Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria

  • Adebayo O Shittu1, 5Email author,

    Affiliated with

    • Kenneth Okon2,

      Affiliated with

      • Solayide Adesida3,

        Affiliated with

        • Omotayo Oyedara1, 4,

          Affiliated with

          • Wolfgang Witte5,

            Affiliated with

            • Birgit Strommenger5,

              Affiliated with

              • Franziska Layer5 and

                Affiliated with

                • Ulrich Nübel5

                  Affiliated with

                  BMC Microbiology201111:92

                  DOI: 10.1186/1471-2180-11-92

                  Received: 22 December 2010

                  Accepted: 5 May 2011

                  Published: 5 May 2011

                  Abstract

                  Background

                  Staphylococcus aureus is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of S. aureus infections, it is crucial to understand the trends in the antibiotic-resistance patterns. In addition, the occurrence and changes in types of S. aureus, clonal identities, and their geographic spread is essential for the establishment of adequate infection control programmes. In this study, 68 S. aureus isolates obtained from clinical and non-clinical sources in Nigeria between January and April 2009 were characterized using phenotypic and molecular methods.

                  Results

                  All the S. aureus isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline. Sixteen percent of the isolates were resistant to oxacillin, while 55% and 72% of isolates were resistant to tetracycline and trimethoprim/sulphamethoxazole (cotrimoxazole), respectively (Table 1). There was excellent correlation between the broth microdilution assay and detection of antibiotic resistance genes by the multiplex PCR, in the determination of S. aureus resistance to erythromycin, gentamicin, methicillin and tetracycline. A total of 28 spa types were identified in the study, and the predominant spa type among the methicillin-susceptible S. aureus (MSSA) isolates was t084 (13 isolates). The t037-ST241-SCCmecIII type was the only clone identified in Maiduguri (North-East Nigeria) while in South-West Nigeria, diversity among the MRSA isolates (t451-ST8-SCCmecV; t008-ST94-SCCmecIV; t002-ST5-SCCmecV; t064-ST8-SCCmecV) was observed. The toxin genes seh and etd were detected in isolates affiliated with clonal complexes CC1, CC80 and sequence type ST25, respectively. The proportion of PVL-positive isolates among MSSA was high (40%). Most of the PVL-positive MSSA isolates were obtained from wound infections and associated with clonal complexes CC1, CC30, CC121 and with sequence type ST152.
                  Table 1

                  Antibiotic resistance profile of S. aureu s (MSSA and MRSA) from Nigeria

                   

                  Number (%) of resistant isolates among:

                  Antibiotic

                  MSSA

                  (n = 57)

                  MRSA

                  (n = 11)

                  Total

                  (n = 68)

                  Penicillin

                  49 (86)

                  11 (100)

                  60 (88.2)

                  Oxacillin

                  0 (0)

                  11 (100)

                  11 (16.2)

                  Teicoplanin

                  0 (0)

                  0 (0)

                  0 (0)

                  Vancomycin

                  0 (0)

                  0 (0)

                  0 (0)

                  Gentamicin

                  1 (1.8)

                  9 (81.8)

                  10 (14.7)

                  Tetracycline

                  27 (47.4)

                  11 (100)

                  38 (55.9)

                  Ciprofloxacin

                  12 (21.1)

                  8 (72.7)

                  20 (29.4)

                  Moxifloxacin

                  0 (0)

                  7 (63.6)

                  7 (10.3)

                  Trimethoprim/sulfamethoxazole

                  39 (68.4)

                  10 (90.9)

                  49 (72.1)

                  Phosphomycin

                  0 (0)

                  0 (0)

                  0 (0)

                  Fusidic acid

                  0 (0)

                  0 (0)

                  0 (0)

                  Erythromycin

                  2 (3.5)

                  6 (54.5)

                  8 (11.8)

                  Clindamycin

                  0 (0)

                  6 (54.5)

                  6 (8.8)

                  Rifampicin

                  0 (0)

                  0 (0)

                  0 (0)

                  Daptomycin

                  0 (0)

                  0 (0)

                  0 (0)

                  Mupirocin

                  0 (0)

                  0 (0)

                  0 (0)

                  Linezolid

                  0 (0)

                  0 (0)

                  0 (0)

                  Tigecycline

                  0 (0)

                  0 (0)

                  0 (0)

                  Conclusions

                  The use of phenotypic and molecular methods provided useful information on antibiotic resistance and molecular diversity of S. aureus in Nigeria. The high proportion of PVL-positive MSSA isolates affiliated to various clonal complexes and detected in all the health institutions is a major concern, both as a source of severe infections and as a potential reservoir that could lead to the emergence of PVL-positive MRSA. This study presents the first baseline information on the nature of the antibiotic resistance genes from S. aureus isolates in Nigeria. There is the need to curtail the spread and establishment of MRSA and PVL-positive MSSA clones in Nigerian health care institutions.

                  Background

                  Staphylococcus aureus is a leading cause of diseases such as skin and soft tissue infections, pneumonia, bloodstream infections, osteomyelitis and endocarditis, as well as toxin-mediated syndromes like toxic shock and food poisoning [1, 2]. It has developed resistance to a wide range of antimicrobial drugs, which complicates the treatment of infections. In particular, methicillin-resistant S. aureus (MRSA) has become a notorious etiologic agent for a wide variety of infections and it is one of the most important nosocomial pathogens worldwide [36]. Methicillin-susceptible S. aureus (MSSA) become MRSA through the acquisition and insertion into their genomes of a large DNA fragment known as staphylococcal chromosome cassette mec (SCCmec), which contains the methicillin resistance determinant, mecA [7]. Several variants of SCCmec have been described, which differ with respect to the composition of their recombinase genes and mec gene complex (containing the mecA gene) [8, 9].

