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.

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.

Effect of a bacteriophage T5virus on growth of Shiga toxigenic Escherichia coli and Salmonella strains in individual and mixed cultures.

A previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P < 0.05) at a lower multiplicity of infection and sampling time (6-10 h), when STEC O157:H7 was also included in the culture. This phage was able to simultaneously reduce numbers of STEC and Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

Eleven High-Quality Reference Genome Sequences and 360 Draft Assemblies of Shiga Toxin-Producing Escherichia coli Isolates from Human, Food, Animal, and Environmental Sources in Canada.

We report high-quality closed reference genomes for 1 bovine strain and 10 human Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from serogroups O26, O45, O91, O103, O104, O111, O113, O121, O145, and O157. We also report draft assemblies, with standardized metadata, for 360 STEC strains isolated from watersheds, animals, farms, food, and human infections.

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.

Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome.

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1ß secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1ß production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.

Multi-Year Persistence of Verotoxigenic Escherichia coli (VTEC) in a Closed Canadian Beef Herd: A Cohort Study.

In this study, fecal samples were collected from a closed beef herd in Alberta, Canada from 2012 to 2015. To limit serotype bias, which was observed in enrichment broth cultures, Verotoxigenic Escherichia coli (VTEC) were isolated directly from samples using a hydrophobic grid-membrane filter verotoxin immunoblot assay. Overall VTEC isolation rates were similar for three different cohorts of yearling heifers on both an annual (68.5 to 71.8%) and seasonal basis (67.3 to 76.0%). Across all three cohorts, O139:H19 (37.1% of VTEC-positive samples), O22:H8 (15.8%) and O?(O108):H8 (15.4%) were among the most prevalent serotypes. However, isolation rates for serotypes O139:H19, O130:H38, O6:H34, O91:H21, and O113:H21 differed significantly between cohort-years, as did isolation rates for some serotypes within a single heifer cohort. There was a high level of VTEC serotype diversity with an average of 4.3 serotypes isolated per heifer and 65.8% of the heifers classified as "persistent shedders" of VTEC based on the criteria of >50% of samples positive and ≥4 consecutive samples positive. Only 26.8% (90/336) of the VTEC isolates from yearling heifers belonged to the human disease-associated seropathotypes A (O157:H7), B (O26:H11, O111:NM), and C (O22:H8, O91:H21, O113:H21, O137:H41, O2:H6). Conversely, seropathotypes B (O26:NM, O111:NM) and C (O91:H21, O2:H29) strains were dominant (76.0%, 19/25) among VTEC isolates from month-old calves from this herd. Among VTEC from heifers, carriage rates of vt1, vt2, vt1+vt2, eae, and hlyA were 10.7, 20.8, 68.5, 3.9, and 88.7%, respectively. The adhesin gene saa was present in 82.7% of heifer strains but absent from all of 13 eae+ve strains (from serotypes/intimin types O157:H7/γ1, O26:H11/ß1, O111:NM/θ, O84:H2/ζ, and O182:H25/ζ). Phylogenetic relationships inferred from wgMLST and pan genome-derived core SNP analysis showed that strains clustered by phylotype and serotype. Further, VTEC strains of the same serotype usually shared the same suite of antibiotic resistance and virulence genes, suggesting the circulation of dominant clones within this distinct herd. This study provides insight into the diverse and dynamic nature of VTEC populations within groups of cattle and points to a broad spectrum of human health risks associated with these E. coli strains.

Occurrence, characterization, and potential predictors of verotoxigenic Escherichia coli, Listeria monocytogenes, and Salmonella in surface water used for produce irrigation in the Lower Mainland of British Columbia, Canada.

Produce has become a major source of foodborne illness, and may become contaminated through surface water irrigation. The objectives of this study were to (i) determine the frequency of verotoxigenic E. coli (VTEC), Listeria monocytogenes, and Salmonella in surface waters used for irrigation in the Lower Mainland of British Columbia, (ii) assess the suitability of fecal coliforms and generic E. coli as hygiene indicators, and (iii) investigate the correlations of environmental factors with pathogen occurrence. Water samples were collected semi-monthly for 18 months from seven irrigation ditches across the Serpentine and Sumas watersheds. VTEC colonies on water filters were detected using a verotoxin colony immunoblot, and the presence of virulence genes vt1 and vt2 was ascertained via multiplex PCR. Detection of L. monocytogenes and Salmonella was completed using standard, Health Canada Compendium of Analytical Methods. Fecal coliforms and generic E. coli were enumerated by 3M™ Petrifilm™ and filtration methods, and meteorological and geographic data were collected from government records. VTEC, L. monocytogenes, and Salmonella were detected in 4.93%, 10.3%, and 2.69% of 223 samples, respectively. L. monocytogenes occurrence was greatest in the Serpentine watershed (χ2; p < 0.05), and was most common during the winter and fall (Fisher exact test; p < 0.05). Site dependence of VTEC and Salmonella occurrence was observed within watersheds (Fisher's exact test; p < 0.10). Pathogen occurrence correlated with fecal coliform counts (r = 0.448), while VTEC occurrence also correlated with precipitation over the five days before sampling (r = 0.239). The density of upstream livestock correlated with VTEC (rs = 0.812), and L. monocytogenes (rs = 0.841) detection. These data show that foodborne pathogens are present in the waters used for irrigation in the Lower Mainland of British Columbia, but their frequency may depend on spatial and temporal factors.

Subtyping of Canadian isolates of Salmonella Enteritidis using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) alone and in combination with Pulsed-Field Gel Electrophoresis (PFGE) and phage typing.

