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

  • Adebayo Shittu1, 2Email author,

    Affiliated with

    • Omotayo Oyedara1, 3,

      Affiliated with

      • Fadekemi Abegunrin1,

        Affiliated with

        • Kenneth Okon4,

          Affiliated with

          • Adeola Raji5,

            Affiliated with

            • Samuel Taiwo6,

              Affiliated with

              • Folasade Ogunsola7,

                Affiliated with

                • Kenneth Onyedibe8 and

                  Affiliated with

                  • Gay Elisha2

                    Affiliated with

                    BMC Infectious Diseases201212:286

                    DOI: 10.1186/1471-2334-12-286

                    Received: 28 May 2012

                    Accepted: 29 October 2012

                    Published: 2 November 2012

                    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

                    Background

                    The staphylococci are part of the normal cutaneous ecosystem, but they are also opportunistic and invasive pathogens that cause a variety of human infections. Staphylococcus aureus is the most important human pathogen in this genus accounting for a high proportion of severe infections in hospitals and outpatient medical care [13]. Although the clinical relevance of coagulase negative staphylococci (CNS) is still controversial, patients at risk of CNS infections include neonates, those with intravascular catheters, prosthetic devices, postoperative sternal wound infections and immunocompromised hosts [47]. The remarkable ability of S. aureus and CNS to acquire antibiotic resistance limits therapeutic options, and morbidity and mortality rates of staphylococcal infections have increased the financial burden on health care systems worldwide [814].

                    Although there are reports on the characterization of staphylococci in Nigeria [1518], many of the studies were limited to antibiotic susceptibility profiles. Moreover, one geographical region or health care institution was investigated, and CNS were not identified to species level. Only a few studies have investigated the molecular epidemiology and diversity of this group of organisms [1923], however, they were not multi-centre surveys on S. aureus and CNS. This work describes a multi-centre study which was conducted in order to provide clinicians and infection control practitioners with information on antibiotic resistance profiles, molecular characteristics of coagulase positive and negative staphylococci, and to alert clinicians to the emergence of new clones in health care institutions (HCIs) in Nigeria.

                    Methods

                    Isolation and identification of staphylococcal isolatess

                    The study commenced with the request for non-duplicate staphylococcal isolates obtained from clinical samples processed in the microbiology laboratories of eight health care institutions in Ile-Ife, Ogbomosho, Iwo, Lagos (two centres) and Ibadan in South-West Nigeria; Jos in North-Central Nigeria, and Maiduguri in North-East Nigeria (Figure 1) during the period January to April, 2010. In this study, only staphylococcal isolates were analyzed and human subjects, clinical samples or human data were not investigated. All the hospitals, except the one located in Iwo, were tertiary health care institutions, each of which were at least 500-bed facilities providing medical care to about 500,000 to one million people. Preliminary verification as staphylococci was based on culture characteristics on blood agar, catalase, coagulase and DNase tests, while identification to species level was performed at the National Health Laboratory Service (NHLS), Groote Schuur Hospital, Cape Town, South Africa using the VITEK 2 system (bioMérieux, France) with the Gram-Positive Identification Card.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2334-12-286/MediaObjects/12879_2012_2114_Fig1_HTML.jpg
                    Figure 1

                    Map indicating the location of the health care institutions in Nigeria.

                    Antibiotic susceptibility testing

                    Antibiotic susceptibility for penicillin G, oxacillin, teicoplanin, vancomycin, gentamicin, tetracycline, ciprofloxacin, moxifloxacin, trimethoprim/sulfamethoxazole (cotrimoxazole), fusidic acid, erythromycin, clindamycin, rifampicin, mupirocin, linezolid and tigecycline was determined by the broth microdilution method (VITEK 2 system, AST 603 cards, bioMérieux). S. aureus ATCC 29213 was the control strain and the VITEK 2 minimum inhibitory concentration (MIC) results were interpreted using the Advanced Expert System of the VITEK 2. Multiresistance was defined as resistance to at least three classes of antibiotics.

                    DNA extraction

                    Before DNA extraction, 5–8 colonies from an 18–24 h old culture on boiled blood agar was pretreated with lysostaphin (Sigma) (20 μg/ml for S. aureus; 50μg/ml for CNS) in 20 mM Tris-Cl, 2mM EDTA at 37°C for 30 min. The cells were harvested and DNA was isolated using a DNeasy tissue kit (Qiagen, Hilden, Germany).

                    PCR detection of the tuf gene

                    Phenotypic identification of the S. aureus isolates was confirmed by detection of the tuf gene as described previously [24]. The PCR products were detected by gel electrophoresis using 2% w/v agarose (Seakem, Whittaker, USA) and run in 1X TAE buffer (pH 8.3) for 1 h at 100 V. Thereafter, the gel was stained with ethidium bromide, visualized under UV light and photographed using a SynGene Bioimaging System.

                    PCR detection of the dfrA gene

                    The presence of the dfrA gene encoding trimethoprim resistance was investigated using the primer pairs: dfr-AF: AAT AGA CGT AAC GTC GTA CT; dfr-AR: AAG AAT GTA TGC GGT ATA GT and subsequent detection of a 289 bp product [25]. The PCR conditions were as follows: Initial denaturation at 95°C for 3min, followed by 30 cycles of amplification at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s and final extension at 72°C for 1min.