                  In the developing world, mortality associated with severe S. aureus infections far exceeds that in developed countries [10, 11]. Recent studies have identified S. aureus as the main etiological agent of many infections in sub-Saharan Africa [1216], and a number of investigations have reported that S. aureus is among the most frequently encountered bacterial species in microbiology laboratories in Nigeria [1722]. However, data on the molecular epidemiology of this pathogen in Nigeria is very limited. Recent reports have indicated that the prevalence of hospital-associated MRSA varies in health care institutions [23, 24]. A community-associated MRSA clone with a unique resistance profile has also been reported from South-West Nigeria [25]. To understand and potentially predict trends in antibiotic-resistance patterns and to establish adequate infection control programs, it is crucial to understand the local epidemiology of S. aureus in Nigeria. Knowledge of the local antimicrobial resistance patterns of bacterial pathogens is essential to guide empirical and pathogen specific therapy. The threat of antibiotic-resistant bacteria has initiated studies on the nature of genes encoding resistance and the mechanism by which these genes spread and evolve. Antibiotic susceptibility testing of S. aureus in Nigeria is based on phenotypic testing especially the disk diffusion technique but recent studies have relied on the PCR detection of the mecA gene for the identification and confirmation of MRSA [2326]. However, no information is available on the nature of antibiotic resistance genes of S. aureus in Nigeria. Our present study provides baseline information on antibiotic resistance and molecular epidemiology of MSSA and MRSA in Nigeria.

                  Results

                  Antibiotic susceptibility testing and detection of antibiotic resistance genes in S. aureus isolates

                  The 68 S. aureus isolates obtained between January and April 2009 were analyzed for antimicrobial resistance (Table 1). All the isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline, and two isolates were susceptible to all the antibiotics tested. In addition to the antibiotics stated above, all MSSA isolates (84%) were susceptible to clindamycin and moxifloxacin and less than 4% were resistant to erythromycin, 21.1% to ciprofloxacin, 47% to tetracycline, 68% to cotrimoxazole and 86% to penicillin. The predominant antibiotypes among the MSSA isolates were resistance to penicillin, tetracycline and cotrimoxazole (15 isolates), and resistance to penicillin and cotrimoxazole (13 isolates). A total of 11 isolates were resistant to oxacillin and confirmed as MRSA based on the detection of the mecA gene (Table 1). The ermA gene was identified in all erythromycin-resistant MRSA isolates, while two erythromycin-resistant MSSA isolates possessed the msrA gene. All the gentamicin-resistant isolates carried the aacA-aphD gene. Moreover, the tetM gene was detected in 11 isolates (7 MRSA and 4 MSSA) and the tetK gene was present in 4 MRSA and 23 MSSA isolates.

                  SCCmec typing

                  The SCCmec type V was identified in four MRSA isolates obtained in Ile-Ife, Ibadan and Lagos, while one MRSA isolate from Ile-Ife possessed the SCCmec type IV element (Table 2). The MRSA isolates from Maiduguri were non-typeable for the SCCmec element based on established protocols [9, 27], and no amplification was observed for the ccrA, ccrB, and ccrh genes. However, these MRSA isolates possessed the ccu gene. The comparison and analysis of the ccu sequences from two selected MRSA isolates in this group with sequences in the GenBank suggested that the MRSA isolates possessed an SCCmec type III element of uncommon organization, which had not been identified using standard protocols.
                  Table 2

                  Characterization of MRSA isolates from Nigeria based on antibiotic susceptibility pattern, detection of antibiotic resistance genes, SCCmec typing, spa typing and MLST

                  Isolate No

                  Location

                  Sample or Clinical Diagnosis

                  Antibiotype

                  Antibiotic resistance genes

                  SCCmec type

                  spa type

                  MLST

                  09-01730

                  Ile-Ife

                  Chronic ulcer

                  PEN, OXA, GEN, OTE, SXT

                  mecA, aacA-aphD, tetK

                  V

                  t451

                  8

                  09-01731

                  Ile-Ife

                  Urinary tract infection

                  PEN, OXA, GEN, OTE

                  mecA, aacA-aphD, tetM

                  IV

                  t008

                  94 (8-slv)

                  09-01739

                  Lagos

                  Wound infection

                  PEN, OXA, OTE, CIP, SXT

                  mecA, tetK

                  V

                  t002

                  5

                  09-01776

                  Ibadan

                  Conjunctivitis

                  PEN, OXA, OTE, SXT

                  mecA, tetK

                  V

                  t064

                  8

                  09-01786

                  Ibadan

                  Wound infection

                  PEN, OXA, GEN, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, tetK

                  V

                  t064

                  8

                  09-01789

                  Maiduguri

                  Wound infection

                  PEN, OXA, GEN, ERY, CLI, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, ermA, tetM

                  III

                  t037

                  241

                  09-01791-1

                  Maiduguri

                  Semen (Infertility)

                  PEN, OXA, GEN, ERY, CLI, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, ermA, tetM

                  IIIa

                  t037

                  241

                  09-01795

                  Maiduguri

                  Throat Infection

                  PEN, OXA, GEN, ERY, CLI, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, ermA, tetM

                  IIIa

                  t037

                  ND

                  09-01809

                  Maiduguri

                  Semen (Infertility)

                  PEN, OXA, GEN, ERY, CLI, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, ermA, tetM

                  IIIa

                  t037

                  241

                  09-01811

                  Maiduguri

                  Wound Infection

                  PEN, OXA, GEN, ERY, CLI, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, ermA, tetM

                  IIIa

                  t037

                  ND

                  09-01812

                  Maiduguri

                  Wound Infection

                  PEN, OXA, GEN, ERY, CLI, OTE, CIP, SXT, MFL

                  mecA, aacA-aphD, ermA, tetM

                  IIIa

                  t037

                  ND

                  aSCCmec type inferred from related isolate 09-01789

                  slv: single locus variant

                  Geographical region: South-West Nigeria (Ile-Ife, Ibadan and Lagos)

                  North-East Nigeria (Maiduguri)

                  KEY

                  PEN: Penicillin G; OX: Oxacillin; GEN: Gentamicin; ERY: Erythromycin; CLI: Clindamycin; OTE: Tetracycline; CIP: Ciprofloxacin; SXT: Trimethoprim/sulfamethoxazole; MFL: Moxifloxacin

                  ND: Not determined

                  Molecular diversity of S. aureus based on spa typing and MLST

                  Twenty-eight spa types were identified in this study. Representative isolates were subsequently selected for MLST (Tables 2 and 3). Results indicated that nine major clonal complexes were represented in our strain collection from Nigeria (Tables 2 and 3). These clonal complexes plus one that we did not find (CC22) seem to predominate the S. aureus population on all continents. In addition, we found sequence type ST152, which has been reported previously in Ibadan and Maiduguri (Nigeria) [24, 25].

