Dataset Information

Whole genome sequencing based typing and characterisation of Shiga-toxin producing Escherichia coli strains belonging to O157 and O26 serotypes and isolated in dairy farms.

ABSTRACT: In the present study, the genetic relationships as well as the virulome and resistome of newly sequenced O26 and O157 Shiga-toxin producing E. coli (STEC) isolates, collected from dairy farms in Italy, were investigated in comparison to publicly available genomes collected worldwide. The whole genome of Italian isolates was sequenced on Illumina MiSeq Platform. Reads quality control, de novo draft genome assembly, species confirmation and the 7- loci Multi-Locus Sequence Type assignment were performed using INNUca pipeline. Reference-based SNPs calling was performed on O157 and O26 genomes, separately, mapping contigs to high-quality finished genomes. Virulence and antimicrobial resistance determinants were detected in silico using the tool ABRicate. Phylogenetic reconstructions revealed that genomes clustered mainly based on their 7-loci MLST type. The virulome of tested genomes included 190 determinants. O157 genomes carried chu genes associated to heme mediated iron uptake, whereas O26 genomes harboured genes ybt associated to siderophore mediated iron uptake. Resistome analysis showed the presence of tet(34) on all but one O157 genomes and on only one O26 genomes. Only 4 genomes carried genes associated to multiresistance. In the present study, the genes chu and ybt were identified as potential biomarker for the differentiation of O157 and O26 serotypes.

SUBMITTER: Pasquali F

PROVIDER: S-EPMC6379698 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Whole genome sequencing based typing and characterisation of Shiga-toxin producing Escherichia coli strains belonging to O157 and O26 serotypes and isolated in dairy farms.

Pasquali Frederique F Palma Federica F Trevisani Marcello M Parisi Antonio A Lucchi Alex A Cesare Alessandra De A Manfreda Gerardo G

Italian journal of food safety 20181201 4

In the present study, the genetic relationships as well as the virulome and resistome of newly sequenced O26 and O157 Shiga-toxin producing E. coli (STEC) isolates, collected from dairy farms in Italy, were investigated in comparison to publicly available genomes collected worldwide. The whole genome of Italian isolates was sequenced on Illumina MiSeq Platform. Reads quality control, de novo draft genome assembly, species confirmation and the 7- loci Multi-Locus Sequence Type assig ...[more]

PMID: 30854339

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Project description:A 12-month longitudinal study was undertaken on two dairy herds to ascertain the Shiga-toxin producing Escherichia coli (STEC) O157 and O26 shedding status of the animals and its impact (if any) on raw milk. Cattle are a recognized reservoir for these organisms with associated public health and environmental implications. Animals shedding E. coli O157 at >10,000 CFU/g of feces have been deemed super-shedders. There is a gap in the knowledge regarding super-shedding of other STEC serogroups. A cohort of 40 lactating cows from herds previously identified as positive for STEC in a national surveillance project were sampled every second month between August, 2013 and July, 2014. Metadata on any potential super-shedders was documented including, e.g., age of the animal, number of lactations and days in lactation, nutritional condition, somatic cell count and content of protein in milk to assess if any were associated with risk factors for super-shedding. Recto-anal mucosal swabs (RAMS), raw milk, milk filters, and water samples were procured for each herd. The swabs were examined for E. coli O157 and O26 using a quantitative real time PCR method. Counts (CFU swab-1) were obtained from a standard calibration curve that related real-time PCR cycle threshold (Ct) values against the initial concentration of O157 or O26 in the samples. Results from Farm A: 305 animals were analyzed; 15 E. coli O157 (5%) were recovered, 13 were denoted STEC encoding either stx1 and/or stx2 virulence genes and 5 (2%) STEC O26 were recovered. One super-shedder was identified shedding STEC O26 (stx1&2). Farm B: 224 animals were analyzed; eight E. coli O157 (3.5%) were recovered (seven were STEC) and 9 (4%) STEC O26 were recovered. Three super-shedders were identified, one was shedding STEC O157 (stx2) and two STEC O26 (stx2). Three encoded the adhering and effacement gene (eae) and one isolate additionally encoded the haemolysin gene (hlyA). All four super-shedders were only super-shedding once during the 1-year sampling period. The results of this study show, low numbers of super-shedders in the herds examined, with high numbers of low and medium shedding. Although four super-shedding animals were identified, no STEC O157 or O26 were recovered from any of the raw milk, milk filter, or water samples. The authors conclude that this study highlights the need for further surveillance to assess the potential for environmental contamination and food chain security.

Project description:Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

Project description:Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.

