Whole-genome Sequencing Of A Multi-drug Resistant Listeria Monocytogenes ST477 And Comparative Genomic Analysis

Listeria monocytogenes is an important foodborne opportunistic pathogen,which can cause listeriosis in humans and animals.Clinical symptoms include septicemia,meningitis,spontaneous abortion,stillbirth etc.Clonal complex 9(CC9)is one of the three predominant clones distributed in foods.However,little is known about the evolutionary relationship and detail genomic traits of this food-associated clone.In this study,we sequenced and constructed the whole genome sequence of a multi-drug resistant L.monocytogenes NH1 isolated from a frozen food sample in China.Comparative genome analysis was performed to investigate phylogenetic relationship of NH1 and potential pathogenicity of different strains.In addition,the genetic features of CC9 isolates were uncovered based on genetic composition,phylogenetic evolution,single nucleotide polymorphism(SNP),and accessory genes.The results are as follows:1.The genomic feature of L.monocytogenes NH1 contained one circular chromosome of 3 002 491 bp and a GC content of 37.92%.A total of 2 995 open reading frame(ORF),a CRISPR system,and 4 genomic islands were identified.2.Mutilocus sequence typing(MLST)analysis showed that the sequence type(ST)of NH1 was 477,which belong to CC9.Phylogenetic analysis based on single-copy orthologs of 59 L.monocytogenes genome sequences showed that ST477 isolate had a close evolutionary relationship with three other ST9 isolates from Canada.Virulence factors analysis showed that Listeria pathogenicity island LIPI-1 was highly conserved,LIPI-3 and the LIPI-4 islands were almost exclusively detected within lineage I.The number of internalins was different in the examined genomes,ranging from two to nine.All CC9 isolates harbored inl and vip genes,which play important roles in host cell invasion and virulent phenotypes in animals.3.Genome alignment of seven CC9 isolates indicated that the ST477 isolate and the three Canadian ST9 isolates show high genomic similarity.Regions of difference contain genes that encode hypothetical proteins,metabolism enzymes,and phagerelated proteins.MLST evolutionary analysis of CC9 strains showed that the ST477 isolate from Chinese food had a close evolutionary relationship with two clinical ST477 isolates.Based on read mapping of each of the CC9 isolates to the ST477 isolate,SNP analysis showed that three Canadian ST9 isolates contained 60 non-synonymous substitutions,these substitutions occurred in important functional genes smc,ftsA,essC and other genes encoding cell surface proteins.According to the clustering analysis of the SNP system,we found that SNPs had no significant relationship with the sequence type and country of strains,but had relationship with the host.Analysis of gene inlA indicated the nucleotide homology of this gene belonging to seven CC9 clones was 29.8%-100% and the ST477 and three Canadian ST9 shared a common subtype(PMSC type 4).4.ST477 genome harbored multiple antibiotic resistance genes and these genes were identical to those found in CC9 isolates.Tn554-like transposon encodes three consecutive transposase genes(tnpABC),five arsenate resistance genes and four genes encoding hypothetical proteins with unknown function,which was exclusively present in CC9 clonal gemomes.CC9 strains contained a same CRISPR system,which was not associated with any cas genes.The ST477 isolate contained four prophages,and the insertion sites included tRNA and comK genes.