                  Detection of markers frequently associated with community-acquired S. aureus

                  A total of 23 of the 57 (40.3%) MSSA isolates (grouped in clonal complexes - CC1, CC5, CC15, CC30, CC121, CC80 and sequence type ST152) were PVL positive (Table 3), while none of the MRSA possessed the PVL gene. The enterotoxin H gene (seh) was detected in the isolates from clonal lineage CC1. Three MSSA isolates (ST25) from nasal samples of healthy individuals and one MSSA (CC80) from a wound infection possessed the etd gene. All the S. aureus isolates were arcA negative.
                  Table 3

                  Characterization of MSSA isolates from Nigeria by antibiotic susceptibility pattern, detection of antibiotic and virulence genes, spa typing and MLST

                  Isolate No

                  Location

                  Sample Or Clinical Diagnosis

                  Antibiotype

                  Antibiotic resistance genes

                  Toxin genes

                  spa type

                  MLST

                  Clonal Complex (CC)

                  09-01760

                  Ife

                  Wound Infection

                  PEN, CIP, SXT

                  -

                  lukPV, seh

                  t127

                   

                  CC1

                  09-01823

                  Ife

                  Wound Infection

                  PEN, OTE

                  tetK

                  lukPV, seh

                  t127

                    

                  09-01785-1

                  Ibadan

                  Conjunctivitis

                  PEN

                  -

                  lukPV, seh

                  t114

                    

                  09-01787

                  Maiduguri

                  Wound Infection

                  PEN, OTE

                  tetK

                  seh

                  t321

                  1

                   

                  09-01733

                  Ife

                  Otitis media

                  PEN, SXT

                  -

                  lukPV

                  t311

                   

                  CC5

                  09-01738

                  Lagos

                  Urinary tract infection

                  PEN, ERY, OTE, CIP, SXT

                  tetM, mrsA

                  -

                  t311

                  5

                   

                  09-01777

                  Ibadan

                  Wound Infection

                  PEN, CIP

                  -

                  -

                  t311

                    

                  09-01815

                  Maiduguri

                  Otitis media

                  PEN, ERY, OTE, SXT

                  tetM, mrsA

                  -

                  t311

                    

                  09-01737

                  Lagos

                  Semen (Infertility)

                  PEN, GEN, OTE, SXT

                  aacA-aphD, tetK

                  -

                  t951

                   

                  CC8

                  09-01742

                  Lagos

                  Unknown

                  PEN, OTE

                  tetK

                  -

                  t1617

                  8

                   

                  09-01780

                  Ibadan

                  Conjunctivitis

                  PEN, SXT

                  -

                  -

                  t064

                    

                  09-01796

                  Maiduguri

                  Sputum (Unknown)

                  PEN, OTE, SXT

                  tetK

                  -

                  t064

                    

                  09-01810

                  Ife

                  Sputum (Unknown)

                  PEN, OTE, CIP, SXT

                  tetK

                  -

                  t1496

                    

                  09-01817-1

                  Maiduguri

                  Urinary tract infection

                  PEN, OTE, SXT

                  tetK

                  -

                  t1496

                    

                  09-01819

                  Maiduguri

                  Semen (Infertility)

                  PEN, OTE, SXT

                  tetK

                  -

                  t2812

                    

                  09-01822

                  Ife

                  Sputum (Unknown)

                  PEN, OTE, SXT

                  tetK

                  -

                  t1496

                    

                  09-01734

                  Ife

                  Unknown

                  PEN, OTE, SXT

                  tetK

                  -

                  t084

                   

                  CC15

                  09-01736

                  Lagos

                  Otitis media

                  OTE

                  tetM

                  -

                  t084

                    

                  09-01750

                  Ife

                  Nasal*

                  PEN, SXT

                  -

                  lukPV

                  t084

                    

                  09-01751

                  Ife

                  Nasal*

                  PEN, SXT

                  -

                  lukPV

                  t084

                  15-slv

                   

                  09-01752

                  Ife

                  Nasal*

                  SXT

                  -

                  lukPV

                  t084

                    

                  09-01755

                  Ife

                  Nasal*

                  PEN, OTE, SXT

                  tetK

                  -

                  t084

                    

                  09-01756

                  Ife

                  Nasal*

                  PEN, OTE, CIP, SXT

                  tetK

                  -

                  t084

                    

                  09-01799

                  Maiduguri

                  Otitis media

                  PEN, OTE, SXT

                  tetK

                  -

                  t084

                    

                  09-01801

                  Maiduguri

                  Otitis media

                  PEN, OTE, SXT

                  tetK

                  -

                  t084

                    

                  09-01805

                  Ife

                  Blood Infection

                  OTE

                  tetM

                  -

                  t084

                  15-slv

                   

                  09-01820

                  Ife

                  Wound Infection

                  PEN, SXT

                  -

                  -

                  t084

                    

                  09-01821

                  Ife

                  Wound Infection

                  PEN, OTE, SXT

                  tetK

                  -

                  t084

                    

                  09-01824

                  Ife

                  Wound Infection

                  CIP, SXT

                  -

                  -

                  t084

                    

                  09-01788

                  Maiduguri

                  Wound Infection

                  PEN, OTE, SXT

                  tetK

                  -

                  t2216

                    

                  09-01798

                  Maiduguri

                  Semen (Infertility)

                  PEN, OTE, SXT

                  tetK

                  -

                  t2216

                  15

                   

