Molecular Subtyping And Virulence Analysis Of Non-O157Shiga Toxin-producing E. Coli And Listeria Monocytogenes Isolates

Non-O157Shiga toxin-producing Escherichia coli （STEC） and Listeria monocytogenesare two important emerging foodborne pathogens that cause human illnesses. In this study,the main virulence factos （stx1, stx2, eae and hlyA and their subtypes）, the serotypes and verocell cytotoxicities （for selected isolates） of359non-O157STEC isolates from food, humanand animals were studied. In the mean time,272isolates were selected from the359STECisolates for PCR Coupled to Electrospray Ionization-Mass Spectrometry （PCR-ESI-MS）analysis; a new plate was developed to identify and subtype them by testing the basecompositions of fragments from two housekeeping genes （mdh and mutS） and five virulencegenes （stx1, stx2, eae, hlyA and aggA）. In addition, the antimicrobial susceptibility andmolecular serogroups of Listeria monocytogenes from retail meats in2008were studied;furthermore, combined with pulsed-field gel electrophoresis （PFGE）, the virulence potentialof them together with extra30isolates from chicken and produce in Maryland from2003were explored by testing the presence of inlC, inlJ and llsX, whether belonging to epidemicclones （EC）, or having full length InlA. Major findings and conclusions were summarized asfollowing:（1） Among the359non-O157STEC isolates,18stx genotypes were observed, and thetop six were stx_1a（61%）, stx_2a（9%）, stx_2dact（8%）, stx_1a+stx_2a（7%）, stx2c（2%） and stx_2a+stx_2dact（2%）. Compared with O157STECs, the percentages of stx2cand multiple alleles were lower.Four eae subtypes eaeβ,eaeε1,eaeγ_1and eaeγ2/θwere observed. Association between eaesubtype and serotype was found and some eae subtypes are flagella associated. Eae positiveisolates and negative isolates differed a lot in their virulence factors in that Shiga toxin type2subtypes in the latter were more diverse than in the former, and stx_2awas the main subtype ineae positive isolates, whereas stx_2dactwas mainly found in eae negative isolates. Isolates withstx_2b, stx_2eand stx_2gshowed lower cell cytotoxicity, whereas isolates with stx_2a, stx2cor stx_2dactshowed higher cell cytotoxicity.（2） The serogroups and virulence genes in isolates from different sources differed a lot.The “top six” non-O157O serogroups O26, O111, O103, O145, O121and O45took up to84% of the STEC serogroups from human. O111, O103and O26encompassed91%of isolatesfrom animals. O serogroups which were other than the top six non-O157accounted for98%of all isolates from food. As for virulence genes, among the99food isolates, the majoritywere Stx2positive （67%）, and21%carried only Stx1, or Stx1and Stx2in combination （11%）.Of those only one isolate carried eae, however almost half （45%） were hlyA positive.Contrary, the majority of human isolates （67%） carried Stx1, and less frequently Stx2（21%）or Stx1+Stx2（12%）, but most were eae （85%）, and hlyA （82%） positive. As for virulencegenotypes, stx_1a+eae+hlyA was prevalent in human （57%）; in food isolates, the genotypeswere more diverse and the most frequently found one was stx_2dact, which was less than18%.（3） The following important serotypes found in food isolates have been reported inhuman illnesses: O113:H21, O91:H21, O104:H21, O22:H8, O45:H2, O73:H18, O113:H21,O116:H21, O121:H19, ONT: H2, ONT: H10and O91:H14. Most of the isolates had stx_2aand/or stx_2dact+hlyA, and some even had vero cell cytotoxicities as high as human isolates.These isolates may have the potential to cause future human diseases and should attach greatimportance.（4） As for PCR-ESI-MS, a total of164well characterized STEC isolates were firstexamined with the assay to build a DNA base composition database. Another panel of108diverse STEC isolates was tested with the established database to evaluate the assay’sidentification capability. Among the108isolates, the assay specificity was100%for three（stx1, eae, and aggA） out of five tested virulence genes, but99%for stx_2and96%for hlyA,respectively. Main stx1/stx2subtypes and multiple alleles of stx1/stx2could be differentiated.The assay successfully identified several clinically significant serotypes, including O91:H14,O103:H25, O145:H28/NM, O113:H21and O104:H4, due to their unique base compositionpatterns of housekeeping genes. Meanwhile, it was able to group isolates with different levelsof pathogenic potential due to the difference in the base compositons of housekeeping genesand virulence genes. The results suggest that this high-throughput method may be useful inclinical and regulatory laboratories for STEC identification, particularly strains with increasedpathogenic potential.（5） Recovery of Listeria spp from about the700raw meat samples was20%, with threedifferent species L. monocytogenes （10%）, L. welshimeri （9%） and L. innocua （2%）. As for L.monocytogenes, the most common molecular serogroup found in the meat samples from2008was1/_2a,3a （33%）, followed by1/_2b,3b,7（22%）,4b,4d,4e （16%）, and4a,4c （2%）. Ingeneral, lineage II isolates （60%） were more commonly found than lineage I isolates （39%）,and only one isolate belonged to lineage III （1%）. Most L.monocytogenes isolates weresusceptible to the antimicrobials tested and low resistance rates to tetracycline （4.5%）, ciprofloxacin（4.5%） and nitrofurantoin （10.5%） were found.61%and85%of isolates wereintermediate resistant to ciprofloxacin and nitrofurantoin respectively. No multiple resistantisolates （resistant to two or more antimicrobials） were found. More resistant isolates werefound in lineage II （9/40） isolates than in lineage I isolates （3/26）.（6） The virulence gene analysis of all97L. monocytogenes isolates including67isolatesfrom retail meats in2008and30isolates from chicken meat and produce in2003showed thatall carried inlC and inlJ except for a lineage III isolate110-1. Most Listeriolysin S （LLS）-carrying isolates （11/12） belonged to lineage I, whereas the remaining one isolate belonged tolineage III. Five4b,4d,4e isolates including two from turkey and three from producebelonged to Epidemic Clone I （ECI）. Four molecular serogroup associated mutation types thatlead to premature stop codons （PMSCs） in inlA were identified. PFGE and inlA sequenceanalysis results were concordant, and different virulence potential within1/_2a,3a and4b,4d,4e isolates were observed. The study revealed that a subset of isolates from meat and producebelonged to ECI, harbored inlC, inlJ and LLS, and produced full length InlA, suggesting thatthey be capable of causing human illness.