                    SCCmec typing

                    SCCmec typing was performed according to a previously published protocol [26] using the control strains S. aureus COL (type I), N315 (type II), ANS46 (type III), MW2 (type IV), WIS (type V) and HDE288 (type VI). For non-typeable SCCmec elements, the investigation of mec complexes and ccr allotypes as described by Kondo et al. [27] was carried out on representative methicillin-resistant S. aureus (MRSA) and CNS isolates based on their spa-MLST profile and location of the health care institutions, respectively. The profiles obtained were characterized and defined according to the current nomenclature used for MRSA [28].

                    Detection of the PVL gene

                    The presence of the PVL gene was determined by PCR using primers luk-PV-1 and luk-PV-2 [29].

                    Genotyping

                    Typing of S. aureus was based on sequencing of the hypervariable region of the protein A gene (spa). The spa types were assigned using the Ridom StaphType (Ridom GmbH, Würzburg, Germany, version 1.5.21) [30]. Multilocus sequence typing (MLST) was carried out according to the protocols described previously [31] on a representative isolate for the predominant and new spa types. PCR products were purified and sequenced at the Central Analytical Facility at the University of Stellenbosch, South Africa. Allelic profiles and sequence types (STs) were assigned using the MLST S. aureus database (http://​www.​mlst.​net). Isolates that were not typed by MLST were assigned STs based on the Based Upon Repeat Pattern (BURP) via the Ridom StaphType software.

                    Results

                    Identification and source of the staphylococcal isolates

                    Preliminary verification of isolates obtained from the microbiology laboratories in eight HCIs in Nigeria identified 91 staphylococcal isolates (51 S. aureus and 40 CNS) and the characteristics of each isolate are presented in Tables 1, 2 and 3. The clinical S. aureus isolates were recovered from wounds, skin and soft tissue infections (SSI), osteomyelitis and burns (36.4%), genitourinary tract infection (GTI)/infertility (18.2%), septicaemia (15.9%), urinary tract infection (UTI) (6.8%), otitis media (6.8%), bronchitis (4.5%), and 11.4% were classified as other infections (Tables 1 and 2). Six species accounted for the CNS isolates: S. haemolyticus (n=21), S. sciuri (n=9), S. saprophyticus (n=5), S. warneri (n=3), S. epidermidis (n=1) and S. hominis (n=1), which were distributed across four HCIs located in Ogbomosho, Lagos (designated as Lagos2), Jos and Maiduguri (Table 3). Most of the S. haemolyticus isolates were from the centres in Lagos2, Jos and Maiduguri, and 47.6% of the isolates were obtained from septicaemia, 14.3% from wound infections, 9.5% each from GTI, UTI, ocular-related infections, and those categorised as other infections (Table 3).
                    Table 1

                    Characterization of MRSA from Nigeria by antibiotic resistance pattern, detection of genes and molecular typing

                    Isolate No

                    Location

                    Sample Or Clinical Diagnosis

                    Antibiogram

                    tuf gene

                    mecA gene

                    dfrA gene

                    SCCmec typing

                    PVL gene

                    spa type

                    MLST (ST)

                    Clonal Complex (CC)

                    50

                    Lagos1

                    Burns

                    PEN, OXA, GEN, CIP, MOX, ERY, CLI*, TET, COT, RIF

                    +

                    +

                    +

                    III

                    -

                    t037

                    ST241

                    CC8

                    2496ψ

                    Maiduguri

                    GTI

                    PEN, OXA, GEN, CIP, ERY, CLI, TET, COT

                    +

                    +

                    -

                    NT/UNT

                    -

                    t037**

                    2589

                    Maiduguri

                    GTI

                    PEN, OXA, GEN, CIP, ERY, CLI, TET, COT

                    +

                    +

                    -

                    NT

                    -

                    t037

                    60ψ

                    Lagos1

                    ASOM

                    PEN, OXA, TET, COT

                    +

                    +

                    -

                    NT/V

                    -

                    t064**

                    ST8

                    CC8

                    69

                    Lagos1

                    Nasal swab/Screening

                    PEN, OXA, GEN, TET, COT

                    +

                    +

                    -

                    NT

                    -

                    t064

                    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

                    TU3ψ

                    Ibadan

                    Wound Infection

                    PEN, OXA, CIP, TET, COT

                    +

                    +

                    -

                    NT/V

                    -

                    t064

                    T5056

                    Ile-Ife

                    Bronchitis

                    PEN, OXA, GEN, CIP, ERY, CLI*, TET, COT, FUS, RIF

                    +

                    +

                    -

                    III

                    -

                    t074

                    ST37

                    CC30

                    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

                    T5843ψ

                    Ile-Ife

                    Osteomyelitis

                    PEN, OXA, TET, COT

                    +

                    +

                    -

                    NT/UNT

                    +

                    t4690**

                    ST152

                    singleton

                    KEY:

                    ψ: 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.

                    Table 2

                    Characterization of MSSA from Nigeria by antibiotic resistance pattern, detection of genes and molecular typing

                    Isolate No

                    Location

                    Sample Or Clinical Diagnosis

                    Antibiotype

                    tuf gene

                    dfrA gene

                    PVL gene

                    spa type

                    MLST (ST)

                    Clonal 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.