                  09-01804

                  Maiduguri

                  Wound Infection

                  PEN, OTE, SXT

                  tetK

                  -

                  t2216

                    

                  09-01806

                  Ife

                  Unknown

                  PEN, SXT

                  -

                  -

                  t328

                    

                  09-01813

                  Ife

                  Wound Infection

                  PEN, SXT

                  -

                  -

                  t5387

                    

                  09-01740

                  Lagos

                  Wound Infection

                  PEN, CIP, SXT

                  -

                  lukPV

                  t318

                   

                  CC30

                  09-01743

                  Lagos

                  Wound Infection

                  CIP, SXT

                  -

                  lukPV

                  t318

                  30

                   

                  09-01747-2

                  Lagos

                  Wound Infection

                  Susceptible to all antibiotics

                  -

                  lukPV

                  t318

                  30

                   

                  09-01779

                  Ibadan

                  Wound Infection

                  PEN, CIP, SXT

                  -

                  lukPV

                  t021

                    

                  09-01825

                  Ife

                  Otitis media

                  PEN, CIP

                  -

                  -

                  t631

                    

                  09-01732

                  Ile-Ife

                  Unknown

                  PEN, SXT

                  -

                  lukPV

                  t159

                   

                  CC121

                  09-01759

                  Ile-Ife

                  Wound Infection

                  PEN, SXT

                  -

                  lukPV

                  t314

                    

                  09-01797

                  Maiduguri

                  Wound Infection

                  OTE

                  tetK

                  lukPV

                  t314

                    

                  09-01826

                  Ile-Ife

                  Otitis media

                  PEN

                  -

                  lukPV

                  t159

                  121

                   

                  09-01745

                  Lagos

                  Semen (Infertility)

                  PEN, SXT

                  -

                  lukPV

                  t2304

                  121

                   

                  09-01781

                  Ibadan

                  Wound Infection

                  PEN, OTE, SXT

                  tetK

                  lukPV

                  t2304

                    

                  09-01761

                  Ife

                  Wound Infection

                  PEN

                  -

                  lukPV

                  t355

                   

                  singleton

                  09-01762

                  Ife

                  Wound Infection

                  PEN

                  -

                  lukPV

                  t355

                    

                  09-01778

                  Ibadan

                  Wound Infection

                  PEN, CIP

                  -

                  lukPV

                  t355

                    

                  09-01790

                  Maiduguri

                  Wound Infection

                  Susceptible to all antibiotics

                  -

                  -

                  t355

                  152

                   

                  09-01793

                  Maiduguri

                  Wound Infection

                  PEN

                  -

                  lukPV

                  t355

                    

                  09-01803

                  Maiduguri

                  Wound Infection

                  PEN, OTE, SXT

                  tetK

                  lukPV

                  t355

                    

                  09-01753

                  Ife

                  Nasal*

                  PEN, SXT

                  -

                  etd

                  t3772

                  25

                  singleton

                  09-01754

                  Ife

                  Nasal*

                  PEN, SXT

                  -

                  etd

                  t3772

                    

                  09-01757

                  Ife

                  Nasal*

                  PEN, SXT

                  -

                  etd

                  t3772

                    

                  09-01802

                  Maiduguri

                  Wound Infection

                  PEN, CIP

                  -

                  -

                  t939

                  45

                  CC45

                  09-01792

                  Maiduguri

                  Wound Infection

                  PEN, OTE

                  tetK

                  -

                  t458

                  97

                  CC97

                  09-01800

                  Maiduguri

                  Wound Infection

                  PEN, OTE, SXT

                  tetK

                  lukPV, etd

                  t934

                  80

                  CC80

                  aClonal complex (CC) inferred from MLST and spa typing

                  *Nasal isolates from apparently healthy male students

                  slv: single locus variant

                  Geographical region: South-West Nigeria (Ile-Ife, Ibadan and Lagos)

                  North-East Nigeria (Maiduguri)

                  Discussion

                  There was excellent correlation between the broth microdilution method and detection of the genetic determinants by multiplex PCR for S. aureus resistance to erythromycin, gentamicin, methicillin and tetracycline (Tables 2 and 3). About 55% (11 MRSA, 27 MSSA) and 70% (10 MRSA, 39 MSSA) of the S. aureus isolates were resistant to tetracycline and cotrimoxazole, and as previous studies from South-West Nigeria had shown [23, 25], it appears that there is a high proportion of S. aureus isolates resistant to these antibiotics in Nigeria. Tetracycline and cotrimoxazole historically had wide clinical application, is inexpensive, orally administered and available from diverse sources where they are sold with or without prescription in Nigeria. Moreover, they are listed in many developing countries as among the antibacterial agents that have been rendered ineffective, or for which there are serious concerns regarding bacterial resistance [28]. It appears that misuse and overuse of these antibiotics could have contributed to this trend in Nigeria. Therefore, to prevent treatment failures in the absence of data on antibiotic susceptibility testing, public enlightenment on the ineffectiveness of these antibiotics against S. aureus infections, and the enactment of effective drug policies in Nigeria are urgently needed. The predominant mechanism of trimethoprim resistance in S. aureus appears to be mutation of the dihydrofolate reductase (DHFR), which is selected even when trimethoprim is used in combination with sulfamethoxazole [29]. In this study, all the trimethoprim-resistant S. aureus isolates were dfrA negative suggesting that mutation of the dihydrofolate reductase (DHFR) is responsible for resistance. Isolates resistant to tetracycline carried either one of the resistance genes tetK or tetM (Tables 2 and 3), which mediate resistance through active drug efflux or ribosomoal protection mechanisms, respectively. This is the first study that provides baseline information on the nature of the antibiotic resistance genes from S. aureus isolates in Nigeria. The multiplex PCR assay was easy to perform, cost-effective and assisted in the prompt characterization of the resistance genes stated above. It could be adapted for use by clinical scientists in the referral health care institutions regarding the antibiotics administered and the prevalent resistance determinants in Nigeria.

