Study of genetic determinants to explain antimicrobial resistance in Escherichia coli and salmonella isolated from food animals, meat and humans in Thailand

Abstract

This study comprised 4 parts. Different bacterial strains with different genotypes were used, depending on the experimental design and objectives. Part 1 - Plasmid profile analysis of E. coli and Salmonella enterica isolated from pigs, pork, and humans. This part aimed to characterize the profile of R plasmids in E. coli (n=1,047) and Salmonella (n=816). Sixteen and 11 Inc groups were identified in E. coli (85.3%) and Salmonella (25.7%), of which IncK-F (23.7%) and IncF (46.2%) were predominant. Plasmid replicon was significantly different among sources of isolates and sampling periods but significantly correlated with resistance phenotype (p<0.05). In conclusion, various plasmids are present in E. coli and Salmonella. The findings in this part form a basis for future studies to explore the possible methodology to counteract horizontal transfer of plasmids. Part 2 - Genomic analysis of Salmonella carrying class 1 integrons with dfrA12-aadA2 gene cassette array isolated from food animals, meat and human in Thailand. This part aimed to investigate the genetic characteristics of transferable plasmid carrying class 1 integrons with dfrA12aadA2 cassette array in 15 Salmonella isolates using Oxford Nanopore Sequencing technologies and Illumina platform. Seven MLST types and 6 clades of phylogenetic trees were identified. IncFIB(K), ColpVC, IncFIB, IncHI2, IncHI2A, IncX1, IncY and IncR plasmids were found. All had 28 AMR genes with the prediction to be resistant to various antibiotic groups. All isolates except B82 which was isolated from human, carried class 1 integrons with dfrA12-DUF1010-aadA2 and qacE-sul1 in conserved region. In conclusion, transferable R plasmids play an important role in the wide distribution of class 1 integrons with dfrA12-aadA2 gene array. Part 3 - Molecular basis of the persistence of chloramphenicol (CHP) resistance among E. coli and Salmonella spp. from food animals, meat and human in Thailand. This study aimed to explore the potential mechanisms associated with the persistence of CHP resistance in E. coli (n=106) and Salmonella (n=57). Most E. coli (67.9%) and Salmonella (64.9%) had ≥4-fold CHP MIC decrease in the presence of PAβN. Ampicillin, tetracycline, and streptomycin selective pressure yielded cmlA-carrying Salmonella and E. coli-transconjugants resistant to CHP (MIC 32-512 µg/mL). IncF plasmids were common in cmlA-carrying Salmonella and E. coli transconjugants. The WGS analysis revealed cmlA1-class1 integrons flanked by IS26 and TnAs1 in IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids, and catA flanked by IS1B and TnAs3 in IncFIA(HI1)/HI1B/Q1. In conclusion, the persistence of CHP-resistance was potentially mediated by cross resistance via multidrug efflux systems using proton motif force (pmf) and co-selection of CHP-resistance genes by other antimicrobials. Part 4 - Resistance to widely-used disinfectants and heavy metals and cross resistance to antibiotics in E. coli isolated from pigs, pork and pig carcass. This study aimed to determine the possible cross resistance between disinfectants/heavy metals and antibiotics in E. coli from pigs (n=643), pork (n=111) and pig carcasses (n=110) in Thailand. Exposure to triclosan (TCS), benzalkonium chloride (BKC) and chlorhexidine (CHX) selected for spontaneousresistant mutants exhibited cross resistance to at least one antibiotic. The presence of PAβN restored MICs of CHP and trimethoprim in BKC- and TCS-spontaneous resistant mutants but PAβN, CCCP and reserpine could not restore ciprofloxacin MIC in ciprofloxacin-resistant mutants with no gyrA and parC mutation. In conclusion, the widely used disinfectants and heavy metals serve as non-antibiotic selective pressure for emergence and spread of AMR via cross-resistance involved in pmf-dependent and/or independent mechanisms. Susceptibilities to disinfectants/heavy metals should be routinely monitored. For the overall conclusion, the objectives of this project were successfully achieved. Further studies are suggested for better understanding of AMR and implementing the strategic actions to contain AMR.