                    Table 3

                    Identification and Characterization of CNS by antibiotic resistance pattern, mecA gene detection and SCC mec typing

                    Isolate No

                    Location

                    Sample/Clinical Condition

                    VITEK 2 Identification

                    Antibiogram

                    mecA gene

                    SCCmec 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

                    14ψ

                    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

                    L2ψ

                    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

                    024ψ

                    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 ψ

                    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

                    J14ψ

                    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:ψ: 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.

                    Antibiotic susceptibility of the staphylococcal isolates and detection of mecA and dfrA genes

                    All the staphylococcal isolates were susceptible to mupirocin, tigecycline, vancomycin and linezolid (Table 4). In addition to the antibiotics stated above, the methicillin-susceptible S. aureus (MSSA) were susceptible to teicoplanin, gentamicin, erythromycin, clindamycin, fusidic acid and rifampicin. About 94% of MSSA were susceptible to the fluoroquinolones and one isolate was susceptible to all the antibiotics tested. However, 80.6% of MSSA were resistant to cotrimoxazole and penicillin, but all the cotrimoxazole-resistant MSSA were dfrA negative. The predominant antibiotype was resistance to penicillin, tetracycline and cotrimoxazole, which was observed in 14 isolates (39%). Resistance to oxacillin was detected in 15 S. aureus isolates and confirmed as MRSA by the detection of the mecA gene. Of the 15 MRSA isolates, 13 were resistant to cotrimoxazole and tetracycline, while 6 and 7 MRSA were resistant to gentamicin and ciprofloxacin, respectively. Two of the 5 erythromycin-resistant MRSA expressed inducible resistance to clindamycin. Overall, 12 of the 15 MRSA were resistant to at least three classes of antibiotics (Table 1). The trimethoprim resistance gene, dfrA, was detected in only 3 MRSA isolates.
                    Table 4

                    Antibiotic resistance profile of staphylococci from Nigeria

                     

                    Number (%) of isolates resistant among

                    Antibiotics

                    MSSA

                    MRSA

                    S. haemolyticus

                    Others (CNS)

                    n=36

                    n=15

                    n=21

                    n=19

                    Penicillin

                    29 (80.6)

                    15 (100)

                    21 (100)

                    18 (94.7)

                    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)

                    Gentamicin

                    0 (0)

                    6 (40)

                    18 (85.7)

                    9 (47.4)

                    Tetracycline

                    21 (58.3)

                    13 (86.7)

                    19 (90.5)

                    9 (47.4)

                    Ciprofloxacin

                    2 (5.6)

                    7 (46.7)

                    17 (80.9)

                    9 (47.4)

                    Moxifloxacin

                    1 (2.8)

                    1 (6.7)

                    6 (28.6)

                    3 (15.8)

                    Erythromycin

                    0 (0)

                    5 (33.3)

                    10 (47.6)

                    0 (0)

                    Clindamycin

                    0 (0)

                    5 (33.3)

                    5 (23.8)

                    0 (0)

                    Fusidic acid

                    0 (0)

                    1 (6.7)

                    1 (4.8)

                    16 (84.2)

                    Tigecycline

                    0 (0)

                    0 (0)

                    0 (0)

                    0 (0)

                    Mupirocin

                    0 (0)

                    0 (0)

                    0 (0)

                    0 (0)

                    Linezolid

                    0 (0)

                    0 (0)

                    0 (0)

                    0 (0)

                    Rifampicin

                    0 (0)

                    2 (13.3)

                    4 (19)

                    0 (0)

                    Cotrimoxazole

                    29 (80.6)

                    13 (86.7)

                    21 (100)

                    8 (42.1)

                    *Intermediate resistance.

                    All the S. haemolyticus isolates were resistant to penicillin, oxacillin and cotrimoxazole, but only one isolate exhibited resistance to fusidic acid. The proportion of S. haemolyticus isolates resistant to ciprofloxacin, gentamicin and tetracycline was 80.9%, 85.7% and 90.5%, respectively (Table 4). Furthermore, 5 (50%) of the 10 erythromycin-resistant S. haemolyticus isolates expressed inducible resistance to clindamycin, and one isolate showed intermediate resistance to teicoplanin (MIC: 16μg/ml). Multiresistant S. haemolyticus isolates were recovered from clinical samples in four HCIs in Lagos2, Maiduguri, Jos and Ogbomosho (Table 3). In contrast, resistance to fusidic acid was common in other species of CNS, and they were susceptible to erythromycin, clindamycin, teicoplanin and rifampicin (Table 4). The S. sciuri isolates were resistant to oxacillin and obtained primarily from various clinical samples in Ogbomosho (Table 3). On the whole, multiresistance was observed in 37 of the 40 CNS isolates, and the CNS isolates identified as methicillin-resistant (VITEK) were mecA positive.