                  The proportion of PVL-positive isolates among MSSA was high (40%). Most of the PVL-positive MSSA isolates were obtained from wound infections and classified in clonal complexes CC1, CC30, CC121 and ST152. Moreover, the detection of the seh gene in CC1 isolates and the identification of the etd gene in ST25 and CC80 isolates is in agreement with previous reports [27, 3032]. PVL is frequently associated with severe and recurrent skin and soft-tissue infections (SSTIs) and has previously been found in S. aureus isolates from various complexes. In particular, PVL-producing MSSA affiliated to CC121 are known to be common in many countries on all continents [30, 33, 34], including Nigeria, Togo and South Africa in sub-Saharan Africa [25, 30, 35]. PVL-positive ST152 was the predominant clone in a study recently conducted in North-Eastern Nigeria [24] and it was the second most prevalent clone in a carriage study from a West-African country (Mali) [36]. Furthermore, the high prevalence of PVL positive MSSA ST152 emerging in the community as well as in hospitals in West Africa has also been described [31]. Hence, ST152 seems to be widespread and frequent in West Africa, whereas it is comparatively rare elsewhere [33, 37], in contrast to many other clonal complexes that display worldwide occurrence. The luk-PV genes are carried on mobile genetic elements (prophages), which may be incorporated into S. aureus lineages through horizontal transfer, either before or after acquisition of the mecA gene [38]. The high proportion of PVL-positive MSSA observed in this study indicate that conditions that increase the risk of inter-individual transmission (e.g skin-to-skin and skin-to-fomite contacts) could represent important routes of spread in the various hospital settings. Contact with colonized and/or infected individuals as well as contaminated fomites in the spread of PVL positive S. aureus have been described as risk factors for community-associated MRSA [39]. Moreover, the detection of PVL-positive MSSA ST152 from members of one family and their relatives with skin infections at the Canary Island underscore the pathogenic and contagious nature of this clone [40]. More detailed investigations on the prevalence of PVL-positive S. aureus are needed in Africa with respect to (i) nasal carriage of S. aureus in the hospitals and community, (ii) cross-transmission from post-operative wound infections acquired during hospital stay, and (iii) cross-transmission from patients admitted to the health institutions for treatment of an SSTI acquired in the community. The detection of PVL-positive MSSA isolates from the various health institutions, indicating their wide geographical distribution, could pose serious problem in the future as potential reservoirs for resistance and virulence factors, and could lead to the emergence and spread of PVL-positive MRSA clones in Nigeria causing severe infections. This could have important implications for the enactment of effective infection control guidelines.

                  MRSA has become a major public health problem worldwide and recent reports have indicated that the prevalence of hospital-associated MRSA (based on the detection of the mecA gene) in health care institutions in Nigeria may vary from 1.5% to 20% [2325]. All the MRSA isolates obtained from Maiduguri (North-East Nigeria) had the same spa type (t037) and MLST profile (ST241), identical to isolates from the same region that had been investigated in a previous study [24]. Another study [25] also reported that the clone was identified in a hospital in Ibadan (South-Western Nigeria). ST241 is a single locus variant (slv) of the ST239 clone, which is prevalent in South East Asia and has also been reported from Europe, the Americas [41], and several countries in Africa [6, 4244]. The multi-resistant nature of this MRSA clone could be explained by the presence of several resistance genes in the SCCmec cassette and it was recently shown to have spread across several continents since the 1960s [41]. MRSA ST239 demonstrating low level resistance to glycopeptides have been reported recently in Russia [45] and New Zealand [46]. In contrast, in South-Western Nigeria, we identified more diversity among the MRSA isolates. In three different hospitals in this region, we observed several different clones of MRSA that can be distinguished on the basis of MLST, SCCmec typing and spa typing, and displayed distinct antimicrobial resistance profiles (Table 2).

                  Conclusions

                  This study showed that the combination of susceptibility testing and various molecular methods provided useful information on the antibiotic resistance and molecular diversity of S. aureus in Nigeria. Although the number of S. aureus isolates available for our investigation and epidemiological information was limited, the high proportion of PVL-positive MSSA observed in this study indicate that adequate measures are needed to curtail the spread and establishment of MRSA and PVL-positive MSSA clones in Nigerian health care institutions.

                  Methods

                  Isolation and identification of S. aureus isolates

                  In this study, a total of 68 non-duplicate consecutive S. aureus isolates (60 - clinical isolates; 8 - nasal isolates; one isolate per sample per individual) obtained between January and April 2009 were characterized. The clinical isolates were obtained from samples processed in the microbiology laboratories of referral health care institutions in Ile-Ife, Ibadan and Lagos (South-West Nigeria), and Maiduguri (North-East Nigeria), each of which are 500-bed facilities providing medical care to about one million people. The clinical isolates were cultured from 30 males (median age: 31 years, range: 1 year-70 years), 21 females (median age: 36 years, range: 1 week-63 years) and 9 unknown gender. In addition, nasal isolates were obtained from apparently healthy male undergraduate students in Ile-Ife. The origin and characteristics of each isolate is stated in Tables 2 and 3. The isolates were cultured on sheep blood agar and phenotypic identification of S. aureus was based on colony morphology and positive plasma coagulase reaction (slide and tube test). The susceptibility testing of the isolates to 18 antibiotics was performed using the broth microdilution assay as described by Deutsches Institut für Normung [47]. The antibiotic panel included penicillin G, oxacillin, teicoplanin, vancomycin, gentamicin, tetracycline, ciprofloxacin, moxifloxacin, trimethoprim/sulfamethoxazole (cotrimoxazole), phosphomycin, fusidic acid, erythromycin, clindamycin, rifampicin, daptomycin, mupirocin, linezolid and tigecycline.

                  DNA extraction

                  Genomic DNA was obtained from a 2 ml overnight culture using a DNeasy tissue kit (Qiagen, Hilden, Germany) with lysostaphin (100 μg/ml) to achieve bacterial lysis.

                  PCR detection of the tuf gene

                  Phenotypic identification of the S. aureus isolates was confirmed by the detection of the tuf gene [48].

