Staphylococcus aureus is an important pathogen in human infections and is implicated in a wide variety of infections, from mild skin infections to more serious and invasive infections, including septicemia, pneumonia, endocarditis, deep-seated abscesses, and toxinoses including food poisoning and toxic shock syndrome.^1,2 The discovery of antimicrobial agents has been a critical element of the therapeutic armamentarium of modern medicine, but the treatment of infections caused by S aureus is still a challenge for clinicians.^3,4 Numerous studies have indicated that S aureus is among the most frequently encountered microorganisms in microbiology laboratories in Nigeria.^5–13 However, data on the antibiotic susceptibility patterns of this pathogen in Southwestern Nigeria are inadequate, and in most cases, isolates are screened against commonly available antibiotics. In addition, information on the prevalence and characterization of methicillin-resistant S aureus (MRSA) in this region is insufficient despite the established fact that MRSA is a significant health problem worldwide. The ability to characterize S aureus and monitor antimicrobial susceptibility patterns is important for clinicians selecting empiric antimicrobial therapy, rational formulation of public healthcare policies, and providing useful information on the global surveillance of this pathogen. The authors report on the antibiotic susceptibility of S aureus isolates obtained from various clinical samples and the characterization of MRSA in Southwestern Nigeria.

Materials and Methods

Staphylococcus aureus isolates. Between June 2002 and August 2004, 200 non-duplicate S aureus isolates were obtained from health institutions in Southwestern Nigeria. Most of the isolates (93%) were obtained from health institutions in Ile-Ife and Ibadan. More than one-third of the total number of isolates was recovered from wound samples, 42 (21%) from blood cultures, 17 (8.5%) from ocular-related infections, 16 (8%) from urine, and 11 (5.5%) from otitis media. Clinical information was unavailable for 30 S aureus isolates. Identification was based on growth and fermentation on mannitol salt agar. Other tests included Gram stain, colonial morphology on nutrient agar, and positive results for catalase, coagulase, and DNase tests. The isolates were preserved in Micro Bank™ (Pro-Lab Diagnostics, Austin, Tex) and stored at -20˚C for further characterization.
Antibiotic susceptibility testing. Antibiotic susceptibility testing was performed using the disk diffusion method. The antibiotics (Mast Diagnostics, UK) included penicillin (10 units), oxacillin (1 µg), gentamicin (10 µg), kanamycin (30 µg), streptomycin (30 µg), neomycin (30 µg), erythromycin (15 µg), clindamycin (2 µg), tetracycline (30 µg), minocycline (30 µg), trimethoprim (2.5 µg), trimethoprim/sulfamethoxazole (25 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), fusidic acid (10 µg), rifampicin (30 µg), teicoplanin (30 µg), vancomycin (30 µg), and mupirocin (5 µg and 200 µg). S aureus ATCC 25923 was the control strain used in every test run. The isolates were considered sensitive, intermediately resistant, and resistant based on the National Committee for Clinical Laboratory Standards (NCCLS; now the Clinical and Laboratory Standards Institute [CLSI]) guidelines.¹⁴ Interpretative zone diameters for resistance to fusidic acid, neomycin, and streptomycin, which are not stated in the CLSI guidelines, were considered as follows: ≤ 18 mm fusidic acid, ≤ 16 mm neomycin, and ≤ 14 mm streptomycin.^15,16 Inducible resistance of clindamycin by erythromycin was determined by the D-test, in which erythromycin and clindamycin disks were placed 15–18 mm apart on the Mueller-Hinton agar medium employed for the antibiotic sensitivity testing. A truncated or blunted clindamycin zone of inhibition (D shape) indicated inducible resistance, and constitutive resistance was recognized by a clindamycin zone diameter of ≤ 14 mm.¹⁷ Growth to the edge of the 200 µg mupirocin disk and within a 14 mm zone of inhibition with the 5 µg mupirocin disk indicated high- and low-level resistance, respectively.¹⁸
The resistance rate to each antibiotic was calculated as the number of intermediate and resistant isolates divided by the total number of isolates. Antibiotyping of methicillin-susceptible and methicillin-resistant S aureus (MSSA and MRSA) was based on their susceptibility to 20 antibiotics. Isolates with similar resistance patterns were grouped in the same antibiotype. Multiresistance was defined as resistance to penicillin along with at least 3 classes of antibiotics.
Molecular detection of the nuc, mecA, and mupA genes by the polymerase chain reaction (PCR). DNA isolation was carried out according to the method of Udo et al.¹⁸ Isolates that indicated intermediate and full resistance to oxacillin were confirmed as S aureus and MRSA by PCR detection of the nuc and the mecA genes, respectively.^19,20 Furthermore, high-level mupirocin resistance was confirmed by the detection of the mupA gene.²¹ The PCR conditions for the detection of these genes were as follows: predenaturation at 95˚C for 5 minutes, 30 cycles of denaturation at 95˚C for 30 seconds, annealing at 55˚C for 30 seconds, extension at 72˚C for 1 minute, and final extension at 72˚C for 5 minutes. Polymerase chain reaction products were detected by gel electrophoresis using 1.5% w/v agarose (SeaKem®, Cambrex, East Rutherford, NJ) in 1X TBE for 2 hours at 80V. Thereafter, the agarose gel was stained in 1 µg/mL ethidium bromide (Sigma-Aldrich, St. Louis, Mo) solution for 45 minutes, visualized under UV, and photographed (SynGene Bioimaging System, SynGene, Cambridge, UK).
Molecular typing. Pulsed field gel electrophoresis (PFGE) typing of SmaI (Fermentas Inc., Ontario, Canada) digested DNA of the MRSA isolates and epidemic MRSA (EMRSA)-15 and -16 was carried out by a modification of the protocol previously described.²² The banding patterns were interpreted visually, and the relatedness of the strains was determined as described previously.^22,23 Strains with banding patterns that differed by at least 3 bands were grouped in a different type and assigned using letters of the alphabet (A, B, C, etc). The staphylococcal cassette chromosome mec (SCCmec) types were determined by a multiplex PCR strategy.²⁴