                    SCCmec typing

                    Fifteen MRSA were obtained from the HCIs in Ile-Ife (n=5); Maiduguri (n=4); Lagos1 (n=3); Ibadan (n=2) and Iwo (n=1). Using the multiplex typing method [26], SCCmec type IV was identified in two MRSA each from Ile-Ife and Maiduguri, SCCmec type III in two MRSA, one each from Ile-Ife and Lagos1, and SCCmec type II in one MRSA from Ile-Ife (Table 1). However, 8 of 15 MRSA and 28 methicillin-resistant CNS (MRCNS) were non-typeable. Based on another PCR-based method [27], two of the four representative MRSA and three S. haemolyticus possessed the SCCmec V element (ccrC/Class C2 mec). However, untypeable SCCmec elements were detected in two MRSA (ccrC/Class A mec and ccr-negative/Class C2 mec) and one isolate each of S. sciuri and S. haemolyticus (ccr-negative/Class A mec and ccr type 4/Class C2 mec gene complex) (Tables 1 and 3).

                    Detection of PVL gene

                    One MRSA from Ile-Ife was PVL positive (Table 1). The gene was also detected in 16 MSSA, of which 10 were isolated from patients with SSI and septicaemia (Table 2).

                    Molecular typing of MSSA and MRSA

                    A total of 21 spa types were identified and representative S. aureus strains assigned as t037, t064, t084, t355, t2331, t4690, and new types t8987 and t9008 were selected for MLST (Tables 1 and 2). In MRSA, seven different spa types were identified and isolates with spa types t037, t064, and t4690 were assigned ST241, ST8 and ST152, respectively. Overall, the clonal complexes for MRSA were distributed across CC8, CC30, CC88 and ST152 (Table 2). In MSSA, fifteen different spa types, including two new types (t8987 and t9008) were identified of which only t064 was identified also in MRSA (Tables 1 and 2). The predominant spa types were t355 (8 isolates), t084 (7 isolates) and t318 (3 isolates) and a diversity of spa types (t318, t1510, t2304, t2331, spa non-typeable) were identified among the MSSA from nasal samples of health care workers (HCWs) (Table 2). MSSA isolates with spa type t084 and t2331 were assigned ST15 and ST8 respectively, while ST152 accounted for the new spa types (t8987, t9008) and t355. Nine and 11 of the 36 MSSA clustered with ST15 and ST152 respectively (Table 2).

                    Discussion

                    The identification of bacterial isolates to species level is of great importance in the clinical microbiology laboratory to obtain information on the diversity and significance of each species in human infection. Moreover, species identification is a prerequisite for epidemiological studies. Many clinical microbiology laboratories in Nigeria do not identify staphylococci (in particular CNS) to species level, which is important in understanding their variety and clinical relevance. Two species, S. haemolyticus and S. sciuri, accounted for the majority of the 40 CNS isolates investigated in this study (Table 3). Most of the S. haemolyticus isolates were obtained from blood culture samples in HCIs in Lagos2 and Jos, and S. sciuri from samples in Ogbomosho (South-West Nigeria). S. haemolyticus is regarded as the second most frequently isolated staphylococci from blood cultures [32] and one of the most clinically relevant CNS, particularly in immunocompromised patients [33]. S. sciuri, principally found in animal species, and although not considered important from the clinical standpoint, has been associated with infections such as endocarditis [34], urinary tract infection [35] and wound infections [19, 36]. Multiresistance to antibiotics was detected in 37 of the 40 CNS isolates, and all the S. haemolyticus isolates were resistant to at least three classes of antibiotics. Apart from the possible clinical importance because of its serious impact on the course of infection, multiresistant CNS are a potential source of genes encoding resistance to antibiotics for other staphylococci pathogenic for man. The isolation of multiresistant mecA positive S. haemolyticus from blood samples (septicaemia) and S. sciuri from various clinical materials (Table 3) in some HCIs clearly indicate that they could be of clinical importance in Nigeria. A number of reports have indicated an increase in the resistance of staphylococci to cotrimoxazole in Nigeria [15, 18, 21, 23, 37]. In this study, 72.5% of CNS and 82.3% of S. aureus isolates were resistant to cotrimoxazole. The antibiotic has wide clinical application, inexpensive, orally administered and available over-the-counter where they are sold with or without prescription in Nigeria. This could possibly explain the high level of staphylococcal resistance observed in this study. Most of the cotrimoxazole-resistant S. aureus were dfrA-negative; hence more studies are needed to understand the molecular mechanism of resistance in Nigeria.

                    Methicillin resistance in staphylococci is due to the expression of the mecA gene, which mediates penicillin binding protein 2a (PBP2a), a transpeptidase with low affinity for beta-lactams [7, 38]. The mecA gene is carried by a mobile genetic element (MGE) termed the staphylococcal cassette chromosome (SCCmec), and though the mec origin remains unknown, it has been suggested that mecA CNS may act as potential SCCmec donor accounting for the rise of new MRSA clones [39]. Eleven SCCmec elements have been described to date: SCCmec I-IV [4042], and V-XI [4348], but few reports exist on the detection of mecA gene and characterization of SCCmec types in Nigeria [2123]. There was excellent correlation between results of antibiotic susceptibility testing (VITEK) and detection of the mecA gene for the confirmation of methicillin-resistant staphylococci. However, the MRCNS were non-typeable using the multiplex PCR protocol [26]. Based on the method of Kondo et al. [27], the SCCmec V element (ccrC/Class C2 mec) was identified in representative S. haemolyticus isolates, as well as untypeable SCCmec elements (ccr-negative/Class A mec and ccr type 4/Class C2 mec gene complex) in one representative isolate each of S. sciuri and S. haemolyticus (Table 3). Recent studies have indicated that SCCmec V is the predominant element among methicillin-resistant S. haemolyticus isolates [33, 49]. To the best of our knowledge, this is the first report on the characterization of SCCmec elements in CNS in Nigeria. It is likely that the mec gene complex and ccr genes in CNS are diverse and distinct, and as indicated in previous reports [7, 28], there is the need to develop new classification schemes for non-typeable SCCmec types in MRCNS.