                  Multiplex PCR for detection of antibiotic resistance genes

                  The antibiotic resistance determinants investigated were the aac-aphD (aminoglycoside resistance) mecA (methicillin resistance) ermA, ermC (erythromycin resistance) and tetK, tetM (tetracycline resistance) genes. PCR primers and conditions were as described in a previously established protocol [49]. Moreover, the detection of the dfrA and msrA genes (trimethoprim resistance and macrolide efflux resistance determinants) were investigated using the following primers tmpI: CTC ACG ATA AAC AAA GAG TCA; tmp II: CAA TCA TTG CTT CGT ATA ACG and msrA f: GAA GCA CTT GAG CGT TCT; msrA r: CCT TGT ATC GTG TGA TGT which amplified a 201bp and 287bp of the dfr and msrA genes, respectively. The PCR conditions were as follows: Initial denaturation at 95°C for 2 minutes followed by 30 cycles of amplification with 94°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 30 seconds and final extension at 72°C for 4 minutes.

                  Multiplex PCR for detection of markers associated with community-acquired S. aureus

                  A multiplex PCR reaction protocol [27] was used to detect markers associated with community-acquired S. aureus. They included the enterotoxin H gene (seh) for community-acquired S. aureus of clonal lineage ST1/USA400, the arginine deiminase gene (arcA) as part of the ACME (arginine catabolic mobile element) cluster for ST8/t008/USA300, the gene for exfoliative toxin D (etd) for ST80, and the Panton-Valentine Leukocidin (PVL) gene.

                  SCCmec typing

                  SCCmec elements were classified by the multiplex PCR strategy [9, 50]. SCCmec elements that could not be typed were characterized based on PCR amplification and sequence analysis of the cassette chromosome recombinases A and B genes (ccrA, ccrB), cassette chromosome helicase (cch) and another gene of unknown function (ccu) [51].

                  Spa typing

                  Spa typing was based on the method described previously [52]. The nucleotide sequences were analyzed using the RIDOM Staph-Type software (Ridom GmbH, Germany) to assign the isolates to the various spa types.

                  Multilocus sequence typing (MLST)

                  MLST was performed according to the previously published protocol [53].

                  Declarations

                  Acknowledgements

                  We would like to thank the management of the hospitals for their support in the collection of the isolates. We gratefully acknowledge the technical assistance of Annette Weller, Mike Henkel, Christa Cuny, Ilona Wermuth and the staff at the Central Sequencing Unit at the Robert Koch Institute. We thank Professor Iruka Okeke for comments and suggestions on the manuscript. The stay of AOS at the Robert Koch Institute was supported by the German Ministry for Economic Cooperation and Development (DAAD award).

                  Authors’ Affiliations

                  (1)
                  Department of Microbiology, Obafemi Awolowo University
                  (2)
                  Department of Medical Microbiology, University of Maiduguri Teaching Hospital
                  (3)
                  Molecular Biology and Biotechnology Division, Nigerian Institute of Medical Research
                  (4)
                  Department of Biological Sciences, College of Science, Engineering and Technology, Osun State University
                  (5)
                  Robert Koch Institute

