Molecular Characteristics Of Key Risk Factors Of Shiga Toxin-producing Escherichia Coli (STEC) From Retail Beef And Optimization Of Detection Method

Shiga toxin-producing Escherichia coli（STEC）is one of the main cause of foodborne disease worldwide.Diarrhea,blood in the stool,hemolytic anemia,and thrombocytopenia are common symptoms of STEC infection,which can lead to hemorrhagic colitis（HC）,hemolytic uremic syndrome（HUS）,and even acute renal failure and death in severe cases.In view of the severity condition of STEC hazards,countries and organizations have formulated relevant standard inspection methods and product limit standards.However,due to the deficient non-O157 STEC standard inspection methods in China,it is difficult to carry out relevant national monitoring and risk assessment work,resulting in a lack of effective and standardized STEC pollution data.About 50%of the outbreaks caused by STEC are attributed to beef.In order to quickly obtain the pollution level of STEC and clarify its related molecular characteristics of key risk factors,retail beef samples collected from eight provinces and cities were sequenced.And the serotype,virulence,drug resistance,molecular typing and phylogenetic characteristics of the STEC strains were analyzed in this study.On that basis,the comparison,optimization and verification of existing international standard methods were carried out,and STEC detection methods were developed and applied in relevant national standard methods.The main research contents and results are as follows:（1）STEC in retail beef samples from different regions were isolated and identified to study the distribution characteristics.A total of 1062 beef samples（808 beef and 254 ground beef）were randomly collected from wet markets（n=890）and supermarkets（n=172）in eight provinces and cities（including Hebei province,Hubei province,Jiangxi province,Shandong province,Shanxi province,shanghai City,Hunan province and Sichuan province）between September 2018 and May2019.Realtime-PCR data from the enrichment broths showed that 50.4%of the samples（535/1062）were stx positive.In total,82 STEC isolated from 79 samples（14.8%,79/535）,including 76 from wet markets and three from supermarkets（P<0.01）,were selected for further study.In addition,the sources of STEC were diverse.（2）Analysis of the characteristics of key risk factors was based on the serotype distribution,virulence,drug resistance,molecular typing and phylogenetic characteristics distribution.（1）Among the 82 STEC isolates,28 distinct serotypes were identified,with non-O157 STEC strains（85.37%,70/82）and O157 STEC strains（14.63%,12/82）.The difference between O157 and non-O157 was statistically significant（P<0.05）.O157:H7 and O26:H11 serotype strains showed diversities in sources.（2）stx1 was identified in 50 isolates,including stx_1a（n=41）and stx_1c（n=9）.On the other hand,stx2 was identified in 53 isolates,including stx_2a（n=8）,stx_2b（n=10）,stx_2c（n=13）,stx_2d（n=11）,stx_2e（n=7）and stx_2g（n=4）.Both stx1 and stx2 was identified in 21 isolates,including stx_1a&stx_2a（n=8）,stx_1a&stx_2c（n=1）,stx_1c&stx_2b（n=9）and stx_1c&stx_2d（n=3）.Among the 36 eae positive isolates,the major serotypes were O157:H7（33.3%,12/36）,O26:H11（33.3%,12/36）and O111:H8（22.2%,8/36）.They all belonged to the―Top seven‖STEC.（3）All STEC isolates were tested for antibiotic resistance.Broth dilution method was used to determine the minimum inhibitory concentration（MIC）of STEC isolates against 14 kinds of antibiotics.Result showed that all isolates were susceptible to imipenem.Among the 82 STEC isolates,56 were susceptible to all the tested antimicrobials.A total of 15 resistance profiles were identified among the 26 resistance isolates,and 22 isolates showed multidrug resistant profiles.Resistance to ampicillin and tetracycline was the most common（23/82,28.0%）,followed by chloramphenicol（21/82,25.6%）,trimethoprim-sulfamethoxazole（20/82,24.4%）,ciprofloxacin（19/82,23.2%）,cefazolin（16/82,19.5%）and cefotaxime（16/82,19.5%）.15cefotaxime resistant isolates showed multi-drug resistant profiles,among which the eleven O111:H8（n=8）and O26:H11（n=3）serotypes were also co-resistant to ciprofloxacin and azithromycin.（4）Drug resistance analysis for STEC revealed that Quinolone resistance factors was present in ciprofloxacin-susceptible or resistant strains.Quinolone resistant determinants（QRDRs）point mutations were identified in 16 isolates,mainly on gyr A and par C regions.Plasmid mediated quinolone resistant mechanisms（PMQR）were identified in 20 isolates.Both PMQR and point mutations in QRDRs were identified in eight O111:H8 and three O26:H11 isolates.（5）Multi-locus sequence type（MLST）typing showed that 82 STEC strains were divided into 26 ST types,among which ST21（12/82,14.63%）,ST11（11/82,13.41%）,ST101（11/82,13.41%）were the dominant sequence types.ST21 and ST11,which was highly correlated with HUS,showed diverse sources.Core genome MLST（cg MLST）result showed that 16 branches and 26 ST subtypes were identified among the 82 isolates.There is a one-to-one correspondence between serotypes and ST types excluded serotypes O157:H7 and ST101.Further,26 antimicrobial resistant isolates were categorized into eight distinct clusters based on the STEC cg MLST.Three dominant ciprofloxacin resistant and eae positive clusters were identified:O111:H8（n=8）,O26:H11（n=7）and O157:H7（n=4）.In addition,the single nucleotide polymorphism（SNP）analysis of O111:H8,O26:H11 and O157:H7 showed that the O26 and O157 isolates had multiple origins,but the O111 isolates were more closely related.（3）Using a strains bank,the standardized methods from four agencies were compared.Recommended screening primers and probes set in the part of four standardized methods had low amplification efficiency for stx_1d subtype（C_t value of FDA/BAM method was 27.3,USDA/MLG method was 36.3,and JAPAN method was>40）.And none of the recommended screening primers and probes set in the four standardized methods could efficiently amplify all the stx2 subtypes because of various mismatches in the primers or the probes sequences.Acriflavine,cefsulodin and novobiocin showed an inhibitory effect to certain STEC isolates at the recommended concentrations,while the other gram-negative bacteria were poorly inhibited.（4）In view of the problems of the current standardized methods,the pre-enrichment step in the STEC detection method and the primers and probes for fluorescent PCR detection were optimized.A new primer and probe system showed high amplification efficiency and specificity for all the stx subtypes.In addition,substitution of the antimicrobials in STEC enrichment broth could improve the detection of STEC isolates.Acid treatment after enrichment was included as an effective step.（5）The optimized STEC testing method was applied in laboratories of relevant departments.Samples for verification covered a total of 12 food samples in five categories,including pre-packaged meat products（cooked beef;salami;cooked mutton）,pre-packaged raw fruit and vegetable products（instant leafy vegetables;fruits chunks;juice）,pre-packaged dairy products（pasteurized milk;cheese）,bulk cooked beef,and primary agricultural products（beef mince;pork mince;lettuce or bean sprouts）.Results showed that the optimized primers and probes used for the stx1 and stx2 genes could detect all verified food sample types（except raw leafy vegetables）with a STEC contamination amount of 10¹CFU/sample.After the antibiotics addition replaced with acid treatment,the recovery rate of STEC for all sample types（except raw leafy vegetables）with a spiked concentration of 10¹CFU/sample was 100%,and the recovery rate could reach 82.9%at a spiked concentration of 10⁰CFU/sample.For primary agricultural products,prepackaged meat products and bulk cooked beef,the recovery rate of STEC with a spiked concentration of 10^-1CFU/sample was46.4%.