Multidrug-resistant Staphylococcus aureus gut as an important reservoir of antibiotic-resistant S. aureus strains (7,8). Most studies on S. aureus have so far been conduct- ed on samples from the nose and throat but only a very few detailed studies on its colonization of the gut have been reported. Thus, there is a need for more studies on S. aureus from the gut, as an important reservoir of multiple antibiotic-resistant bacterial strains, especially in developing countries where the control of antibiotic use is inadequate. We report here the prevalence of multidrug-resist- ant faecal S. aureus isolates from healthy inhabit- ants in Amassoma, Nigeria. MATERIALS AND METHODS Study subjects In total, 120 subjects comprising an equal number of villagers and students of the Niger Delta Univer- sity, Wilberforce Island, Amassoma, Bayelsa state, Nigeria, were randomly recruited into the study for three months from March 2009. Amassoma is a very small village in Bayelsa state, south-southern Nigeria, where the new growing state university ex- ists. Trading, farming of crops, and fishing are the occupations of the villagers. Willingness of the subjects to participate in the study was a strong criterion for the study. The vol- unteers gave informed consents by providing de- mographic data and a completed questionnaire on the evaluation of their previous use of antibiotics. They were not on any antibiotic for at least two weeks at the time of the sampling nor had been ad- mitted to any hospitals in the last one year before the survey. They were aged 15-35 years. Sampling and isolation of S. aureus Sterile cotton swabs were given to the volunteers to deep into their freshly-produced faeces and re- turn into the swab’s case. These stool samples were collected from the volunteers within 24 hours and transported within an hour to the laboratory in iced packs, where these were inoculated directly onto prepared sterilized mannitol salt agar (MSA) plates (Oxoid, UK) and streaked before incubated at 37 oC for 24 hours. Identification of S. aureus S. aureus was identified and differentiated from re- lated organisms based on the colony morphology on the MSA plates. Two discrete yellow colonies on each of the MSA plates were subcultured on sterile nutrient agar (Oxoid, UK) slants aseptically and incubated at 37 oC for 24 hours. The prolifer- ated organisms were characterized by Gram stain- Volume 29 | Number 5 | October 2011 Onanuga A and Temedie TC. ing, productions of catalase, DNase, and coagulase (using human plasma). The organisms that were Gram-positive clustered-shaped cocci, positive to catalase, DNase and coagualse tests were confirmed as S. aureus. These were stored on slant nutrient agar at 4 oC and were used for antibiotic-suscepti- bility testing. Antibiotic-susceptibility testing Agar diffusion method (zone of inhibition measurement) Each isolate of S. aureus was standardized using colo- ny suspension method, and the strain’s suspen- sion was matched with 0.5 McFarland standards to give a resultant concentration of 1.5x108 cfu/mL (colony-forming unit per millilitre) (9). The suscep- tibility testing of commonly-used antibiotics was determined using the modified Kirby-Bauer diffu- sion technique by swabbing each of the Mueller– Hinton agar (Oxoids U.K) plates with the resultant saline suspension of each strain, and the following six antibiotic discs from Oxoids, UK, were placed on the plate after 20 minutes of inoculation: cefoxi- tin 30 μg (as a substitute for methicillin) (9), gen- tamicin 10 μg, ofloxacin 5 μg, ciprofloxacin 5 μg, doxycycline 30 μg, and trimethoprim/sulphame- thoxazole (co-trimoxazole) 1.25/23.75 μg (10). The plates were then allowed to stand for at least 30 minutes before incubated at 30 °C for 24 hours to favour the growth of any methicillin-resistant strains (11). The diameter of the zone of inhibition produced by each antibiotic disc after incubation was measured and interpreted using the zone dia- meter interpretative standards of the Clinical and Laboratory Standard Institute (CLSI) (9). Agar dilution method (MIC test) Standardized solutions of six other commonly-used antibiotics—vancomycin (Novaplus, USA), ampi- cillin (Merck, Germany), erythromycin (Merck, Germany), chloramphenicol (Merck, Germany), amoxicillin-clavulanicacid(augmention)(Glaxo- wellcome, UK), and cefuroxime (Glaxowellcome, UK)—were aseptically prepared. These solutions were used for preparing Mueller-Hinton agar plates of breakpoints of varying CLSI minimum inhibi- tory concentrations (MICs): vancomycin 2-12 μg/ mL, cefuroxime 8 μg/mL, ampicillin 0.25 μg/mL, erythromycin 0.5 μg/mL, chloramphenicol 8 μg/ mL, and amoxicillin-clavulanic acid (augmentin) 4/2 μg/mL. The plates were spot-inoculated with each strain’s standardized suspension and incubat- ed at 30 oC for 24 hours (11). Strains that showed growth on each of the antibiotic agar plates were regarded as resistant to the antibiotic using the CLSI breakpoint standards (9). 447