Results

Antibiotic susceptibility of S aureus isolates in Southwestern Nigeria. The antibiotic susceptibility of 200 S aureus isolates in Southwestern Nigeria is described in Table 1. All the isolates (MSSA and MRSA) were susceptible to teicoplanin, vancomycin, fusidic acid, and rifampicin, while less than 3% were resistant to oxacillin, erythromycin, clindamycin, neomycin, and minocycline. About 90% of MSSA isolates were resistant to penicillin, and resistance to sulfonamides and tetracycline was 47.2% and 51.3%, respectively. In addition, MSSA resistance to gentamicin, kanamycin, chloramphenicol, and ciprofloxacin was less than 10%. One MSSA exhibited high-level resistance to mupirocin, and 3 MSSA displayed the inducible macrolide-lincosamide-streptogramin B (MLSB) phenotype.
A total of 28 antibiotypes were identified among the MSSA isolates (Table 2). Overall, 4.6% of MSSA were sensitive to all antibiotics tested, and 5% were sensitive to penicillin but resistant to a number of antibiotics. Resistance only to penicillin; resistance to penicillin, tetracycline, and trimethoprim; resistance to penicillin and tetracycline; and resistance to penicillin and trimethoprim accounted for 25.4%, 20.8%, 14.2%, and 10.7% of the total number of MSSA isolates, respectively. Furthermore, the proportion of multi-drug resistant MSSA was 13.7% (27 of 197 isolates).
Only 3 isolates (obtained from wound samples) were confirmed as MRSA by the detection of the mecA gene. The MRSA isolates were resistant to tetracycline but susceptible to gentamicin, kanamycin, neomycin, chloramphenicol, and mupirocin (Table 1). The inducible MLSB phenotype was detected in 2 MRSA isolates. In addition, the MRSA isolates were grouped into different antibiotypes, and only 2 were multi-resistant (Table 2). The MRSA isolates were grouped into 3 pulsotypes by PFGE. In addition, the banding patterns of 1 MRSA strain obtained in Ibadan, Nigeria (28IDA), differed from EMRSA-15 by 2 to 3 bands, suggesting that they are closely related. SCCmec typing indicated that the MRSA strains belonged to types I, III, and IV (Figure 1).

