Study On The Predominant Serotype And Antimicrobial Resistance Of Pathogenic Salmonella From Chickens In Guangxi Province

Salmonella, one of the important bacteria, can cause pullorum disease, gallinarum disease and fowl typhoid to chickens, characterized with horizontal and vertical transmission, and cause serious economic losses to the poultry industry. More than one billion chickens were bred in Guangxi each year, so it is very importmant to study the predominant serotypes and antimicrobial resistence of currently pathogenic Salmonella from chickens.1. Establishment and application of a multiple PCR assay for detection of pathogenic Chicken SalmonellaTo establish a rapid identification method for detection of pathogenic chicken salmonella, a multiple PCR assay was established. In this assay, three pairs of primers were designed to specifically amplify 285 bp of invA gene from pathogenic Salmonella,600 bp of fliC gene from chicken Salmonella,507 bp of spvR gene from virulence plasmid of Salmonella, respectively. The assay could only react with pathogenic chichen Salmonella, but not with E. coli, Pasteurella multocida, Sh. dysenteriae and Proteus vulgaris. The detection limit of the method was as little as 4.0×10~2 CFU/mL of Salmonella pullorum from cell culture and 1.67×10~3 copies/?L of recombinant plasmids. The established assay were successfully used to detect 310 clinical samples and 34 samples were positive for invA gene, of which 22 samples were positive for invA and spvR gene,2 samples were positive for invA and fliC genes, while 12 samples were positive for invA, fliC and spvR genes. The results showed that the multiple PCR assay could be used for differential detection and epidemiological investigation of pathogenic Salmonella from chichens.2. Identification, serotype distribution and antimicrobial susceptibility of pathogenic Salmonella isolated from chickensTo investigate the situations of predominant strain and antimicrobial resistance of pathogenic Salmonella from chickens in Guangxi province, Salmonella was pre-enriched and isolated from tissues of clinical suspected chickens, and the Salmonella isolates were identified by biochemical test using ID 32E System for the identification of Enterobacteriaceae of VITEK System ATB Expression, determined serotypes by slid agglutination test, analyzed antimicrobial susceptibility by ATB VET Susceptibility Test Strip of enterobacteriaceae antimicrobial drugs. The results showed that thirty-four Salmonella strains were obtained from 310 clinical samples, of which 1 strain belonged to A serogroup,1 to C2 serogroup,14 to B serogroup,14 to D serogroup and 3 to untyped serogroup, and S. typhimurium of B serogroup and S. pullorum of D serogroup were the predominant serotypes. All Salmonella isolates were sensitive to spectinomycine and apramycine, and the resistance rates to chloramphenicol, kanamycine and gentamicine were less than 10%, and the resistance rates to amoxic/clavsre, streptomycine, flumequine, oxolinique, sulfamethizol, tetracycline and nitrofurantoine ranged between 50% and 89%, while the resistance rates to penicilline, oxacilline, fiisidique, rifampicine and metronidazole reached to 100%. The results indicated that the serotype distribution of pathogenic Salmonella from chickens exhibited regional characteristics, and S. typhimurium and S. pullorum were the predominant serotypes in Guangxi province, and antimicrobial resistance of Salmonella isolates was very serious which should be highly concerned.3. Analysis of antimicrobial resistance, resistance genes and serogroup of Salmonella isolated from chickensTo investigate the relationship among antimicrobial resistance, resistance genes and serogroup,29 pathogenic Salmonella isolates from chickens, belonging to B and D serogroup by slid agglutination test, were tested for the antimicrobial resistance against 20 antimicrobials by ATBVET method and were detected for 19 relevant drug-resistant genes by PCR method. The results showed that the resistant rate over 50% of all the 29 isolates was most frequently found to penicilline and oxacilline (100%), followed by to sulfamethizol (82.76%), streptomycine (75.86 %), oxolinique (75.86%), flumequine (65.52%), enrofloxacine (55.17 %), tetracycline (55.17%) and amoxicilline (51.72%), while all the 29 isolates were susceptible to spectinomycine, gentamicine, apramycine, chloramphenicol and polymyxin E. In addition, all the 29 isolates were resistant to at least 6 antimicrobials, and 6-7 multidrug resistance was the most common (51.73%), even 14 antimicrobials (3.45%). Of all the 19 resistance genes detected,14 resistance genes could be detected, and the positive rate over 40%was most frequently carried with blaTEM (100%), followed by with aadA1 (62.07%), Sul2 (62.07%), Sul3 (51.72%), tetA (48.28%), qnrA (44.83%), while blaPSE, aadA2, tetG, tetM and tetX could not be detected. At least 2 resistance genes were detected in all the 29 isolates, and most isolates harboured 4-6 resistance genes (79.31%), even 10 genes (3.45%). There was no obvious difference of antimicrobial resistance and resistance genes between B and D serogroup. The results suggested that antimicrobial resistance and multidrug resistance of pathogenic Salmonella isolated from chickens in Guangxi province were very common, and the antimicrobial resistant phenotype was positively correlated with resistance genes, while no significant correlation with serogroup.4. Establishment and application of TaqMan-MGB real-time PCR for detection of blaNDM-1 gene of superbugIn this study, a TanMan-MGB real-time PCR assay was established for rapid detection of blaNDM-1 gene which codes New Delhi metallo-?-lactamase-1 (NDM-1) of superbug. One pair of primers and one TanMan-MGB probe were designed according to the sequence of blaNDM-1 gene, and a recombinant plasmid harbouring blaNDM-1 gene was constructed and used as the positive control. After optimizing the reaction conditions, a real-time PCR based on TanMan-MGB probe for detection of blaNDM-1 gene was established. The results showed that the assay was specific and had no cross-reaction with four standard bacterial strains, i.e. S. pollorum, E. coli, P. multocida and S. typhimurium. It was also sensitive and the detection limit was 2.59 copies/?L. Furthermore, it had high reproducibility and the coefficient of variation was less than 1.5%. Then, the established assay was used to detect blaNDM-1 gene of 34 pathogenic Salmonella isolates from chickens and no isolate was found to be positive for blaNDM-1 gene. The result indicated that the established real-time PCR assay was highly specific, sensitive and reproducible and could be used to monitor the superbug harbouring blaNDM-1 gene.In conclusion, a multiplex PCR based on invA, fliC and spvR genes for differential detection of pathogenic salmenalla from chickens was established. The serotype distribution of pathogenic Salmonella from chickens exhibited regional characteristics, and S. typhimurium and S. pullorum were the predominant serotypes in Guangxi province. Antimicrobial resistance and multidrug resistance of Salmonella isolates were very common and serious, and the antimicrobial resistant phenotype was positively correlated with resistance genes, while no significant correlation with serogroup. One TanMan-MGB real-time PCR assay was established for rapid detection of blaNDM-1 gene which codes New Delhi metallo-?-lactamase-1 (NDM-1) of superbug.