| The antimicrobial resistance of S.aureus and E.coli isolated from different food-related hosts, including pigs, bovines, chicken, fish, food poisoning patients and person of food processing, was detected, and tracing to the source was researched by several kinds of methods-disk diffusion method, PCR-AGE test, PCR-DHPLC test, real-time PCR and MALDI-TOF MS. PCR methods of detecting antimicrobial resistance genes of MRSA for aminoglycoside, erythromycin, tetracycline were established. PCR methods of detecting species identification and class I integron, tetracycline and common antimicrobial resistance genes of E.coli were developed. PCR detection methods of aminoglycoside, erythromycin, tetracycline-resistant genes was developed for MRSA. PCR detection methods of species identification, class I integron, tetracycline and common resistance genes was developed for E.coli. MALDI-TOF MS method was used to research antimicrobial resistance and traceability for S.aureus and E.coli. Theoretical basis was provided in this study for entry and exit quarantine inspection and food safety testing in China.Antimicrobial resistance phenotype of S.aureus and E.coli was tested using disk diffusion testing. The drug resistance results of S.aureus for OXA, CFX, AMP, GEN, KAN, TOB, ERY, TET, CIP, LEV, VAN were obtained. ESBLs strains of E.coli were screened and the drug resistance results of E.coli for TET, CIP, GEN, TOB, CFX, AZT, FEP, IPM were obtained.Antimicrobial resistance of 12 antibiotics for S.aureus and 8 antibiotics for E.coli was tested by disk diffusion method. The results showed that detection rate of drug-resistant and intermediate drug-resistant S.aureus strains of OXA, CFX, AMP, GEN, KAN, TOB, ERY, PMC, TET, CIP, LEV respectively were 50.51%,52.58%,85.57%,40.21%,44.33%,42.27%,76.29%,71.13%,58.76%,59.79%,56.70% in the 97 S.aureus strains, except all VAN susceptibility. There was 29 ESBL strains in all 47 E.coli. The detection rate of drug-resistant and intermediate drug-resistant E.coli strains of TET, CIP, GEN, TOB, CFX, AZT, FEP respectively were 70.21%,20.27%,19.14%,14.89%,25.53%,4.3%,70.21%, except all IPM susceptibility.The PCR-AGE and DHPLC methods of drug resistance genes of MSSA/MRSA for aminoglycosides, erythromycin, tetracycline were studied. Multiplex-PCR detection method of aminoglycoside resistance genes for MRSA/MSSA was established, multiplex-PCR detection method of erythromycin resistance genes for MRSA/MSSA was established, and multiplex-PCR detection method of tetracycline resistance genes for MRSA/MSSA was established, and the results were compared with phenotype. It indicated that these methods developed in this assay had high specificity, sensitivity and practicability. Compared with the resistant genetype and resistant phenotype results of testing practical samples by the established methods, it was above 90% of coincidence rate. So it indicated that it has good practicability for foodborne S.aureus by the established methods in this assay. The specificity and sensitivity of established methods were studied, isolateds from practical samples were tested. The results of PCR test were compared with drug-resistant phenotype, and practicability of methods was verified. The results showed that it was high specificity and sensitivity of resistant genotypes detection method in this assay. Drug resistance gene detection results were compared with resistant phenotype of isolateds from practical samples by established methods, and the coincidence rate were all more than 90%. It indicated that these methods a good practicability for foodborne S.aureus.The detection method of antimicrobial resistance genes, ermA, ermC, aph(3’)-Ⅲa and aacA-aphD genes, was established firstly for foodborne S.aureus in China. The real-time PCR methods of common drug resistance genes of MSSA/MRSA were developed, and the specificity and sensitivity were studied. Test results were were compared with conventional PCR. It indicated that these methods developed in this assay had high specificity, sensitivity and practicability. And this method was more rapid than conventional PCR.Species identification, class I integron, tetracycline, and common drug resistance genes of E.coli were studied. Duplex-PCR of detecting species identification and class I integron genes, duplex-PCR of detecting tetracycline resistance genes, and multiplex-PCR of detecting common drug resistance genes were respectively established. Then the specificity and sensitivity were studied, and isolates from practical samples were tested. The results of PCR test were compared with drug-resistant phenotype, and practicability of methods was verified. The results showed that it was high specificity and sensitivity of resistant genotypes detection method in this assay. Drug resistance gene detection results were compared with resistant phenotype of isolateds from practical samples by established methods. There was effective for identifying E.coli and detecting drug resistance genes, and the negative ESBL strains but hiding blaCTX-M-1 and blaCTX-M-9 genes were tested. So it indicated that it has good practicability for foodborne E.coli by the established methods in this assay.After MALDI-TOF MS datum were added into database, identification results were compared between the original database and the new. In addition, S.aureus and E.coli were tracing analyzed by cluster analysis. It showed that antimicrobial resistance of S.aureus and E.coli had not be confirmed directly, but drug-resistant strains screening.Antimicrobial resistance strains of S.aureus and E.coli were tested and tracing analyzed firstly by Database searches and cluster analysis methods via MALDI-TOF MS in China. The strains of S.aureus and E.coli were tested in this study, and MALDI-TOF MS data of S.aureus were added in the database. The identification result of S.aureus and E.coli were compared between original and new database. The results showed that resistant/sensitive strains could be distinguished, and the accuracy rate of distinguishing MRSA/MSSA could be above 70%. There was a better prompt function for E.coli resistant strain identification, the accuracy rate of distinguishing resistant/sensitive strains was above 70%. Traceability of S.aureus and E.coli were researched by cluster analysis, it indicated that antimicrobial resistance of S.aureus and E.coli could not be directly identified now, but it could be used to screen reseach. |
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