Microbial water quality at contrasting recreational areas in a mixed-use watershed in eastern Canada.

Recreational water use is an important source of human enteric illness. Enhanced (episodic) surveillance of natural recreational waters as a supplement to beach monitoring can enrich our understanding of human health risks. From 2011 to 2013, water sampling was undertaken at recreational sites on a watershed in eastern Canada. This study compared the prevalence and associations of human enteric pathogens and fecal indicator organisms. Beach water samples had lower pathogen presence than those along the main river, due to different pollution sources and the hydrological disposition. Pathogen profiles identified from the beach sites suggested a more narrow range of sources, including birds, indicating that wild bird management could help reduce public health risks at these sites. The presence and concentration of indicator organisms did not differ significantly between beaches and the river. However, higher concentrations of generic Escherichia coli were observed when Salmonella and Cryptosporidium were present at beach sites, when Salmonella was present at the river recreational site, and when verotoxigenic E. coli were present among all sites sampled. In this watershed, generic E. coli concentrations were good indicators of potential contamination, pathogen load, and elevated human health risk, supporting their use for routine monitoring where enhanced pathogen testing is not possible.

Antimicrobial-Resistant E. coli from Surface Waters in Southwest Ontario Dairy Farms.

Untreated surface waters can be contaminated with a variety of bacteria, including , some of which can be pathogenic for both humans and animals. Therefore, such waters need to be treated before their use in dairy operations to mitigate risks to dairy cow health and milk safety. To understand the molecular ecology of , this study aimed to assess antimicrobial resistance (AMR) in recovered from untreated surface water sources of dairy farms. Untreated surface water samples ( = 240) from 15 dairy farms were collected and processed to isolate . A total of 234 isolates were obtained and further characterized for their serotypes and antimicrobial susceptibility. Of the 234 isolates, 71.4% were pan-susceptible, 23.5% were resistant to one or two antimicrobial classes, and 5.1% were resistant to three or more antimicrobial classes. Whole genome sequence analysis of 11 selected multidrug-resistant isolates revealed AMR genes including and that confer resistance to the critically important extended-spectrum cephalosporins, as well as a variety of plasmids (mainly of the replicon type) and class 1 integrons. Phylogenetic and comparative genome analysis revealed a genetic relationship between some of the sequenced and Shiga toxin-producing O157:H7 (STEC), which warrants further investigation. This study shows that untreated surface water sources contain antimicrobial-resistant which may serve as a reservoir of AMR that could be disseminated through horizontal gene transfer. This is another reason why effective water treatment before usage should be routinely done on dairy farm operations.

Complete genome sequence of Actinobacillus suis H91-0380, a virulent serotype O2 strain.

Here, we report the first complete genome sequence of Actinobacillus suis, an important opportunistic pathogen of swine. By comparing the genome sequence of A. suis with those of other members of the family Pasteurellaceae, we hope to better understand the role of these organisms in health and disease in swine.

Characterization of antibiotic-resistant and potentially pathogenic Escherichia coli from soil fertilized with litter of broiler chickens fed antimicrobial-supplemented diets.

The objective of this study was to characterize antimicrobial resistance and virulence determinants of Escherichia coli from soil amended with litter from 36-day-old broiler chickens ( Gallus gallus domesticus ) fed with diets supplemented with a variety of antimicrobial agents. Soil samples were collected from plots before and periodically after litter application in August to measure E. coli numbers. A total of 295 E. coli were isolated from fertilized soil samples between August and March. Antibiotic susceptibility was determined by Sensititre, and polymerase chain reaction was performed to detect the presence of resistance and virulence genes. The results confirmed that E. coli survived and could be quantified by direct plate count for at least 7 months in soil following litter application in August. The effects of feed supplementation were observed on E. coli numbers in November and January. Among the 295 E. coli, the highest antibiotic resistance level was observed against tetracycline and ß-lactams associated mainly with the resistance genes tetB and bla(CMY-2), respectively. Significant treatment effects were observed for phylogenetic groups, antibiotic resistance profiles, and virulence gene frequencies. Serotyping, phylogenetic grouping, and pulsed-field gel electrophoresis confirmed that multiple-antibiotic-resistant and potentially pathogenic E. coli can survive in soil fertilized with litter for several months regardless of antimicrobials used in the feed.

Virulence Genotype and Phenotype of Multiple Antimicrobial-Resistant Escherichia coli Isolates from Broilers Assessed from a "One-Health" Perspective.

ABSTRACT: Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context.