                  References

                  1. Richards MJ, Edwards JR, Culver DH, Gaynes RP: Nosocomial infections in medical intensive care units in the United States, National Nosocomial Infections Surveillance System. Crit Care Med 1999, 27:887–892.PubMedView Article
                  2. Perez-Vazquez M, Vindel A, Marcos C, Oteo J, Cuevas O, Trincado P, Bautista V, Grundmann H, Campos J, on behalf of the EARSS spa-typing Group: Spread of invasive Spanish Staphylococcus aureus spa-type 067 associated with a high prevalence of the aminoglycoside-modifying enzyme gene ant (4')-Ia and the efflux genes msrA / msrB . J Antimicrob Chemother 2009, 63:21–31.PubMedView Article
                  3. Tiemersma EW, Bronzwaer SL, Lyytikainen O, Degener JE, Schrijnemakers P, Bruinsma N, Monen J, Witte W, Grundman H, European Antimicrobial Resistance Surveillance System Participants: Methicillin-resistant Staphylococcus aureus in Europe, 1999–2002. Emerg Infect Dis 2004, 10:1627–1634.PubMed
                  4. Huang YC, Su LH, Wu TL, Lin TY: Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates from a teaching hospital in Northern Taiwan. J Clin Microbiol 2006, 44:2268–2270.PubMedView Article
                  5. Sola C, Cortes P, Saka HA, Vindel A, Bocco JL: Evolution and molecular characterization of methicillin-resistant Staphylococcus aureus epidemic and sporadic clones in Cordoba, Argentina. J Clin Microbiol 2006, 44:192–200.PubMedView Article
                  6. Shittu AO, Nübel U, Udo EE, Lin J, Gaogakwe S: Characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates from hospitals in KwaZulu-Natal (KZN) province, Republic of South Africa. J Med Microbiol 2009, 58:1219–1226.PubMedView Article
                  7. Hiramatsu K, Cui L, Kuroda M, Ito T: The emergence and evolution of methicillin-resistant Staphylococcus aureus . Trends Microbiol 2001, 9:486–493.PubMedView Article
                  8. Chongtrakool P, Ito T, Ma XX, Kondo Y, Trakulsomboon S, Tiensasitorn C, Jamklang M, Chavalit T, Song JH, Hiramatsu K: Staphylococcal cassette chromosome mec (SCC mec ) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCC mec elements. Antimicrob Agents Chemother 2006, 50:1001–1012.PubMedView Article
                  9. Oliveira DC, Milheirico C, de Lencastre H: Redefining a structural variant of staphylococcal cassette chromosome mec , SCC mec type VI. Antimicrob Agents Chemother 2006, 50:3457–3459.PubMedView Article
                  10. Nickerson EK, West TE, Day NP, Peacock : Staphylococcus aureus disease and drug resistance in resource-limited countries in South and East Asia. Lancet Infect Dis 2009, 9:130–135.PubMedView Article
                  11. Nickerson EK, Hongsuwan M, Limmathurotsakul D, Wuthiekanun V, Shah KR, Srisomang P, Mahavanakul W, Wacharaprechasgul T, Fowler VG, West TE, Teerawatanasuk N, Becher H, White NJ, Chierakul W, Day NP, Peacock SJ: Staphylococcus aureus bacteraemia in a tropical setting: patient outcome and impact of antibiotic resistance. PLoS ONE 2009, 4:e4308.PubMedView Article
                  12. Mulu A, Moges F, Tessema B, Kassu A: Pattern and multiple drug resistance of bacterial pathogens isolated from wound infection at University of Gondar Teaching Hospital, Northwest Ethiopia. Ethiop Med J 2006, 44:125–131.PubMed
                  13. Feleke Y, Mengistu Y, Enquselassie F: Diabetic infections: clinical and bacteriological study at Tikur Anbessa Specialized University Hospital, Addis Ababa, Ethiopia. Ethiop Med J 2007, 45:171–179.PubMed
                  14. Olatunji , Fadeyi A, Ayanniyi AA, Akanbi AA: Non-gonococcal bacterial agents of conjunctivitis and their antibiotic susceptibility patterns in Ilorin, Nigeria. Afr J Med Med Sci 2007, 36:243–247.PubMed
                  15. Anguzu JR, Olila D: Drug sensitivity patterns of bacterial isolates from septic post-operative wounds in a regional referral hospital in Uganda. Afr Health Sci 2007, 7:148–154.PubMed
                  16. Nantanda R, Hildenwall H, Peterson S, Kaddu-Mulindwa D, Kalyesubula I, Tumwine JK: Bacterial aetiology and outcome in children with severe pneumonia in Uganda. Ann Trop Paediatr 2008, 28:253–260.PubMedView Article
                  17. Ambe JP, Gasi IS, Mava Y: Review of neonatal infections in University of Maiduguri Teaching Hospital: common bacterial pathogens seen. Niger J Clin Pract 2007, 10:290–293.PubMed
                  18. Legbo JN, Legbo JF: Bacterial isolates from necrotizing fasciitus: a clinico-pathological perspective. Niger J Med 2007, 16:143–147.PubMed
                  19. Anah MU, Udo JJ, Ochigbo SO, Abia-Bassey LN: Neonatal septicaemia in Calabar, Nigeria. Trop Doct 2008, 38:126–128.PubMedView Article
                  20. Odetoyin WB, Aboderin AO, Ikem RT, Kolawole BA, Oyelese AO: Asymptomatic bacteriuria in patients with diabetes mellitus in Ile-Ife, South-West, Nigeria. East Afr Med J 2008, 85:18–23.PubMed
                  21. Obidike EO, Anigbo G, Igbodo C: Sensitivity pattern of bacterial isolates in childhood sepsis in clinical practice at Onitsha. Niger J Clin Pract 2009, 12:302–305.PubMed
                  22. Ubani UA: Bacteriology of external ocular infections in Aba, South Eastern Nigeria. Clin Exp Optom 2009, 92:482–489.PubMedView Article
                  23. Shittu AO, Lin J, Kolawole DO: Antimicrobial susceptibility patterns of Staphylococcus aureus and characterization of MRSA in Southwestern Nigeria. WOUNDS 2006, 18:77–84.
                  24. Okon KO, Basset P, Uba A, Lin J, Oyawoye B, Shittu AO, Blanc DS: Co-occurrence of predominant Panton Valentine leukocidin-positive sequence type (ST) 152 and multidrug-resistant ST241 Staphylococcus aureus clones in Nigerian hospitals. J Clin Microbiol 2009, 47:3000–3003.PubMedView Article
                  25. Ghebremedhin B, Olugbosi MO, Raji AM, Layer F, Bakare RA, Konig B, Konig W: Emergence of a community-associated methicillin-resistant Staphylococcus aureus with unique resistance profile in Southwest of Nigeria. J Clin Microbiol 2009, 47:2975–2980.PubMedView Article
                  26. Adesida S, Boelens H, Babajide B, Kehinde A, Snijders S, van Leeuwen W, Coker A, Verbrugh H, van Belkum A: Major epidemic clones of Staphylococcus aureus in Nigeria. Microb Drug Resist 2005, 11:115–121.PubMedView Article
                  27. Strommenger B, Braulke C, Pasemann B, Schmidt C, Witte W: Multiplex PCR for rapid detection of Staphylococcus aureus isolates suspected to represent community-acquired strains. J Clin Microbiol 2008, 46:582–587.PubMedView Article
                  28. Okeke IN: Factors contributing to the emergence of resistance. In The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment - Workshop Summary. Edited by: Knobler SL, Lemon SM, Najafi M, Burroughs T. Washington, DC: The National Academies Press; 2003:132–139.
