Open Access

Antibiotic resistance and molecular epidemiology of Staphylococcus aureusin Nigeria

  • Adebayo O Shittu1, 5Email author,
  • Kenneth Okon2,
  • Solayide Adesida3,
  • Omotayo Oyedara1, 4,
  • Wolfgang Witte5,
  • Birgit Strommenger5,
  • Franziska Layer5 and
  • Ulrich Nübel5
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. aureusisolates

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.

SCCmectyping

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

SCCmectype

spatype

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 spatyping 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

spatype

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. aureusisolates

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 tufgene

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.

SCCmectyping

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].

Spatyping

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. 10.1097/00003246-199905000-00020.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. 10.1093/jac/dkp078.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.PubMedPubMed CentralView Article
  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. 10.1128/JCM.00776-06.PubMedPubMed CentralView 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. 10.1128/JCM.44.1.192-200.2006.PubMedPubMed CentralView 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. 10.1099/jmm.0.011452-0.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. 10.1016/S0966-842X(01)02175-8.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 (SCCmec) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCCmec elements. Antimicrob Agents Chemother. 2006, 50: 1001-1012. 10.1128/AAC.50.3.1001-1012.2006.PubMedPubMed CentralView Article
  9. Oliveira DC, Milheirico C, de Lencastre H: Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI. Antimicrob Agents Chemother. 2006, 50: 3457-3459. 10.1128/AAC.00629-06.PubMedPubMed CentralView 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. 10.1016/S1473-3099(09)70022-2.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: e4308-10.1371/journal.pone.0004308.PubMedPubMed CentralView 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.PubMedPubMed Central
  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. 10.1179/146532808X375404.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. 10.1258/td.2006.006037.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.PubMedView Article
  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. 10.1111/j.1444-0938.2009.00425.x.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. 10.1128/JCM.01119-09.PubMedPubMed CentralView 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. 10.1128/JCM.00648-09.PubMedPubMed CentralView 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. 10.1089/mdr.2005.11.115.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. 10.1128/JCM.01600-07.PubMedPubMed CentralView Article
  28. Okeke IN: Factors contributing to the emergence of resistance. 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. 2003, Washington, DC: The National Academies Press, 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. 10.1006/jmbi.1996.0770.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. 10.1086/652008.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. 10.1128/JCM.00204-07.PubMedPubMed CentralView 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. 10.1128/JCM.02094-09.PubMedPubMed CentralView 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. 10.1086/517503.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. 10.1128/JCM.00521-08.PubMedPubMed CentralView 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. 10.1128/JB.01947-07.PubMedPubMed CentralView 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. 10.1128/JB.00493-09.PubMedPubMed CentralView 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. 10.1086/524684.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. 10.1128/JCM.00622-09.PubMedPubMed CentralView 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. 10.1126/science.1182395.PubMedPubMed CentralView 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. 10.1128/AAC.50.3.1083-1085.2006.PubMedPubMed CentralView 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, 10.1111/j.1469-0691.2010.03219.x.
  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. 10.1128/JCM.01704-10.PubMedPubMed CentralView 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. 10.1111/j.1469-0691.2009.02891.x.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. 10.1128/JB.00951-10.PubMedPubMed CentralView 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. 10.1128/JCM.39.7.2541-2547.2001.PubMedPubMed CentralView 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. 10.1128/JCM.41.9.4089-4094.2003.PubMedPubMed CentralView 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. 10.1007/s10096-004-1262-x.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. 10.1111/j.1469-0691.2006.01548.x.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. 10.1128/JCM.41.12.5442-5448.2003.PubMedPubMed CentralView 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.PubMedPubMed Central

Copyright

© Shittu et al; licensee BioMed Central Ltd. 2011

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.