                    Molecular typing of the MRSA isolates indicated that two clones (CC8-SCCmecnon-typeable and CC88-SCCmec IV) existed in Maiduguri, North-East Nigeria, and four clones in South-West Nigeria (CC8-SCCmecIII/IV/V; CC30-SCCmecII/III; CC88-SCCmecIV and ST152-SCCmecnon-typeable) (Table 1). Previous studies on the epidemiology of MRSA in Nigeria identified MRSA clones t064-ST8-CC8 and t037-ST241-CC8 in South-West and North-East Nigeria, respectively [22, 23]. A community-associated clone, CC88-MRSA-IV, previously reported in Ibadan, Nigeria [21] was identified from wound and blood samples in Maiduguri and Ile-Ife, however, we were unable to determine whether the isolates were community-associated. Untypeable SCCmec types (ccrC/Class A mec; ccr-negative/Class C2 mec) were detected in two representative MRSA isolates, of which one was a PVL-positive isolate (t4690-ST152) from a patient with osteomyelitis in Ile-Ife (South-West Nigeria).

                    The MSSA were assigned mainly to clonal complexes CC5, CC8, CC15, CC30, CC45, CC80 and ST152 (Table 2). A high proportion of MSSA from Maiduguri were grouped in ST15 or ST152 indicating that these clones were successful in North East Nigeria. PVL-encoding genes were detected in 16 MSSA (44.4%) isolates belonging to almost all clonal complexes identified in this study. Moreover, 56% of these strains were associated with ST152 and 10 of 16 (62.5%) PVL positive MSSA isolates were associated with SSI and septicaemia. These observations confirm previous studies that PVL-positive MSSA ST152 are widespread in African countries [5052] including Nigeria [22, 23]. Furthermore, MSSA isolates (ST121) from nasal samples of HCWs were PVL positive. Our data suggests that the sequence types for pandemic PVL-positive MSSA identified in this study did not function as a reservoir for PVL-positive MRSA supporting the argument of Schaumburg et al. [52] that the intimate inter-relation between PVL-positive MSSA and MRSA, which could lead to the emergence and spread of PVL-positive MRSA clones has not been established. However, the detection of an ST152-PVL-positive MRSA in this study suggests that surveillance for this clone is strongly advocated in Nigeria.

                    Conclusions

                    This is the first multi-centre study on the characterization of coagulase positive and negative staphylococci using phenotypic and molecular methods in Nigeria. However, there were a number of limitations in this study. The number of isolates from the HCIs was low and variable which did not allow for comparison of data among the participating centres. Moreover, the diversity of the staphylococci analysed in this study does not represent their distribution patterns in health care institutions in Nigeria. Lastly, we could not conduct full characterization of the mecA-positive CNS isolates, and only representative isolates were selected for SCCmec typing and MLST. However, this study has highlighted the importance of species identification for CNS, their clinical relevance, and the need for longitudinal multi-centre surveys to provide data on antibiotic resistance and epidemiology of the staphylococci in Nigeria. Characterization of multiresistant mecA positive S. haemolyticus and S. sciuri from clinical samples is important in understanding their epidemiology within hospitals in Nigeria. The identification of untypeable SCCmec types in the study will be further evaluated and characterized; however, there is the need to develop new SCCmec classification schemes for non-typeable methicillin-resistant staphylococci. There is an urgent need to curtail the spread and establishment of PVL-positive MSSA, in particular, the MSSA ST152 clone, in Nigeria.

                    Abbreviations

                    SCCmec

                    Staphylococcal Cassette Chromosome mec

                    CC: 

                    Clonal Complex

                    PVL: 

                    Panton Valentine Leukocidin

                    ST: 

                    Sequence type

                    CNS: 

                    Coagulase negative staphylococci

                    HCIs: 

                    Health Care Institutions

                    MIC: 

                    Minimum Inhibitory Concentration

                    MLST: 

                    Multilocus sequence typing

                    ASOM: 

                    Acute suppurative otitis media

                    CSOM: 

                    Chronic suppurative otitis media

                    SLE: 

                    Systemic lupus erythematosus

                    GTI: 

                    Genitourinary tract infection

                    UTI: 

                    Urinary tract infection

                    SSI: 

                    Skin and soft tissue infection

                    MSSA: 

                    Methicillin-susceptible Staphylococcus aureus

                    MRSA: 

                    Methicillin-resistant Staphylococcus aureus

                    MRCNS: 

                    Methicillin-resistant coagulase negative staphylococci

                    NT: 

                    Non-typeable

                    ND: 

                    Not determined

                    UNT: 

                    Untypeable

                    Antibiotics:; PEN: 

                    Penicillin

                    OXA: 

                    Oxacillin

                    TEICO: 

                    Teicoplanin

                    TET: 

                    Tetracycline

                    GEN: 

                    Gentamicin

                    CIP: 

