Molecular Investigation On Acquired And Adaptive Colistin Resistance In Salmonella

BackgroundAntimicrobial resistance amongst pathogenic bacteria is a global crucial problem.Till now,very little knowledge has been attained in the therapeutic industry to address this issue.This has directed the medical field to re-introduce the past drugs that were believed to be toxic for clinical use.Especially,the cyclic cationic peptides such as colistin,which is distinctive for multidrug resistant Gram-negative bacteria,have been used as "last resort" antimicrobials.Before the 1980s,this drug was postponed for their neural toxicities;however,new clinical practices and possibly improved manufacturing have made it safer to use.Colistin was previously suggested to primarily attack the Gram-negative bacterial membranes,which lead to membranes disruption.Recent research exploring antimicrobial resistance and the mechanisms of action has provided new perspectives.Historically,colistin resistance was believed to be strictly limited to adaptive intrinsic mutations in bacterial regulation system,including two-component system i.e.PhoP/Q and PmrA/B.Lately,Liu et al.described an upsurge of colistin resistance among commensal E.coli across China.They recognized this gene mainly located in certain plasmids and named it as mobilized colistin resistance(mcr)-1 gene.The gene product,mcr-1,was found to be phosphoethanolamine transferases signifying resistance by modifying the phosphate groups of lipid A in Lipopolysaccharides(LPS).The recent global dissemination of the acquired mobilized colistin resistance(mcr)genes is an urgent public health threat.A)Molecular investigation of the acquired colistin resistance in Salmonella Objectives:An accurate estimation of the global prevalence of mcr genes,their reservoirs especially mcr-carrying Salmonella isolates and an updated knowledge of potential pathways for human transmission are required to implement control,prevention strategies,yet such data are lacking.To address this question,we collected,analyzed global prevalence of mcr-carrying bacteria by using evidence-based data and investigated the colistin-resistant Salmonella isolates from various origins by using whole genome sequencing approach.Methods:Publications from four English(PubMed,Scopus,the Cochrane Database of Systematic Reviews and Web of Science)and two Chinese(CNKI and WANFANG)databases published between 18 November 2015 and 30 December 2018 were identified.In this systematic review and meta-analysis,the prevalence of mcr genes in bacteria isolated from humans,animals,the environment and food products were investigated.A total of 974 publications were identified.202 observational studies were included in the systematic review and 71 in the meta-analysis.Different Salmonella isolates from different serovars isolated from apparently healthy finishing pigs obtained from pig farms and slaughterhouses in addition to healthy and infected humans from different provinces in China have been screened for colistin resistance and mcr genes existence.Further investigations have been conducted on the positive isolates including genomic characterization.Results:1)A meta-analysis study of the global burden of mcr-carrying bacteriamcr genes were reported from more than 47 countries across six continents and the overall average prevalence was 4.7%(0.1-9.3%).China reported the highest number of mcr-positive strains.Pathogenic Escherichia coli(54%),isolated from animals(52%)and harboring an IncI2 plasmid(34%)were the bacteria with highest prevalence of mcr genes.The estimated prevalence of mcr-1 pathogenic E.coli was higher in food-animals than in humans and food products,which suggests a role for the foodborne transmission.This study provides a comprehensive assessment of prevalence of the mcr gene by source,organism,genotype and type of plasmid.2)Prevalence of colistin resistant Salmonella isolates carrying mcr genes2.1)We investigated 337 Salmonella isolates from apparently healthy finishing pigs,which is rarely studied,obtained from pig farms and slaughterhouses in China.The mcr-1 gene was found in four colistin-resistant S.enterica 4,[5],12:i:-isolates.Notably,all four isolates belonged to sequence type 34(ST34)with multidrug resistance phenotype.Further genomic sequencing and antimicrobial resistance characterization confirmed that mcr was responsible for the colistin resistance,and the conjugation assay demonstrated that three of four isolates carried mcr-1 in IncHI2 plasmid.Importantly,mcr-1 and class-1 integron in two strains were found to co-localize with IncHI2 plasmid.By collecting all the mcr-1-carrying Typhimurium and monophasic variant strains across the food