Screening And Characterization Of Antibiotic-resistant Plasmids In Salmonella

Salmonella, as an important foodborne pathogen, widely distributed, has serious threat to public health and food safety. Due to the prolonged and extensive use of antibiotics, the drug resistance of Salmonella is more and more serious, new drug-resistant strains are emerging. Plasmid, as a selfreplicated movable element, is widespread in Salmonella isolates, and participating in horizontal transfer of antibiotic-resistant genes. Therefore, it is significant to understand the distribution of antibiotic-resistant plasmids in Salmonella, and explore the relationship between the plasmid types and the antibiotic-resistance phenotypes to prevent and control the outbreak of antibiotic-resistant Salmonella.According to the molecular basis of serovars, O antigen(rfb gene cluster), H1 antigen(fliC gene) and H2 antigen(fljB gene), PCR assays were conducted, combining with DNA sequencing to determine the serovars of 78 Salmonella isolates. Consequently, these isolates were detected into 21 serovars, mainly representing S. Typhimurium(25/78, 32.1%) and S. Enteritidis(12/78, 15.4%). The accuracy was as high as 98.7%(77/78) with the only one exception that happened to the false positive result of S. Typhi H2 antigen; moreover, the self-curing Salmonella SJTUF10023 was identified as S. Typhimurium.A total of 24 sequence types(STs) were found, ST34 was the dominant sequence type, accounting for 23.1%(18/78) of all isolates. MLST was used to predict different serovars accurately, and it turned out that each clustering branch was substantially corresponded with one single serovar(75/78, 96.2%). However, three exceptions were found in one S. Agona, one S. Aberdeen and one S. Stanley. According to the antibiotic-resistance phenotypes and antibiotic-resistant gene carryings of these isolates obtained in previous work, and combining with MLST typing results, it is found that more than half of the blaOXA-1 gene carriers were ST34(18/25, 72.0%). Since ST34 is mainly representing the serovar of S. Typhimurium, the antibioticresistance of S. Typhimurium may be more serious than other serovars. The distribution of antibiotic-resistance phenotypes and antibiotic-resistant genes in 78 Salmonella isolates is diversed, indicating that no significant correlation among the MLST types, antibiotic-resistance phenotypes and antibiotic-resistant genes in Salmonella.The incompatibility groups(Inc) of 78 Salmonella isolatess were typed by 5 multiplex PCRs and 3 single PCRs, with total 18 pairs of primers. It turned out that 74 isolates were carrying common plasmid incompatibility groups. The types of these plasmids were comprising of nine plasmid incompatibility groups(Inc P, Inc HI2, Inc N, Inc A/C, Inc FIIS, Inc FIA, Inc FIB, Inc FIC, Inc I1). Among them, the Inc P group and Inc HI2 group were the largest group. Combining with MLST typing results, it was found that all ST34 isolates were carrying Inc HI2 plasmids with the absence of Inc F plasmids(including Inc FIIS, Inc FIC, Inc FIA and Inc FIB); All the ST11 isolates did not carry Inc HI2 plasmids.Based on the carrying of antibiotic-resistant genes and the typing of plasmids, it was found that all the isolates which carrying blaCMY-2 gene contained Inc I1 plasmids and Inc HI2 plasmids; the isolates which carrying qnrA gene basically contained Inc A/C plasmids(4/5, 80.0%); 88.0% of the isolates which carrying blaOXA-1 gene contained Inc HI2 plasmids(22/25); 12.0% of the isolates which carrying blaOXA-1 gene contained Inc P plasmids(3/25), and 12.0% of the isolates which carrying blaOXA-1 gene contained Inc I1 plasmids(3/25); the plasmid types contained by the isolates which carrying blaTEM-1 gene were complex, including Inc HI2, Inc P, Inc I1 and Inc A/C plasmids.By combining the antibiotic-resistance phenotypes with the plasmid incompatibility groups of 32 antibiotic-resistant Salmonella isolates, it was found that all of them contained a total of 5 plasmid families(Inc HI2, Inc P, Inc F family, Inc I1 and Inc A/C). Among them, Inc HI2 were the most common plasmid group(19/32, 59.4%). In addition, all the isolates carrying Inc N plasmids were antibiotic-susceptible(20/20, 100%), as well as the majority of Inc P plasmids carrying isolates(39/45, 86.7%).The plasmid elimination assay was conducted with 32 antibioticresistant Salmonella isolates in 5% SDS + LB broth at 44.5oC, 48 h, and consequently, 15 isolates were changed from antibiotic-resistant to antibiotic-susceptible isolates, and the elimination efficiency was 46.9%(15/32). Later, the plasmids of 15 antibiotic-resistant mutants were extracted, and the plasmid profiles were significantly changed after the plasmid elimination assay. Inc HI2, Inc I1 and Inc F plasmids had diverse losses(the elimination rate was 87.5%, 100%, and 100%, respectively). Accordingly, the antibiotic-resistant genes acc(6’)-ib-cr, blaOXA-1, bla TEM-1, blaCMY-2 and qnrA also had diverse losses(the elimination rate was 42.9%, 80.0%, 83.3%, 100%, and 100%, respectively).Combing the changes of antibiotic-resistance phenotypes, antibioticresistant genes and plasmid incompatibility groups after the plasmid elimination, it was shown that the lost plasmids were mainly Inc HI2 group, and the β-lactamases antibiotic-resistant genes(blaOXA-1, blaTEM-1 and blaCMY-2) were lost accordingly with the loss of Inc HI2 plasmids, as well as the antibiotic-resistance phenotypes. The β-lactamases antibiotic-resistant genes were lost accordingly with the loss of Inc I1 plasmids. The antibioticresistant genes of the isolates carrying Inc F plasmid family were lost. Inc P plasmids were not eliminated in 5 isolates, however, the antibiotic-resistance phenotype and antibiotic-resistant genes were somehow lost. Therefore, the horizontal transfer of antibiotic-resistant genes in Salmonella was mainly related to Inc HI2 plasmids, but not Inc P plasmids.In this study, 78 Salmonella isolates from different sources were subtyped by serotyping, MLST typing to understanding the relationships among the antibiotic-resistant genes, serovars and ST types. Moreover, plasmid typing by using PCR-based replicon amplification was taken and then a plasmid elimination assay was used to locate the antibiotic-resistant genes in antibiotic-resistant plasmids in these Salmonella isolates, and to determine the prevalence of Salmonella with different antibiotic-resistant plasmid types and antibiotic-resistant genes, which could provide support date for monitoring the horizontal transfer of Salmonella antibiotic-resistant genes.