                    Ciprofloxacin

                    MOX: 

                    Moxifloxacin

                    ERY: 

                    Erythromycin

                    CLI: 

                    Clindamycin

                    FUS: 

                    Fusidic acid

                    RIF: 

                    Rifampicin

                    COT: 

                    Cotrimoxazole

                    Declarations

                    Acknowledgments

                    We would like to thank the management of the hospitals for their support in the collection of the staphylococcal isolates. We also appreciate the assistance of Professor Mark Nicol, Dr. Eliya Madikane, Melissa Jansen van Rensburg, Malefu Moleleki (Division of Medical Microbiology, University of Cape Town, South Africa), staff of the National Health Laboratory Service (NHLS, Groote Schuur Hospital, Cape Town, South Africa), Dr Franziska Layer (Staphylococcal Reference Centre, Robert Koch Institute, Wernigerode, Germany), Professor Teruyo Ito and Dr. Xiao Han of the Department of Bacteriology, Juntendo University, Japan. We appreciate the comments and suggestions of Drs Edet Udo, Birgit Strommenger, Ulrich Nübel and Professor Iruka Okeke in the preparation of the manuscript. The research visit of AOS to the Division of Medical Microbiology, University of Cape Town, South Africa was supported by the visitor’s award by Clinical Infectious Disease Research Initiative (CIDRI).

                    Authors’ Affiliations

                    (1)
                    Department of Microbiology, Obafemi Awolowo University
                    (2)
                    Division of Medical Microbiology, University of Cape Town
                    (3)
                    Department of Biological Sciences, College of Science, Engineering and Technology, Osun State University
                    (4)
                    Department of Medical Microbiology, University of Maiduguri Teaching Hospital
                    (5)
                    Department of Medical Microbiology and Parasitology, Lagos State University Teaching Hospital
                    (6)
                    Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology Teaching Hospital
                    (7)
                    Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos
                    (8)
                    Department of Medical Microbiology and Parasitology, Jos University Teaching Hospital

