular characterization of the isolates. All authors read and 12. Jain A, Agarwal A, Verma RK, Awasthi S, Singh KP: Intravenous device approved the final manuscript. associated blood stream staphylococcal infection in paediatric patients. Indian J Med Res 2011, 134(2):193–199. Acknowledgments 13. Jean-Baptiste N, Benjamin DK Jr, Cohen-Wolkowiez M, Fowler VG Jr, We would like to thank the management of the hospitals for their support in Laughon M, Clark RH Smith PB: Coagulase negative staphylococcal the collection of the staphylococcal isolates. We also appreciate the infections in the neonatal intensive care unit. Infect Control Hosp assistance of Professor Mark Nicol, Dr. Eliya Madikane, Melissa Jansen van Epidemiol 2011, 32(7):679–686. Rensburg, Malefu Moleleki (Division of Medical Microbiology, University of 14. 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