Discussion

Antimicrobial resistance among nosocomial pathogens is a significant problem in clinical settings that adds to the cost of medical care and the morbidity and mortality of patients.²⁵ The adaptation of S aureus to the modern hospital environment has been marked by the acquisition of drug resistance genes soon after antibiotic introduction.²⁶ In this study, all isolates (MSSA and MRSA) were susceptible to teicoplanin, vancomycin, fusidic acid, and rifampicin. A limitation in this study was using the disk diffusion method to determine the susceptibility of isolates to glycopeptides, which is unreliable due to its low sensitivity.²⁷ However, more than 95% of all isolates studied were susceptible to erythromycin, clindamycin, neomycin, and minocycline.
Studies on antibiotic susceptibility in Nigeria have been unable to classify the susceptibility patterns of MSSA and MRSA isolates based on the detection of the mecA gene. The prevalence of MSSA resistance to sulfonamides (trimethoprim and trimethoprim-sulfamethoxazole) and tetracycline was 47.2% and 51.3%, respectively. This trend was relatively higher compared with surveys on MSSA resistance to these antibiotics in South Korea16 and Poland.²⁸ Data from these surveys are greater in scale compared to the present study; however, it appears that there is a high level of MSSA resistance to sulfonamides and tetracycline in Southwestern Nigeria. These antibacterial agents have wide clinical application, are inexpensive in Nigeria, and are available from diverse sources where they are sold with or without prescription. Trimethoprim-sulfamethoxazole and tetracycline are listed among antibacterial agents that have been rendered ineffective or for which there are serious concerns regarding bacterial resistance in Nigeria.^29,30 Apart from full susceptibility to oxacillin, teicoplanin, vancomycin, rifampicin, and fusidic acid, the proportion of MSSA resistant to erythromycin, clindamycin, neomycin, and minocycline was less than 3%. Hence, these antibacterial agents could also be considered as options for the treatment of MSSA infections in Nigeria.
The prevalence of MRSA in this study (1.5%) was lower than previous studies in Southwestern Nigeria, which ranged from 9% to 50%.^7,31–34 It should be noted, however, that detection of the mecA gene, which is the “gold standard” for determining methicillin resistance, was not investigated in the previous studies. A recent multicenter study in Southwestern Nigeria³⁵ confirmed resistance to methicillin by the detection of the mecA gene by PCR and reported a prevalence of 1.4%, which is in agreement with the present data. In this study, 2 of the 3 MRSA isolates demonstrated intermediate resistance to oxacillin (zone sizes: 12 mm). Although the disk diffusion method using oxacillin and the recently introduced cefoxitin disks are still considered useful, isolates exhibiting intermediate or full resistance to these antibiotics should be confirmed by PCR detection of the mecA gene.
Although the prevalence of MRSA was low in the present study, it is evident that multi-resistant MSSA occurred frequently in Southwestern Nigeria (Table 2). In the hospital environment, the acquisition of the SCCmec (in its various forms) by multi-resistant MSSA could make infection control measures extremely difficult and could have serious consequences. A recent study on multilocus sequence typing (MLST) of S aureus strains in Nigeria indicated that certain major clones of MSSA are extremely successful in Nigeria.³⁵ They include sequence type (ST) 25, 30, 120/121, which has been recognized as internationally well-disseminated clones³⁶ along with the ST8 MSSA clone, which appeared to possess some epidemic potential and had acquired the mecA gene.³⁵ Therefore, strict antibiotic and infection control policies are important factors to be considered in order to forestall the emergence and dissemination of multi-resistant MRSA in Southwestern Nigeria.
Mupirocin resistance is an emerging trend in health institutions worldwide. The origin of high-level mupirocin resistance is still a matter of speculation. Three high-level mupirocin-resistant isolates of S aureus and S epidermidis from Nigeria³⁷ were described in 1956, and a coagulase negative staphylococcal isolate from the UK³⁸ was reported in 1967, before mupirocin was used clinically. High-level mupirocin-resistant strains cannot be eradicated with mupirocin and constitute a serious clinical problem, especially when they are resistant to methicillin.³⁹ One of the MSSA isolates, obtained from a blood culture in a hospital in Ibadan, Southwestern Nigeria, exhibited high-level resistance to mupirocin. This was confirmed by PCR detection of the mupA gene. It is interesting to note that mupirocin is not administered or prescribed in this health facility, and this is the first report of the isolation and molecular confirmation of a mupirocin-resistant MSSA in Southwestern Nigeria. Therefore, the authors suggest that MSSA and MRSA should be routinely tested for in clinical microbiology laboratories in Nigeria to detect resistant isolates early and to facilitate the prompt institution of infection control measures.
Analysis of the DNA banding patterns by PFGE of MRSA strains and worldwide clones (EMRSA-15 and -16) indicated that 1 MRSA strain obtained in Ibadan, Nigeria (28IDA), differed from EMRSA-15 by only 2 to 3 bands, suggesting that they are closely related (Figure 1). The PFGE patterns of this MRSA strain and the B5 variant of EMRSA-15 reported by O’Neill et al⁴⁰ were indistinguishable. EMRSA-15 is among the most prevalent MRSA clones in hospitals in the United Kingdom and has been detected in northern Berlin, Germany, the Czech Republic, Spain, and Kuwait.^40–45 Antibiotic susceptibility patterns of the MRSA strain from Ibadan, Nigeria, and EMRSA-15 were similar. In addition, the strain from Nigeria was resistant to tetracycline. Furthermore, SCCmec typing indicated that the strain belonged to the SCCmec type IV. To the best of the authors’ knowledge, this is the first report of the detection and genetic characterization of a MRSA strain closely related to the worldwide clone EMRSA-15 in Nigeria. Therefore, epidemiological studies on the clonal relationship of MRSA strains in Nigeria with worldwide clones would be useful and important in understanding the global dissemination of this pathogen.

Conclusion

Information from this survey could be valuable for programs and policy decisions to forestall the emergence and spread of antimicrobial resistance of S aureus in Southwestern Nigeria. Continuous surveillance on antibiotic susceptibility of S aureus is essential for the detection of emerging trends and the development of appropriate therapeutic strategies. Surveillance programs on the prevalence and characterization of MRSA would be of great importance in understanding its epidemiology in Nigeria.

Acknowledgments

The authors thank Bongi Sigwebela (University of Zululand, South Africa) for assistance in the acquisition of research articles, A.O. Komolafe, A. Olaosun, and the microbiology laboratory staff of the various health institutions for their valuable contributions and assistance in the collection of the S aureus isolates.