                    References

                    1. Chambers HF, Deleo FR: Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009,7(9):629–641.PubMedView Article
                    2. Nickerson EK, West TE, Day NP, Peacock SJ: Staphylococcus aureus disease and drug resistance in resource-limited countries in South and East Asia. Lancet Infect Dis 2009,9(2):130–135.PubMedView Article
                    3. Fry DE, Barie PS: The changing face of Staphylococcus aureus : a continuing surgical challenge. Surg Infect (Larchmt) 2011,12(3):191–203.View Article
                    4. Huebner J, Goldmann DA: Coagulase-negative staphylococci: role as pathogens. Ann Rev Med 1999, 50:223–236.PubMedView Article
                    5. O’Gara JP, Humpreys H: Staphylococcus epidermidis biofilms: importance and implications. J Med Microbiol 2001,50(7):582–587.PubMed
                    6. Deepa S, Kumari A, Venkatesha D: Increasing trends of methicillin resistant coagulase negative Staphylococcus in neonatal septicaemia – a study in a tertiary care hospital, Mysore, South India. OJHAS 2010,9(4):1–3.
                    7. Zong Z, Peng C, Lu X: Diversity of SCC mec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. PLoS One 2011,6(5):e20191.PubMedView Article
                    8. Venkatesh MP, Placencia F, Weisman LE: Coagulase negative staphylococcal infections in the neonate and child: an update. Semin Pediatr Infect Dis 2006,17(3):120–127.PubMedView Article
                    9. Anderson-Berry A, Brinton B, Lyden E, Faix RG: Risk factors associated with development of persistent coagulase negative staphylococci bacteremia in the neonate and associated short-term and discharge morbidities. Neonatology 2011,99(1):23–31.PubMedView Article
                    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 Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2011. Epub ahead of print
                    11. de Kraker ME, Davey PG, Grundmann H, on behalf of the BURDEN study group: Mortality and hospital stay associated with resistant Staphylococcus aureus and Escherichia coli bacteremia: estimating the burden of antibiotic resistance in Europe. PLoS Med 2011,8(10):e1001104.PubMedView Article
                    12. Jain A, Agarwal A, Verma RK, Awasthi S, Singh KP: Intravenous device associated blood stream staphylococcal infection in paediatric patients. Indian J Med Res 2011,134(2):193–199.PubMed
                    13. Jean-Baptiste N, Benjamin DK Jr, Cohen-Wolkowiez M, Fowler VG Jr, Laughon M, Clark RH Smith PB: Coagulase negative staphylococcal infections in the neonatal intensive care unit. Infect Control Hosp Epidemiol 2011,32(7):679–686.PubMedView Article
                    14. Marra AR, Camargo LF, Pignatari AC, Sukiennik T, Behar PR, Medeiros EA, Ribeiro J, Girão E, Correa L, Guerra C, Brites C, Pereira CA, Carneiro I, Reis M, de Souza MA, Tranchesi R, Barata CU, Edmond MB, Brazilian SCOPE Study Group: Nosocomial bloodstream infections in Brazilian hospitals: analysis of 2,563 cases from a prospective nationwide surveillance study. J Clin Microbiol 2011,49(5):1866–1871.PubMedView Article
                    15. Akinkunmi EO, Lamikanra A: Species distribution and antibiotic resistance in coagulase negative staphylococci colonizing the gastrointestinal tract of children in Ile-Ife, Nigeria. Trop J Pharm Res 2010,9(1):35–43.View Article
                    16. Akinjogunla OJ, Enabulele IO: Virulence factors, plasmid profiling and curing analysis of multidrug resistant Staphylococcus aureus and coagulase negative Staphylococcus spp. isolated from patients with acute otitis media. J American Sci 2010,6(11):1022–1033.
                    17. Efuntoye MO: Study of antibiotic sensitivity pattern and enterotoxigenicity of staphylococci isolated from swimming pools in Ibadan, Nigeria. World Appl Sci J 2010,9(11):1324–1327.
                    18. Jombo GTA, Akpan S, Epoke J, Akaa PD, Eyong KI, Gyuse AN, Okwori EE, Etukumana EA: Antimicrobial susceptibility profile of community acquired and nosocomial isolates of Staphylococcus aureus and that of coagulase negative staphylococci from clinical blood culture specimens at a Nigerian University Teaching Hospital. J Clin Med Res 2010,2(6):83–90.
                    19. Shittu AO, Lin J, Morrison D, Kolawole DO: Isolation and molecular characterization of multiresistant Staphylococcus sciuri and Staphylococcus haemolyticus associated with skin and soft-tissue infections. J Med Microbiol 2004,53(Pt1):51–55.PubMedView Article
                    20. Shittu AO, Lin J, Morrison D, Kolawole DO: Identification and molecular characterization of mannitol positive, coagulase negative staphylococci from nasal samples of medical personnel and students. J Med Microbiol 2006,55(Pt3):317–324.PubMedView Article
                    21. Ghebremedhin B, Olugbosi MO, Raji AM, Layer F, Bakare RA, König B, König W: Emergence of a community-associated methicillin-resistant Staphylococcus aureus strain with a unique resistance profile in Southwest Nigeria. J Clin Microbiol 2009,47(9):2975–2980.PubMedView Article
                    22. Okon KO, Basset P, Uba A, Lin J, Shittu AO, Blanc DS: Co-occurrence of predominant Panton-Valentine leukocidin-positive sequence type (ST) 152 and multi-drug resistant ST241 Staphylococcus aureus clones in Nigerian hospitals. J Clin Microbiol 2009,47(9):3000–3003.PubMedView Article
                    23. Shittu AO, Okon K, Adesida S, Oyedara O, Witte W, Strommenger B, Layer F, Nübel U: Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria. BMC Microbiol 2011, 11:92.PubMedView Article
                    24. Martineau F, Picard FJ, Ke D, Paradis S, Roy PH, Ouellette M, Bergeron MG: Development of a PCR assay for identification of staphylococci at genus and species levels. J Clin Microbiol 2001,39(7):2541–2547.PubMedView Article
                    25. Ender M, Berger-Bächi B, McCallum N: Variability in SCCmec N1 spreading among injection drug users in Zurich, Switzerland. BMC Microbiol 2007, 7:62.PubMedView Article
                    26. Milheiriço C, Oliveira DC, de Lencastre H: Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus . Antimicrob Agents Chemother 2007,51(9):3374–3377. Epub 2007 Jun 18. Erratum in: Antimicrob Agents Chemother 2007, 51(12):4537PubMedView Article
                    27. Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, Hiramatsu K: Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec , ccr and major differences in junkyard regions. Antimicrob Agents Chemother 2007,51(1):264–274.PubMedView Article
                    28. International Working Group on the classification of Staphylococcal Cassette Chromosome elements (IWG-SCC): Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCC mec elements. Antimicrob Agents Chemother 2009,53(12):4961–4967.View Article
