Identification And Characterization Of Escherichia Coli Strains Producing A Novel Shiga Toxin 2k Subtype

Shiga toxin-producing Eschrichia coli(STEC)strains are significant enteric foodborne pathogens that are associated with illnesses ranging from mild diarrhea to hemorrhagic colitis(HC)and life-threatening hemolytic uremic syndrome(HUS)complications in humans.Ruminants,especially cattle and goat,are the dominant reservoir of STEC.STEC is mainly transmitted through faecal-oral route.It has been estimated that there are more than 470 STEC serotypes,among which O1 57:H7 serotype is usually associated with more severe clinical outcomes.However,sporadic infections or outbreaks caused by non-O157 STEC have been frequently reported in some countries in recent years.Shiga toxin(Stx),including Shiga toxin 1(Stxl)and Shiga toxin 2(Stx2),is considered to be the primary virulence factor of STEC.Three Stxl and nine Stx2 subtypes have been reported in E.coli.Different Stx subtypes have been reported to vary in receptor preference and toxin potency.At present,Stx1/Stx2 subtyping is mainly based on the specific PCR method.In our previous study,we found that a stx2-positive strain,STEC388,could not be typed by the specific PCR methods.A phylogenetic tree constructed with the amino acid sequences of different Stx2 subtypes,showed that STEC388 formed a separate branch,suggesting the strain contained a novel Stx2 subtype and we designed it as Stx2k.The sequences identity between Stx2k and Stx2a-Stx2i ranged from 62.5%to 96.3%in nucleotide level,and from 72.7%to 97.0%in amino acid level,respectively.We established a Stx2k-specific PCR method,and screened 787 non-O157 STEC strains collected from April 2009 to November 2017.Eight additionalStx2k-STEC strains were identified.The nine Stx2k-STEC strains showed different Stx2 expression levels and cytotoxicity after MMC induction,which was lower compared to STEC 0157:H7 strain Xuzhou21.The complete genome size of the nine stx2k-containing strains varied from 4,857,389 bp to 5,326,462 bp,with an average genomic GC content range from 51.73%to 51.84%.The CDSs ranged from 4,753 to 5,291.Whole-genome sequencing analysis revealed that the nine stx2k-containing strains are genetically diverse.Seven rare serotypes,i.e.,O8:H19,048:H21,O100:H19,O159:H16,ONT:H30,O22:H8,and O93:H28,were identified among nine strains,which were classified into seven different sequence types(ST),i.e.,ST162,ST155,ST1611,ST3630,ST993,ST446,and ST403.The size of nine Stx2k prophages ranged from 28,694 bp to 54,005 bp,with predicted CDSs ranging from 53 to 86.Three Stx2k prophages were integrated into the coding sequence of dusA,three were integrated into the yccA gene,two were inserted in zur gene and one was inserted in Ser-tRNA gene.The high-resolution phylogenetic analyses indicated that stx2k-containing strains formed two major clusters.Seven strains clustered together with strains belonging to STEC,ETEC,and STEC/ETEC hybrid pathotypes.Two strains clustered closely with STEC/ETEC hybrid strains,implying Stx2k phage could integrate into different Escherichia coli strains.Stx alone is insufficient to cause severe illness,intimin also plays a critical role in intestinal colonization,which is encoded by the eae gene that resides on the locus of enterocyte effacement (LEE)pathogenicity island.There are limited studies about eae genes in non-O157 STEC.In this study,we investigated the prevalence of eae gene and analyzed eae subtypes and polymorphisms in non-O157 STEC strains.Among 735 non-O157 STEC strains,eae was present in 70(9.5%)strains isolated from cattle(n=41),yak(n=2),raw beef(n=3),raw mutton(n=1),and diarrheal patients(n=9).Fourteen eae-positive strains were identified from unknown sources.62 eae gene sequences were obtained and 18 unique eae sequences were identified.Seven new eae genotypes was identified in this study when compared with all available eae sequences in the Genbank database.The nucleotide sequences identity among the 18 unique eae sequences ranged from 86.01%to 99.97%based on pairwise comparisons.Five eae subtypes,namely,?1,?1,?1,?3 and ?,were assigned based on phylogenic analysis.We found that the eae subtype/genotype is related to the serotype and host.Serotypes of O26:H11,O103:H2,and O111:H8 were associated with specific eae subtypes,corresponding to ?1,?1 and ?,respectively.Most STEC strains from diarrhea patients(7/9,77.8%)carried the eae-?1 subtype,while most cattle STEC strains(23/26,88.5%)harbored the eae-?3 subtype.In summary,this study depicted the discovery of a novel Shiga toxin 2 subtype,designated as Stx2k,from E.coli strains circulating in China.The study enlarged the pool of Stx subtypes,contributed to a better understanding of plasticity of Stx-converting phages.The prevalence and genetic diversity of eae gene in non-O157 STEC strains from different sources in China was also investigated.