Using whole-genome sequence data to examine the epidemiology of antimicrobial resistance in Escherichia coli from wild meso-mammals and environmental sources on swine farms, conservation areas, and the Grand River watershed in southern Ontario, Canada.

Antimicrobial resistance (AMR) threatens the health of humans and animals and has repeatedly been detected in wild animal species across the world. This cross-sectional study integrates whole-genome sequence data from Escherichia coli isolates with demonstrated phenotypic resistance that originated from a previous longitudinal wildlife study in southern Ontario, as well as phenotypically resistant E. coli water isolates previously collected as part of a public health surveillance program. The objective of this work was to assess for evidence of possible transmission of antimicrobial resistance determinants between wild meso-mammals, swine manure pits, and environmental sources on a broad scale in the Grand River watershed, and at a local scale-for the subset of samples collected on both swine farms and conservation areas in the previous wildlife study. Logistic regression models were used to assess potential associations between sampling source, location type (swine farm vs. conservation area), and the occurrence of select resistance genes and predicted plasmids. In total, 200 isolates from the following sources were included: water (n = 20), wildlife (n = 73), swine manure pit (n = 31), soil (n = 73), and dumpsters (n = 3). Several genes and plasmid incompatibility types were significantly more likely to be identified on swine farms compared to conservation areas. Conversely, internationally distributed sequence types (e.g., ST131), extended-spectrum beta-lactamase- and AmpC-producing E. coli were isolated in lower prevalences (<10%) and were almost exclusively identified in water sources, or in raccoon and soil isolates obtained from conservation areas. Differences in the odds of detecting resistance genes and predicted plasmids among various sources and location types suggest different primary sources for individual AMR determinants, but, broadly, our findings suggest that raccoons, skunks and opossums in this region may be exposed to AMR pollution via water and agricultural sources, as well as anthropogenic sources in conservation areas.

Rural Raccoons (Procyon lotor) Not Likely to Be a Major Driver of Antimicrobial Resistant Human Salmonella Cases in Southern Ontario, Canada: A One Health Epidemiological Assessment Using Whole-Genome Sequence Data.

Non-typhoidal Salmonella infections represent a substantial burden of illness in humans, and the increasing prevalence of antimicrobial resistance among these infections is a growing concern. Using a combination of Salmonella isolate short-read whole-genome sequence data from select human cases, raccoons, livestock and environmental sources, and an epidemiological framework, our objective was to determine if there was evidence for potential transmission of Salmonella and associated antimicrobial resistance determinants between these different sources in the Grand River watershed in Ontario, Canada. Logistic regression models were used to assess the potential associations between source type and the presence of select resistance genes and plasmid incompatibility types. A total of 608 isolates were obtained from the following sources: humans (n = 58), raccoons (n = 92), livestock (n = 329), and environmental samples (n = 129). Resistance genes of public health importance, including bla CMY-2, were identified in humans, livestock, and environmental sources, but not in raccoons. Most resistance genes analyzed were significantly more likely to be identified in livestock and/or human isolates than in raccoon isolates. Based on a 3,002-loci core genome multi-locus sequence typing (cgMLST) scheme, human Salmonella isolates were often more similar to isolates from livestock and environmental sources, than with those from raccoons. Rare instances of serovars S. Heidelberg and S. Enteritidis in raccoons likely represent incidental infections and highlight possible acquisition and dissemination of predominantly poultry-associated Salmonella by raccoons within these ecosystems. Raccoon-predominant serovars were either not identified among human isolates (S. Agona, S. Thompson) or differed by more than 350 cgMLST loci (S. Newport). Collectively, our findings suggest that the rural population of raccoons on swine farms in the Grand River watershed are unlikely to be major contributors to antimicrobial resistant human Salmonella cases in this region.

Complete Genome Sequences for 36 Canadian Salmonella enterica Serovar Typhimurium and I 1,4,[5],12:i:- Isolates from Clinical and Animal Sources.

Here, we report the complete genome sequences for 36 Canadian isolates of Salmonella enterica subsp. enterica serovar Typhimurium and its monophasic variant I 1,4,[5]:12:i:- from both clinical and animal sources. These genome sequences will provide useful references for understanding the genetic variation within this prominent serotype.

ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data.

