Characterization Of Human-derived Non-O157 Shiga Toxin-producing Escherchia Coli Isolates And Genotyping Of The Enterohaemolysin Gene (EhxA)

Shiga toxin-producing Escherichia coli(STEC)refers to a pathotype of E.coli producing one or more types of Shiga toxin(Stx).STEC is a well-known pathogen as a cause of diarrhea,hemorrhagic colitis(HC)and hemolytic uremic syndrome(HUS)with high mortality.Stx is the most critical virulence factor of STEC,besides,other factors such as intimin and enterohemolysin(ehxA)also play a role in the pathogenicity.Although STEC O157:H7 has been regarded as the most predominant and important serotype,sporadic infections or outbreaks caused by non-0157 STEC have been more reported compared to STEC 0157 in many countries in recent years.This study was designed to investigate the prevalence of non-0157 STEC in stools of diarrheal patients and healthy carriers in China,and to analyze the serotypes,virulence genes/genotypes,Shiga toxin-coverting phage by whole genome sequencing and comparative analysis.Twenty-seven non-0157 STEC strains were isolated from diarrheal patients or healthy carriers from 2010 through 2014 in five regions(sui county of Henan province,Zigong of Sichuan province,Yushu of Qinghai province,Longgang of Shenzhen,and Shanghai).The isolation rate of non-0157 STEC in diarrheal patients was about 1.3%,while only about 0.2%in healthy carriers.Twenty-seven non-0157 STEC strains were typed into 16 O:H serotypes,the predominant serotypes was 0130:H8,including 6 isolates;followed by 026:H11 and 0117:H8 serotypes including 3 isolates each.O91:H14 and 0107:H7 serotypes include 2 isolates each,the remaining 11 serotypes(0111:H8,0112;H8,021:H25,084:H2,05:HNT,020:H30,ONT:H30,0104;H7,048:H21,0149:H10 and 043:H2)comprise only one isolates each.The 27 strains were typed into 16 different sequence types(STs),the most predominat ST type was ST13,including 9 isolates,followed by ST21 and ST504 types which include 3 and 2 isolates,respectively;the other 13 isolates belong to different ST types.Eleven isolates carry stx1a subtype,12 isolates carry stx1c subtypes,1 isolate carrys stx2d subtype,3 isolates carry stx2e subtype,and 1 isolate carrys both stx1a and stx2b subtypes.The prevalence of other virulence genes eae.efal,saa,paa,toxB,astA and ehxA were 18.5%(5),18.5%(5),29.6%(8),22.2%(6),11.1%(3),11.1%(3)and 25.9%(7),respectively.Stx induction and cytotoxicity assay showed that the two stx2e-positive isolates STEC413 and STEC509 had lower cytotoxicity compared to STEC 0157:H7 strain Xuzhou21.Stx phage carried by STEC413 and STEC509 were 32133 bp and 61142 bp in size,respectively;C+G%were 48%and 49%;the insertion sites were opmW and yecE,respectively;and the number of CDS predicted was 58 and 105,respectively.By comparison with the Stx2e phage of the pig-derived reference strain S1191,the Stx2e phage of STEC413 had a similarity of up to 50.0%,and possessed the same integrase and cleavase,but the insertion site and the regulatory gene Q were different.The Stx2e phage of STEC509 is very specific and there is no similar phage sequence reported,indicating that the Stx2e phage genome is highly diverse.Comparative genomic analysis showed that STEC413 closely related to commensal E.coli;while STEC509 closely grouped with non-0157 STEC strains.Our study showed that Stx2e phage can be integrated into different genetic background strains.In addition to the bacteriophage-encoded Shiga toxin and LEE island-encoded intimin,enterohaemolysin,which is located on a large toxic plasmids plays an important role in pathogenicity in STEC 0157:H7.Of 434 non-0157 STECs,138(31.8%)ehxA-positive isolates were detected,including 10 human-derived isolates(1 healthy carrier,9 diarrheal patients),108 animal-derived isolates,and 20 food-derived isolates.The full-length ehxA gene was obtained by PCR amplification and 36 unique ehxA sequences were identified.Based on ehxA sequence analysis,three phylogenetic ehxA groups(? ?,and ?)were determined.Strains of diarrheal patients belonged to Group ?,and most of them carried the eae gene,while animals(97.1%)and food(80%)strains are mostly distributed in Group ?.In summary,this study depicted the occurrence and molecular feactures of human non-0157 STEC strains in China.The stx2e subtypes strains that rarely reported in humans were further analysed by whole genome sequencing and comparative analysis.The ehxA gene typing based on sequence and phylogenetic analysis was proposed,which may predict the pathogenetic potential of non-0157 STEC strains.