chain(farm animals,animal-origin food,and humans),our phylogenomic analysis of available 66 genomes,including four strains in this study,demonstrated an independent phylogenetic cluster of all eight Chinese swine-originated isolates and one human isolate.Together,this study provides direct evidence for clonal and pork-borne transmission of mcr-1 by Salmonella 4,[5],12:i:-ST34 in China and highlighted a domestication pathway by acquisition of additional antimicrobial resistance determinants in Chinese ST34 isolates.2.2)A retrospective study of 287 S.Newport clinical risolates collected during 1997-2018 was undertaken for characterization of antimicrobial-resistant profiles using the micro-dilution assay.We found a recent emergence of colistin resistance in four Chinese clinical isolates,including mcr-1-positive isolates.Importantly,phylogenomic and microbiological investigations indicate multiple independent clonal transmission of colistin-resistant S.Newport isolates of different seafood origins.It highlights potential reservoirs for transmission of colistin resistance and suggests that the global food supply chain may facilitate this dissemination.2.3)Moreover,a retrospective study was conducted to detect the presence of the mcr genes in the large animal outbreak isolates.We report the detection of mcr-9 harboring ESBL-producing Salmonella enterica serovar Newport sequence type 45(ST45)in equine from an outbreak in large animals in the US through a retrospective study.Reports of mcr-carry ESBL-producing and colistin resistant Salmonella in large animals are infrequent and of critical public concern.B)Molecular investigation of the adaptive colistin resistance in SalmonellaObjectives:Known colistin resistance mechanisms include mutational and adaptive resistance have been detected through surveillance and epidemiological studies.The knowledge about the mechanisms of resistance to colistin and the genetic determinants involved in these mechanisms through bacterial evolution cycle remain obscure.Methods:To address these concerns,we did serial passages experimental evolution assay for S.Typhimurium ATCC 14028 under aerobic and anaerobic conditions.Each passage was obtained in 24 hours.Aerobic evolved isolates were conducted to Genome wide association analysis(GWAS)and untargeted metabolomic assays to discover the novel associated genes and potential novel mechanisms.Results:We noticed that the evolved bacteria become resistant to colistin under anaerobic condition faster than under aerobic condition through the serial passages of experimental evolution assay to S.Typhimurium ATCC 14028.Genome wide association analysis(GWAS)and untargeted metabolomic assays were conducted on the aerobic evolved isolates.Due to the limited number of the anaerobic passages,further genomic and metabolomic assays were not conducted.Novel associated genes with colistin resistance were discovered in evolved S:Typhimurium ATCC 14028.These genes are found to carry statistically significant Single Nucleotide Polymorphisms(SNPs)output from GWAS analysis.mhbR,bccG,rfbJ,dgcQ,are also involved in cell membranes synthesis,tufA and fusA might play in protecting bacterial ribosomes during the different kinds of stress.C)Conclusion and implicationThe thesis provides a molecular investigation into the acquired colistin resistance through a comprehensive picture of mcr genes from animal hosts,bacterial species,bacterial genotype,and plasmid types worldwide.Besides,it demonstrated the significant role of the food-chain and/or the environment in mcr gene dissemination and their relationship with other suitable vectors(animal hosts,bacteria species,bacterial genotype,plasmid type),and Co-resistance of mcr-positive pathogen with other antimicrobials that are critical for the treatment of antimicrobial resistant bacterial infections is an increasing concern,both in human or veterinary medicine.In addition,it highlights the importance of the occurrence of IncHI2 plasmids in Salmonella serovars which may act as a vehicle for the mcr gene and multiple antimicrobial-resistant genes during their dissemination through the food chain which emphasizes the requirements for internationally coordinated strategies and continuing surveillance to mitigate mcr-carrying bacteria dissemination.Additionally,it highlights the role of the oxygen tension in the adaptive bacterial colistin resistance and hypothesizes novel mechanisms and newly detected genes which might play a crucial role in adaptive colistin resistance during the bacterial evolution cycle,and these results provided new avenue for understanding of the development of antimicrobial resistance,including acquired and adaptive colistin resistance.