Escherichia coli is a priority foodborne pathogen of public health concern and phenotypic serotyping provides critical information for surveillance and outbreak detection activities. Public health and food safety laboratories are increasingly adopting whole-genome sequencing (WGS) for characterizing pathogens, but it is imperative to maintain serotype designations in order to minimize disruptions to existing public health workflows. Multiple in silico tools have been developed for predicting serotypes from WGS data, including SRST2, SerotypeFinder and EToKi EBEis, but these tools were not designed with the specific requirements of diagnostic laboratories, which include: speciation, input data flexibility (fasta/fastq), quality control information and easily interpretable results. To address these specific requirements, we developed ECTyper (https://github.com/phac-nml/ecoli_serotyping) for performing both speciation within Escherichia and Shigella, and in silico serotype prediction. We compared the serotype prediction performance of each tool on a newly sequenced panel of 185 isolates with confirmed phenotypic serotype information. We found that all tools were highly concordant, with 92-97 % for O-antigens and 98-100 % for H-antigens, and ECTyper having the highest rate of concordance. We extended the benchmarking to a large panel of 6954 publicly available E. coli genomes to assess the performance of the tools on a more diverse dataset. On the public data, there was a considerable drop in concordance, with 75-91 % for O-antigens and 62-90 % for H-antigens, and ECTyper and SerotypeFinder being the most concordant. This study highlights that in silico predictions show high concordance with phenotypic serotyping results, but there are notable differences in tool performance. ECTyper provides highly accurate and sensitive in silico serotype predictions, in addition to speciation, and is designed to be easily incorporated into bioinformatic workflows.

Population structure, case clusters, and genetic lesions associated with Canadian Salmonella 4,[5],12:i:- isolates.

Monophasic Salmonella 4,[5]:12:i:- are a major public health problem because they are one of the top five Salmonella serotypes isolated from clinical cases globally and because they can carry resistance to multiple antibiotics. A total of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium whole genome sequences (WGS) were generated. The various genetic lesions causing the Salmonella 4,[5]:12:i:- genotype were identified and assessed with regards to their distribution in the population of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium isolates, their geographical and temporal distribution, and their association with non-human sources. Several clades were identified in the population structure, and the largest two were associated almost exclusively with a short prophage insertion and insertion of a mobile element carrying loci encoding antibiotic and mercury resistance. IS26-mediated deletions and fljB point mutants appeared to spread clonally. 'Inconsistent' Salmonella 4,[5]:12:i:- isolates associated with specific, single amino acid changes in fljA and hin were found in a single clade composed of water, shellfish, and avian isolates. Inclusion of isolates from different case clusters identified previously by PFGE validated some of the clusters and invalidated others. Some wgMLST clusters of clinical isolates composed of very closely related isolates contained an isolate(s) with a different genetic lesion, suggesting continuing mobility of the implicated element responsible. Such cases may need to be left out of epidemiological investigations until sufficient numbers of isolates are included that statistical significance of association with sources is not impaired. Non-human sources were frequently found in or near clinical case clusters. Prospective surveillance and WGS of non-human sources and retrospective analysis by WGS of isolates from existing culture collections provides data critical for epidemiological investigations of food- and waterborne outbreaks.

Antibiotic Resistance in Shiga Toxigenic Escherichia coli Isolates from Surface Waters and Sediments in a Mixed Use Urban Agricultural Landscape.

Antibiotic resistance (AR) phenotypes and acquired resistance determinants (ARDs) detected by in silico analysis of genome sequences were examined in 55 Shiga toxin-producing Escherichia coli (STEC) isolates representing diverse serotypes recovered from surfaces waters and sediments in a mixed use urban/agricultural landscape in British Columbia, Canada. The isolates displayed decreased susceptibility to florfenicol (65.5%), chloramphenicol (7.3%), tetracycline (52.7%), ampicillin (49.1%), streptomycin (34.5%), kanamycin (20.0%), gentamycin (10.9%), amikacin (1.8%), amoxicillin/clavulanic acid (21.8%), ceftiofur (18.2%), ceftriaxone (3.6%), trimethoprim-sulfamethoxazole (12.7%), and cefoxitin (3.6%). All surface water and sediment isolates were susceptible to ciprofloxacin, nalidixic acid, ertapenem, imipenem and meropenem. Eight isolates (14.6%) were multidrug resistant. ARDs conferring resistance to phenicols (floR), trimethoprim (dfrA), sulfonamides (sul1/2), tetracyclines (tetA/B), and aminoglycosides (aadA and aph) were detected. Additionally, narrow-spectrum ß-lactamase blaTEM-1b and extended-spectrum AmpC ß-lactamase (cephalosporinase) blaCMY-2 were detected in the genomes, as were replicons from plasmid incompatibility groups IncFII, IncB/O/K/Z, IncQ1, IncX1, IncY and Col156. A comparison with surveillance data revealed that AR phenotypes and ARDs were comparable to those reported in generic E. coli from food animals. Aquatic environments in the region are potential reservoirs for the maintenance and transmission of antibiotic resistant STEC, associated ARDs and their plasmids.