                    29. Lina G, Piémont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, Vandenesch F, Etienne J: Involvement of Panton-Valentine Leukocidin-Producing Staphylococcus aureus in Primary Skin Infections and Pneumonia. Clin Infect Dis 1999,29(5):1128–1132.PubMedView Article
                    30. Harmsen D, Claus H, Witte W, Rothgänger J, Claus H, Turnwald D, Vogel U: Typing of methicillin-resistant Staphylococcus aureus in a university setting by using novel software for spa repeat determination and database management. J Clin Microbiol 2003,41(12):5442–5448.PubMedView Article
                    31. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG: Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus . J Clin Microbiol 2000,38(3):1008–1015.PubMed
                    32. Ing MB, Baddour LM, Bayer AS: Bacteremia and infective endocarditis: pathogenesis, diagnosis, and complications. In The Staphylococci In Human Disease. Edited by: Crossley KB, Archer GL. New York: Churchill Livingstone; 1997:331–354.
                    33. Bouchami O, Ben Hassen A, de Lencastre H, Miragaia M: High prevalence of mec complex C andccrCis independent of SCC mec type V in Staphylococcus haemolyticus . Eur J Clin Microbiol Infect Dis 2012,31(4):605–614.PubMedView Article
                    34. Wallet F, Stuit L, Boulanger E, Roussel-Delvallez M, Dequiedt P, Courcol RJ: Peritonitis due to Staphylococcus sciuri in a patient on continuous ambulatory peritoneal dialysis. Scand J Infect Dis 2000,32(6):697–698.PubMedView Article
                    35. Stepanovic S, Jezek P, Vukovic D, Dakic I, Petras P: Isolation of members of the Staphylococcus sciuri group from urine and their relationship to urinary tract infections. J Clin Microbiol 2003,41(11):5262–5264.PubMedView Article
                    36. Coimbra DG, Almeida AGCS, Júnior JBO, da Silva AF, Pimentel BJ, Gitaí DLG, Moreira LS, Silva-Filho EA, de Andrade TG: Wound infection by multiresistant Staphylococcus sciuri identified by molecular methods. New Microbiol 2011,34(4):425–427.PubMed
                    37. Shittu AO, Lin J, Kolawole DO: Antimicrobial susceptibility patterns of Staphylococcus aureus and characterization of MRSA in Southwestern Nigeria. WOUNDS 2006,18(4):77–84.
                    38. Garza-González E, Morfin-Otero R, Llaca-Diaz JM, Rodriguez-Noriega E: Staphylococcal cassette chromosome (SCC mec ) in methicillin-resistant coagulase negative staphylococci. A review and the experience in a tertiary-care setting. Epidemiol Infect 2010,138(5):645–654.PubMedView Article
                    39. Hanssen AM, Ericson Sollid JU: SCC mec in staphylococci: genes on the move. FEMS Immunol Med Microbiol 2006,46(1):8–20.PubMedView Article
                    40. Ito T, Katayama Y, Hiramatsu K: Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob Agents Chemother 1999,43(6):1449–1458.PubMed
                    41. Ito T, Katayama Y, Asada K, Mori N, Tsutsumimoto K, Tiensasitorn C, Hiramatsu K: Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 2001,45(5):1323–1336.PubMedView Article
                    42. Ma XX, Ito T, Tiensasitorn C, Jamklang M, Chongtrakool P, Boyle-Vavra S, Daum RS, Hiramatsu K: Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains. Antimicrob Agents Chemother 2002,46(4):1147–1152.PubMedView Article
                    43. Ito T, Ma XX, Takeuchi F, Okuma K, Yuzawa H, Hiramatsu K: Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Chemother 2004,48(7):2637–2651.PubMedView Article
                    44. Oliveira DC, Milheiriço C, de Lencastre H: Redefining a structural variant of staphylococcal cassette chromosome mec , SCC mec type VI. Antimicrob Agents Chemother 2006,50(10):3457–3459.PubMedView Article
                    45. Berglund C, Ito T, Ikeda M, Ma XX, Söderquist B, Hiramatsu K: Novel type of staphylococcal cassette chromosome mec in a methicillin-resistant Staphylococcus aureus strain isolated in Sweden. Antimicrob Agents Chemother 2008,52(10):3512–3516.PubMedView Article
                    46. Zhang K, McClure JA, Elsayed S, Conly JM: Novel staphylococcal cassette chromosome mec type, tentatively designated type VIII, harboring class A mec and type 4 ccr gene complexes in a Canadian epidemic strain of methicillin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 2009,53(2):531–540.PubMedView Article
                    47. Li S, Skov RL, Han X, Larsen AR, Larsen J, Sørum M, Wulf M, Voss A, Hiramatsu K, Ito T: Novel types of staphylococcal cassette chromosome mec elements identified in clonal complex 398 methicillin resistant Staphylococcus aureus strains. Antimicrob Agents Chemother 2011,55(6):3046–3050.PubMedView Article
                    48. Shore AC, Deasy EC, Slickers P, Brennan G, O’Connell B, Monecke S, Ehricht R, Coleman DC: Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA , mecI , mecR1 , blaZ , and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 2011,55(8):3765–3773.PubMedView Article
                    49. Barros EM, Ceotto H, Bastos MCF, dos Santos KRN, Giambiagi-deMarval M: Staphylococcus haemolyticus as an important hospital pathogen and carrier of methicillin resistance genes. J Clin Microbiol 2012,50(1):166–168.PubMedView Article
                    50. Ruimy R, Maiga A, Armand-Lefevre L, Maiga I, Diallo A, Koumaré AK, Ouattara K, Soumaré S, Gaillard K, Lucet JC, Andremont A, Feil EJ: The carriage population of Staphylococcus aureus from Mali is composed of a combination of pandemic clones and the divergent Panton-Valentine leukocidin-positive genotype ST152. J Bacteriol 2008,190(11):3962–3968.PubMedView Article
                    51. Breurec S, Fall C, Pouillot R, Boisier P, Brisse S, Diene-Sarr F, Djibo S, Etienne J, Fonkoua MC, Perrier-Gros-Claude JD, Ramarokoto CE, Randrianirina F, Thiberge JM, Zriouil SB, Garin B, Laurent F, Working Group on Staphylococcus aureus Infections: Epidemiology of methicillin-susceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes. Clin Microbiol Infect 2010,17(4):633–639.View Article
                    52. Schaumburg F, Ngoa AU, Kösters K, Köck R, Adegnika AA, Kremsner PG, Lell B, Peters G, Mellmann A, Becker K: Virulence and genotypes of Staphylococcus aureus from infection and carriage in Gabon. Clin Microbiol Infect 2011,17(10):1507–1513.PubMedView Article
                    53. Pre-publication history

                      1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​12/​286/​prepub

                    Copyright

                    © Shittu et al.; licensee BioMed Central Ltd. 2012

                    This article is published under license to 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.