                  29. Dale GE, Broger C, D'Arcy A, Hartman PG, DeHoogt R, Jolidon S, Kompis I, Labhardt AM, Langen H, Locher H, Page MG, Stuber D, Then RL, Wipf B, Oefner C: A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance. J Mol Biol 1997, 266:23–30.PubMedView Article
                  30. Rasigade JP, Laurent F, Lina G, Meugnier H, Bes M, Vandenesch F, Etienne J, Tristan A: Global distribution and evolution of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus , 1981–2007. J Infect Dis 2010, 201:1589–1597.PubMedView Article
                  31. 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, the Working Group on Staphylococcus aureus infections, Garin B, Laurent F: Epidemiology of methicillin-susceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes. Clin Microbiol Infect 2010.
                  32. Holtfreter S, Grumann D, Schmudde M, Nguyen HT, Eichler P, Strommenger B, Kopron K, Kolata J, Giedrys-Kalemba S, Steinmetz I, Witte W, Bröker BM: Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates. J Clin Microbiol 2007, 45:2669–2680.PubMedView Article
                  33. Masiuk H, Kopron K, Grumann D, Goerke C, Kolata J, Jursa-Kulesza J, Giedrys-Kalemba S, Broker BM, Holfreter S: Association of recurrent furunculosis with Panton-Valentine Leukocidin and the genetic background of Staphylococcus aureus . J Clin Microbiol 2010, 48:1527–1535.PubMedView Article
                  34. Wiese-Posselt M, Heuck D, Draeger A, Mielke M, Witte W, Ammon A, Hamouda O: Successful termination of a furunculosis outbreak due to lukS-lukF-positive, methicillin-susceptible Staphylococcus aureus in a German village by stringent decolonization, 2002–2005. Clin Infect Dis 2007, 44:e88–95.PubMedView Article
                  35. Goering RV, Shawar RM, Scangarella NE, O'Hara FP, Amrine-Madsen H, West JM, Dalessandro M, Becker JA, Walsh SL, Miller LA, van Horn SF, Thomas ES, Twynholm ME: Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from global clinical trials. J Clin Microbiol 2008, 46:2842–2847.PubMedView Article
                  36. Ruimy R, Maiga A, Armand-Lefevre L, Maiga I, Diallo A, Koumare AK, Ouattara K, Soumare 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, 190:3962–3968.PubMedView Article
                  37. Ruimy R, Armand-Lefevre L, Barbier F, Ruppe E, Cocojaru R, Mesli Y, Maiga A, Benkalfat M, Benchouk S, Hassaine H, Dufourcq JB, Nareth C, Sarthou JL, Andremont A, Feil EJ: Comparisons between geographically diverse samples of carried Staphylococcus aureus . J Bacteriol 2009, 191:5577–5583.PubMedView Article
                  38. O'Hara FP, Guex N, Word JM, Miller LA, Becker JA, Walsh SL, Scangarella NE, West JM, Shawar RM, Amrine-Madsen H: A geographic variant of the Staphylococcus aureus Panton-Valentine Leukocidin toxin and the origin of community-associated methicillin-resistant S. aureus USA300. J Infect Dis 2008, 197:187–194.PubMedView Article
                  39. Cataldo MA, Taglietti F, Petrosillo N: Methicillin-resistant Staphylococcus aureus : a community health threat. Postgrad Med 2010, 122:16–23.PubMedView Article
                  40. Perez-Roth E, Alcoba-Florez J, Lopez-Aquilar C, Gutierrez-Gonzalez I, Rivero-Perez B, Mendez-Alvarez S: Familial furunculosis associated with community-acquired leukocidin-positive methicillin susceptible Staphylococcus aureus ST152. J Clin Microbiol 2010, 48:329–332.PubMedView Article
                  41. Harris SR, Feil EJ, Holden MT, Quail MA, Nickerson EK, Chantratita N, Gardete S, Tavares A, Day N, Lindsay JA, Edgeworth JD, de Lencastre H, Parkhill J, Peacock SJ, Bentley SD: Evolution of MRSA during hospital transmission and intercontinental spread. Science 2010, 327:469–474.PubMedView Article
                  42. Ramdani-Bouguessa N, Bes M, Meugnier H, Forey F, Reverdy ME, Lina G, Vandenesch F, Tazir M, Etienne J: Detection of methicillin-resistant Staphylococcus aureus strains resistant to multiple antibiotics and carrying the Panton-Valentine leukocidin genes in an Algiers hospital. Antimicrob Agents Chemother 2006, 50:1083–1085.PubMedView Article
                  43. Breurec S, Zriouil SB, Fall C, Boisier P, Brisse S, Djibo S, Etienne J, Fonkoua MC, Perrier-Gros-Claude JD, Pouillot R, Ramarokoto CE, Randrianirina F, Tall A, Thiberge JM, the Working Group on Staphylococcus aureus infections, Laurent F, Garin B: Epidemiology of methicillin-resistant Staphylococcus aureus lineages in five major African towns: emergence and spread of atypical clones. Clin Microbiol Infect 2010.
                  44. Moodley A, Oosthuysen WF, Dusé AG, Marais E, the South African MRSA Surveillance Group: Molecular Characterization of Clinical Methicillin-Resistant Staphylococcus aureus Isolates in South Africa. J Clin Microbiol 2010, 48:4608–4611.PubMedView Article
                  45. Baranovich T, Zaraket H, Shabana II, Nevzorova V, Turcutyuicov V, Suzuki H: Molecular characterization and susceptibility of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from hospitals and the community in Vladivostok, Russia. Clin Microbiol Infect 2010, 16:575–582.PubMedView Article
                  46. Howden BP, Seemann T, Harrison PF, McEvoy CR, Stanton JA, Rand CJ, Mason CW, Jensen SO, Firth N, Davies JK, Johnson PD, Stinear TP: Complete genome sequence of Staphylococcus aureus JKD6008, an ST239 clone of methicillin-resistant Staphylococcus aureus with intermediate-level vancomycin resistance. J Bacteriol 2010, 192:5848–5849.PubMedView Article
                  47. Deutsches Institut für Normung DIN 58940: Medical Microbiology-susceptibility testing of pathogens to antimicrobial agents. Part 8. Microdilution. General method specific requirements. 2004, 342–353.
                  48. 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, 39:2541–2547.PubMedView Article
                  49. Strommenger B, Kettlitz C, Werner G, Witte W: Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus . J Clin Microbiol 2003, 41:4089–4094.PubMedView Article
                  50. Witte W, Braulke C, Cuny C, Strommenger B, Werner G, Heuck D, Jappe U, Wendt C, Linde HJ, Harmsen D: Emergence of methicillin-resistant Staphylococcus aureus with Panton-Valentine Leukocidin genes in Central Europe. Eur J Clin Microbiol Infect Dis 2005, 24:1–5.PubMedView Article
                  51. Lina G, Durand G, Berchich C, Short B, Meugnier H, Vandenesch F, Etienne J, Enright MC: Staphylococcal chromosome cassette evolution in Staphylococcus aureus inferred from ccr gene complex sequence typing analysis. Clin Microbiol Infect 2006, 12:1175–1184.PubMedView Article
                  52. 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, 41:5442–5448.PubMedView Article
                  53. 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, 38:1008–1015.PubMed

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                  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.

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