Rapid and accurate SNP genotyping of clonal bacterial pathogens with BioHansel.

Hierarchical genotyping approaches can provide insights into the source, geography and temporal distribution of bacterial pathogens. Multiple hierarchical SNP genotyping schemes have previously been developed so that new isolates can rapidly be placed within pre-computed population structures, without the need to rebuild phylogenetic trees for the entire dataset. This classification approach has, however, seen limited uptake in routine public health settings due to analytical complexity and the lack of standardized tools that provide clear and easy ways to interpret results. The BioHansel tool was developed to provide an organism-agnostic tool for hierarchical SNP-based genotyping. The tool identifies split k-mers that distinguish predefined lineages in whole genome sequencing (WGS) data using SNP-based genotyping schemes. BioHansel uses the Aho-Corasick algorithm to type isolates from assembled genomes or raw read sequence data in a matter of seconds, with limited computational resources. This makes BioHansel ideal for use by public health agencies that rely on WGS methods for surveillance of bacterial pathogens. Genotyping results are evaluated using a quality assurance module which identifies problematic samples, such as low-quality or contaminated datasets. Using existing hierarchical SNP schemes for Mycobacterium tuberculosis and Salmonella Typhi, we compare the genotyping results obtained with the k-mer-based tools BioHansel and SKA, with those of the organism-specific tools TBProfiler and genotyphi, which use gold-standard reference-mapping approaches. We show that the genotyping results are fully concordant across these different methods, and that the k-mer-based tools are significantly faster. We also test the ability of the BioHansel quality assurance module to detect intra-lineage contamination and demonstrate that it is effective, even in populations with low genetic diversity. We demonstrate the scalability of the tool using a dataset of ~8100 S. Typhi public genomes and provide the aggregated results of geographical distributions as part of the tool's output. BioHansel is an open source Python 3 application available on PyPI and Conda repositories and as a Galaxy tool from the public Galaxy Toolshed. In a public health context, BioHansel enables rapid and high-resolution classification of bacterial pathogens with low genetic diversity.

Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes.

BACKGROUND: Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. RESULTS: We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. CONCLUSIONS: Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Food reservoir for Escherichia coli causing urinary tract infections.

Closely related strains of Escherichia coli have been shown to cause extraintestinal infections in unrelated persons. This study tests whether a food reservoir may exist for these E. coli. Isolates from 3 sources over the same time period (2005-2007) and geographic area were compared. The sources comprised prospectively collected E. coli isolates from women with urinary tract infection (UTI) (n = 353); retail meat (n = 417); and restaurant/ready-to-eat foods (n = 74). E. coli were evaluated for antimicrobial drug susceptibility and O:H serotype and compared by using 4 different genotyping methods. We identified 17 clonal groups that contained E. coli isolates (n = 72) from >1 source. E. coli from retail chicken (O25:H4-ST131 and O114:H4-ST117) and honeydew melon (O2:H7-ST95) were indistinguishable from or closely related to E. coli from human UTIs. This study provides strong support for the role of food reservoirs or foodborne transmission in the dissemination of E. coli causing common community-acquired UTIs.

Lineage and host source are both correlated with levels of Shiga toxin 2 production by Escherichia coli O157:H7 strains.

Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx(2) flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx(2)) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx(2), whereas all LII strains carried variant stx(2c) and 4 of 14 LI/II strains had copies of both stx(2) and variant stx(2c). Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx(2) mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

Assessing the genomic relatedness and evolutionary rates of persistent verotoxigenic Escherichia coli serotypes within a closed beef herd in Canada.

Verotoxigenic Escherichia coli (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher's exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 (n=22), O22:H8 (n=30), O108:H8 (n=39), O139:H19 (n=44) and O157:H7 (n=106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for E. coli; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes (P≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.

Genomic regions conserved in lineage II Escherichia coli O157:H7 strains.

Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (<6%) of the 17 lineage I strains. Several of these lineage II-related fragments contain insertions/deletions that may play an important role in virulence. These lineage II-related loci were also shown to be useful markers for genotyping of E. coli O157:H7 strains isolated from human and animal sources.