Project description:Cattle are a reservoir for Shiga toxin-producing Escherichia coli (STEC), zoonotic pathogens that cause serious clinical disease. Scotland has a higher incidence of STEC infection in the human population than the European average. The aim of this study was to investigate the prevalence and epidemiology of non-O157 serogroups O26, O103, O111, and O145 and Shiga toxin gene carriage in Scottish cattle. Fecal samples (n = 2783) were collected from 110 herds in 2014 and 2015 and screened by real-time PCR. Herd-level prevalence (95% confidence interval [CI]) for O103, O26, and O145 was estimated as 0.71 (0.62, 0.79), 0.43 (0.34, 0.52), and 0.23 (0.16, 0.32), respectively. Only two herds were positive for O111. Shiga toxin prevalence was high in both herds and pats, particularly for stx2 (herd level: 0.99; 95% CI: 0.94, 1.0). O26 bacterial strains were isolated from 36 herds on culture. Fifteen herds yielded O26 stx-positive isolates that additionally harbored the intimin gene; six of these herds shed highly pathogenic stx2-positive strains. Multiple serogroups were detected in herds and pats, with only 25 herds negative for all serogroups. Despite overlap in detection, regional and seasonal effects were observed. Higher herd prevalence for O26, O103, and stx1 occurred in the South West, and this region was significant for stx2 at the pat level (P = 0.015). Significant seasonal variation was observed for O145 prevalence, with the highest prevalence in autumn (P = 0.032). Negative herds were associated with Central Scotland and winter. Herds positive for all serogroups were associated with autumn and larger herd size and were not housed at sampling.IMPORTANCE Cattle are reservoirs for Shiga toxin-producing Escherichia coli (STEC), bacteria shed in animal feces. Humans are infected through consumption of contaminated food or water and by direct contact, resulting in serious disease and kidney failure in the most vulnerable. The contribution of non-O157 serogroups to STEC illness was underestimated for many years due to the lack of specific tests. Recently, non-O157 human cases have increased, with O26 STEC of particular note. It is therefore vital to investigate the level and composition of non-O157 in the cattle reservoir and to compare them historically and by the clinical situation. In this study, we found cattle prevalence high for toxin, as well as for O103 and O26 serogroups. Pathogenic O26 STEC were isolated from 14% of study herds, with toxin subtypes similar to those seen in Scottish clinical cases. This study highlights the current risk to public health from non-O157 STEC in Scottish cattle.

Project description:Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni are notable health hazards associated with the consumption of raw milk. These bacteria may colonize the intestines of asymptomatic cattle and enter bulk tank milk via fecal contamination during milking. We studied the frequency of STEC O157:H7 and C. jejuni contamination in tank milk (n = 785) and the in-line milk filters of milking machines (n = 631) versus the frequency of isolation from cattle feces (n = 257) on three Finnish dairy farms for 1 year. Despite simultaneous isolation of STEC O157:H7 (17%) or C. jejuni (53%) from cattle, these bacteria were rarely isolated from milk filters (2% or <1%, respectively) and milk (0%). As revealed by phylogenomics, one STEC O157:H7 strain at a time was detected on each farm and persisted for ≤12 months despite rigorous hygienic measures. C. jejuni strains of a generalist sequence type (ST-883 and ST-1080) persisted in the herds for ≥11 months, and several other C. jejuni types were detected sporadically. The stx gene carried by STEC was detected more frequently from milk filters (37%) than from milk (7%), suggesting that milk filters are more suitable sampling targets for monitoring than milk. A questionnaire of on-farm practices suggested lower stx contamination of milk when major cleansing in the barn, culling, or pasturing of dairy cows was applied, while a higher average outdoor temperature was associated with higher stx contamination. Because pathogen contamination occurred despite good hygiene and because pathogen detection from milk and milk filters proved challenging, we recommend heat treatment for raw milk before consumption.IMPORTANCE The increased popularity of raw milk consumption has created demand for relaxing legislation, despite the risk of contamination by pathogenic bacteria, notably STEC and C. jejuni However, the epidemiology of these milk-borne pathogens on the herd level is still poorly understood, and data are lacking on the frequency of milk contamination on farms with cattle shedding these bacteria in their feces. This study suggests (i) that STEC contamination in milk can be reduced, but not prevented, by on-farm hygienic measures while fecal shedding is observable, (ii) that milk filters are more suitable sampling targets for monitoring than milk although pathogen detection from both sample matrices may be challenging, and (iii) that STEC and C. jejuni genotypes may persist in cattle herds for several months. The results can be utilized in developing and targeting pathogen monitoring and risk management on the farm level and contributed to the revision of Finnish legislation in 2017.

Dataset Information

Whole genome sequencing based typing and characterisation of Shiga-toxin producing Escherichia coli strains belonging to O157 and O26 serotypes and isolated in dairy farms.

Publications

Whole genome sequencing based typing and characterisation of Shiga-toxin producing <i>Escherichia coli</i> strains belonging to O157 and O26 serotypes and isolated in dairy farms.

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