Analysis Of Antibiotic Resistance Of Staphylococcus Aureus Isolated From Foods Of Animal Origin And Mutiplex PCR For Detection Of BlaZ,mecA And Nuc Genes

In recent years,the danger of drug-resistant bacteria in food aroused attention, and researches on the drug-resistance of food-borne pathogens were increasing.Some studies indicated that enterotoxins-producing and drug-resistant Staphylococcus aureus widely existed in food.In particular,a large quantity of antibiotics used in livestock-raising,animal foods were very likely to be the reservoirs of drug-resistant bacteria.To analyze the prevalence and antibiotic resistance of S.aureus in animal food,samples of pork,milk,beef,chicken and eggs were collected from seven places in Sichuan Province,and a multiplex PCR method was set up for rapid detection ofβ-lactam resistance genes,especially targeting methicillin-resistant S.aureus(MRSA).1.Isolation and Identification of Staphylococcus aureusA total of 2560 samples were collected from December 2006 to September 2007. Strains were isolated and identified using conventional methods with reference to GB and manual of biochemical identification of Staphylococcus(TH-16S).118 suspicious strains were initially screened with Baird-Parker plate,then 108 of them were identified as S.aureus by GB according to the characteristics on CHROMagar medium, staining,hemolysis and plasma coagulation results,whereas 113 coagulase positive strains were S.aureus by TH-16S fork index,and only 76 strains were coincidental by the results of 16 biochemical experiments.The highest separation rate of S.aureus was in raw milk(10.54%),followed by pork(7.11%),chicken(4.12%),few egg samples were positive for S.aureus.In all,the biochemical phenotype of those strains were complex and presented differences,so phenotypic analysis might have some defects. The prevalence of S.aureus in samples from various places were different,and compared with other reports,the pollution was low in pork,raw milk and eggs in Sichuan Province.2.Analysis of Antimicrobial Resistance113 coagulase positive S.aureus were tested for susceptibility to 25 kinds of antibiotics using broth micro-dilution method,following the guidelines of National Committee on Clinical Laboratory Standard(NCCLS).The rates of resistance to penicillin G(93.81%),ampicillin(92.92%),trimethoprim(92.92%) and sulfisoxazole (88.5%) were high,the rate of resistance to lincomycin,erythromycin,azithromycin and tetracycline were 41.59%～47.79%,and 21.24%～29.20%to gentamicin, kanamycin and tilmicosin,few strains were resistant to chloramphenicol, danofloxacin and ciprofloxacin(2.65%～7.08%),no strain was resistant to oxacillin, cephazolin,ceftriaxone,ceftiofur,amikacin,doxycycline,enrofloxacin,lomefloxacin, norfloxacin.A total of 47 kinds of antibiotic-resistance spectrum were presented,all the strains had a resistance pattern against at least 2 of the antibiotics,and multi-drug resistance showed mainly to penlcillins,sulfonamides and macrolides.The antimicrobial resistance of S.aureus from animal food in Sichuan showed no obvious difference compared with other reports,and there was no detection of MRSA.The results could be taken as reference for veterinary medicine as well as proof for the safety assessment of drug-resistance S.aureus of food source.In addition,49.56%of the 113 coagulase positive strains showedβ-lactamase activity.68 of multi-drug resistant strains were induced in the presence of increasing concentrations of oxacillin(OXA),8 drug-resistant strains SCI1～SCI8 were obtained when the drug concentration reached 5μg/mL,then three suspected MRSA,SCI3, SCI6 and SCI8,were screened by M-H agar plate with 6μg/mL OXA and 4%NaCl, requested further verification through gene analysis.3.Polymerase Chain Reaction(PCR) Methods and Applications3 pairs of specific primers for S.aureus thermonuclease gene nuc,β-lactamase gene blaZ and methicillin-resistance gene mecA,were designed using Primer 5 according to the sequences registered in GenBank.Through optimization of conditions,a triple-multiplex PCR method for assay onβ-lactam antibiotic resistance was established,the fragments of 229bp(nuc),787bp(blaZ) and 1459bp(mecA) could be simultaneously amplified in one PCR reaction.79 strains were tested by this multiplex PCR.97.47%of the strains were nuc positive,73.42%blaZ positive,and all mecA negative.The concordance of the nuc PCR results with conventional method including thermonuclease and coagulase activity was 97.47%,and 75.95%with biochemical identification.The concordances between blaZ amplification with results ofβ-lactamase activity and susceptibility to penicillins were 62.34%and 77.92%,respectively,and the degree of coincidence of the three results was 50.65%.The results of multiplex PCR would be reference for the rapid detection and analysis of S.aureus and its antibiotic resistance in future.8 OXA induced strains(SCI1～SCI8) were also analysed by multiplex PCR.The original strain SCI6 was nuc and blaZ positive but mecA negative,however 8,10,12,16μg/mL OXA induced strains(SCI6-8,SCI6-10,SCI6-12,SCI6-16)were all nuc and blaZ negative and mecA positive;they might obtain mecA by OXA abduction, and could be served as materials for further studies of the acquisition of mecA gene and the genetic background of MRSA;the 'missing' or 'variation' of nuc and blaZ, and the origin of mecA should be further confirmed.The multiplex PCR results of the other seven induced strains had no change.The nuc,blaZ and mecA PCR products of ATCC33591 and mecA fragment of SCI6-8 strains were sequenced,fragments' size consisted with the size designed by software.The homology of genes and corresponding amino acid sequences were very high.These sequences were registered in GenBank,the numbers were FJ809757, FJ809758,FJ810876 and FJ810877,respectively.