Genotype, serotype, and antibiotic resistance of sorbitol-negative Escherichia coli isolates from feedlot cattle.

Rectal fecal samples from 80 steers receiving Rumensin, Revalor-S, and Liquamycin alone or in combination for growth promotion and disease prevention were examined for the presence of non-O157:H7 Shiga toxin-producing Escherichia coli. All isolates were identified with the API 20E test, virulence genes were detected with a PCR assay, and antibiotic susceptibilities were determined with the Sensititre system. Of the 153 E. coli isolates recovered 126 (82.3%) were sorbitol negative. Isolates were classified into 14 biochemical E. coli groups; 51.6% were negative for arginine dihydrolase, ornithine decarboxylase, sorbitol, and saccharose reactions but positive for lysine decarboxylase, indole production, and rhamnose reactions. Twenty-one O:H serotypes were detected in the 153 E. coli isolates. The most frequent serotypes were O2:H42 (49.7% of isolates), O49:NM (13.7%), O?:H25 (9.2%), and O10:NM (7.2%). One isolate of E. coli O172:H25 and one of E. coli O157: H39 were found. The stx1 gene was found in the two E. coli O98:H25 isolates. The eaeA and e-hlyA genes were detected in 21, 14, and 10 isolates of serotypes O49:NM, O?:H25, and O10:NM, respectively, and in each isolate of serotype O156:H25 and O172:H25. Four E. coli O132:H18 isolates were multiresistant to ampicillin, chloramphenicol, kanamycin, streptomycin, and sulfisoxazole. Tetracycline resistance due to the tet(B) gene was observed in 74 of the 76 E. coli O2:H42 isolates. Except for one isolate, all tetracycline-resistant isolates were negative for the virulence genes eaeA and e-hlyA or stx1. Pulsed-field gel electrophoresis typing revealed that the tetracycline-resistant serotypes were genetically diverse. Our data illustrate that cattle are a potential source of some atypical antibiotic-resistant E. coli isolates that harbor virulence genes.

General detection of Shiga toxin 2 and subtyping of Shiga toxin 1 and 2 in Escherichia coli using qPCR.

Shiga toxin-producing E. coli (STEC) is a gastrointestinal pathogen and has been recognized as one of the serious problems in public health. Shiga toxin genes (stx) can be grouped into different types according to their differences in sequence and biological activities. The two main groups of stx are stx1 and stx2 with each group containing several subtypes. It is essential to develop rapid stx1/stx2 subtyping assays and accurate stx2 general detection assays to provide a quicker turn-around time to predict disease outcome and also to provide data for surveillance purposes. The stx2 general detection qPCR assay developed in this study showed 100% sensitivity and 100% specificity with no cross reactivity with stx2 negative STEC and non-STEC isolates. This stx2 general detection assay was able to detect all seven different stx2 subtypes at low level of detection and with good PCR efficiency. In addition, stx1/stx2 subtyping qPCR assays were succesfully developed to detect all stx1 subtypes and stx2 subtypes with the exception of one stx2b subtype carried by one strain. The qPCR stx1/stx2 subtyping assays showed 100% specificity with no cross reactivity on subtypes not targeted by each assay. The rapidity with faster turn-around time along with high throughput volume of the stx1/stx2 subtyping and stx2 general detection qPCR assays will have great value as tools for STEC associated risk assessment, outbreak monitoring, epidemiology studies, and clinical management.

Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR.

We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico, modified to be RNase H2-compatible, and then optimized by laboratory testing. Optimization often required repeated cycling through variations in primer design, assay conditions, reagent concentrations and selection of alternative SNP targets. The final rhPCR assay uses 28 independent rhPCR reactions to target 14 DNA bases that can distinguish 15 possible lineages and sub-lineages of SH. On evaluation, the assay correctly identified the 12 lineages and sub-lineages represented in a panel of 75 diverse SH strains. Non-specific amplicons were observed in 160 (15.2%) of the 1050 reactions, but due to their low intensity did not compromise assay performance. Furthermore, in silico analysis of 500 closed genomes from 103 Salmonella serovars and laboratory rhPCR testing of five prevalent Salmonella serovars including SH indicated the assay can identify Salmonella isolates as SH, since only SH isolates generated amplicons from all 14 target SNPs. The genotyping results can be fully correlated with whole genome sequencing (WGS) data in silico. This fast and economical assay, which can identify SH isolates and classify them into related or unrelated lineages and sub-lineages, has potential applications in outbreak identification, source attribution and microbial source tracking.