ISOLATION AND MOLECULAR CHARACTERIZATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM POULTRY AND POULTRY FARM WORKERS IN NIGERIA
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- NGN 3000
ABSTRACT
The increasing rate of drug resistance associated with methicillin resistant Staphylococcus aureus is not only a problem in the clinical sector but also in livestock disease treatment and management. Methicillin resistant Staph. aureus is now a leading cause of staphylococcal infections in human and animals. In view of the present serious problem of resistance to antibiotics from Staph. aureus, the present study was undertaken to investigate the incidence of methicillin resistance in Staph. aureus. The study was carried out in Kano State, Nigeria to evaluate the incidence of methicillin resistant Staph aureus from poultry and poultry farm workers. Cloacae and nostril of 1200 poultry birds selected randomly in 12 farms from the three senatorial zones of Kano State and 60 nostrils of poultry farm workers were screened for the presence of Staph aureus using standard microbiological techniques. Antibiotic susceptibility pattern was determined using disc agar diffusion (DAD) method. Vancomycin resistance was determined using vancomycin agar screening method. Molecular studies of 16SrRNA, nuc,mecA and PVL gene were carried out using multiplex PCR, the PCR may permit sufficient sensitivity and specificity for the direct detection of Staph. aureus.Twenty two isolates were tested for Panton Valentine Leukocidin (PVL) using PCR. Ninety eight isolates (8.2%) were confirmed and characterized as Staph aureus, sixty six of the isolates were from broiler and 32 from layers. Cloacae yielded high number of Staph aureus than nostril. The result of antibiotic susceptibility test showed general resistance to β-lactam antibiotics; ampicillin and oxacillin and oxytetracycline at 71.4% each, chloramphenicol (61.2%) and sulfamethaxazole/trimethroprim (51 %). However higher percentage of sensitivity was recorded against vancomycin (74.5 %), AugmentinR and cefoxitin (69.4 %), ciprofloxacin (64.3 %), gentamicin 60.2% and neomycin 54.1%. Thirty percent (30.6 %) of the Staph aureus isolates were phenotypically identified as methicillin resistant using cefoxitin 30 µg., and one from poultry farm workers. Determination of multiple antibiotics resistance index showed that 80 (81.6 %) were resistant to 3 or more antibiotics MARI ≥ 0.3 and 5.1 % had MARI 0.3. Eighty three point three percent of the MRSA (83.3 %) were multidrug resistant. Eighty nine point eight percent (83.3 %) of the total staphylococcal (89.8 %) isolates produced β-lactamase and 19/30(63.3%) phenotypic MRSA were β-lactamase hyper producers. Vancomycin resistance determined using vancomycin screening agar showed that 90% of the MRSA were vancomycin resistant (VRSA). The molecular analysis vii of the isolate using PCR showed that all the isolates were Staph aureus of 800bp. PCR showed a correlation between phenotype and recovery of MRSA and genotypic detection of mecA gene. The prevalence of mecA mediated methicillin resistance in Staph aureus is high as 40.7% of the total MRSA isolate carried mecA gene. The amplified product of Staph aureus mecA gene showed a correlation between the staphylococcal penicillin binding protein (PBP2a) .Sixteen (53.3%) were PBP2a positive. Only one isolate from farm worker had mecA gene. Twenty two isolates were tested for Panton Valentine Leukocidin (PVL) but only 14 (63.36%) were positive. Analysis showed that PVL was not associated with truly community acquired as all PVL positive isolates in this study were from poultry. Further analysis showed that 3 of the seven housekeeping genes (pta,gmk,and yqil) were present. 35 % expressed Spa typing at variable regions. Multilocus sequence typing of 8 isolates selected showed that the main sequence type (ST398) of livestock associated methicillin resistant Staph. aureus (LA-MRSA) was not present in the poultry used in this study. The multidrug resistant nature of most of the isolates, and the high resistance level to especially β-lactam antibiotics is a sign of misuse and overuse of the agents in the environment (poultry farms). These call for rapid and accurate detection of multi drug resistant methicillin resistant Staph. aureus,and drawing up of guidelines for the prompt, effective and appropriate use of antibiotic therapy and for control of CA-MRSA and LA-MRSA.
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