Salmonella enterica subspecies enterica serovar Enteritidis (SE) is one of the most common causes of human salmonellosis and in Canada currently accounts for over 40% of human cases. Reliable subtyping of isolates is required for outbreak detection and source attribution. However, Pulsed-Field Gel Electrophoresis (PFGE), the current standard subtyping method for Salmonella spp., is compromised by the high genetic homogeneity of SE. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) was introduced to supplement PFGE, although there is a lack of data on the ability of MLVA to subtype Canadian isolates of SE. Three subtyping methods, PFGE, MLVA and phage typing were compared for their discriminatory power when applied to three panels of Canadian SE isolates: Panel 1: 70 isolates representing the diversity of phage types (PTs) and PFGE subtypes within these PTs; Panel 2: 214 apparently unrelated SE isolates of the most common PTs; and Panel 3: 27 isolates from 10 groups of epidemiologically related strains. For Panel 2 isolates, four MLVA subtypes were shared among 74% of unrelated isolates and in Panel 3 isolates, one MLVA subtype accounted for 62% of the isolates. For all panels, combining results from PFGE, MLVA and PT gave the best discrimination, except in Panel 1, where the combination of PT and PFGE was equally as high, due to the selection criteria for this panel. However, none of these methods is sufficiently discriminatory alone for reliable outbreak detection or source attribution, and must be applied together to achieve sufficient discrimination for practical purposes. Even then, some large clusters were not differentiated adequately. More discriminatory methods are required for reliable subtyping of this genetically highly homogeneous serovar. This need will likely be met by whole genome sequence analysis given the recent promising reports and as more laboratories implement this tool for outbreak response and surveillance.

Evidence for the benefits of food chain interventions on E. coli O157:H7/NM prevalence in retail ground beef and human disease incidence: A success story.

OBJECTIVES: Human infection with Escherichia coli O157:H7/NM has historically been associated with consumption of undercooked ground beef. The purpose of this paper is to investigate the correlation of the decline in E. coli O157:H7/NM infections in Canada with the introduction of control efforts in ground beef by industry. METHODS: The human incidence of E. coli O157:H7/NM, prevalence in ground beef and interventions from 1996 to 2014 were analyzed. Pathogen prevalence data were obtained from federal government and industry surveillance and inspection/compliance programs. A survey of the largest ground beef producers in Canada was conducted to identify when interventions were implemented. RESULTS: The incidence of E. coli O157:H7/NM infections in Canada declined from ≈4 cases/100 000 to ≈1 case/100000 from 2000 to 2010. Verotoxigenic Escherichia coli (VTEC) prevalence in ground beef sold at retail declined from about 30% around the year 2000 to <2% since 2012. Other measures of the prevalence of E. coli, VTEC, and E. coli O157:H7/NM in beef and ground beef also declined. The number and types of interventions implemented in the major beef processing establishments in Canada increased from 1996 to 2016. CONCLUSION: The observed decline in human illnesses and pathogen levels in relation to retail meats was associated with the introduction of control efforts by industry, federal and provincial/territorial governments, and the general population. Industry-led changes in beef processing along with the introduction of food safety policies, regulations, and public education have led to improved food safety in Canada.

Complete Genome Sequences of 17 Canadian Isolates of Salmonella enterica subsp. enterica Serovar Heidelberg from Human, Animal, and Food Sources.

Salmonella enterica subsp. enterica serovar Heidelberg is a highly clonal serovar frequently associated with foodborne illness. To facilitate subtyping efforts, we report fully assembled genome sequences of 17 Canadian S Heidelberg isolates including six pairs of epidemiologically related strains. The plasmid sequences of eight isolates contain several drug resistance genes.

Influence of Season and Feedlot Location on Prevalence and Virulence Factors of Seven Serogroups of Escherichia coli in Feces of Western-Canadian Slaughter Cattle.

Pooled feces collected over two years from 1749 transport trailers hauling western-Canadian slaughter cattle were analysed by PCR for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157. Sequential immunomagnetic separation was then used to collect bacterial isolates (n = 1035) from feces positive for target serogroups. Isolated bacteria were tested by PCR to confirm serogroup and the presence of eae, ehxA, stx1, and stx2 virulence genes. Based on PCR screening, serogroup prevalence in feces ranged from 7.0% (O145) to 94.4% (O103) with at least 3 serogroups present in 79.5% of samples. Origin of cattle affected serogroup PCR prevalence and O157 was most prevalent in feces from south-west Alberta (P < 0.001). All serogroups demonstrated seasonal variations in PCR prevalence, with O26, O45, O103, O121, and O157 least prevalent (P < 0.001) in cooler winter months, while uncommon serogroups O111 and O145 increased in prevalence during winter (P < 0.001). However, isolates collected during winter were predominantly from serogroups O103 and O45. No seasonal variation was noted in proportion of isolates which were Shiga toxin containing E. coli (STEC; P = 0.18) or positive for Shiga toxin and eae (enterohemorrhagic E. coli; EHEC; P = 0.29). Isolates of serogroups O111, O145, and O157 were more frequently EHEC than were others, although 37.6-54.3% of isolates from other serogroups were also EHEC. Shiga-toxin genes present also varied by geographic origin of cattle (P < 0.05) in all serogroups except O157. As cattle within feedlots are sourced from multiple regions, locational differences in serogroup prevalence and virulence genes imply existence of selection pressures for E. coli and their virulence in western-Canadian cattle. Factors which reduce carriage or expression of virulence genes, particularly in non-O157 serogroups, should be investigated.

Yersinia enterocolitica-Specific Infection by Bacteriophages TG1 and ϕR1-RT Is Dependent on Temperature-Regulated Expression of the Phage Host Receptor OmpF.

UNLABELLED: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB_YenM_TG1 (TG1) and vB_YenM_ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface. IMPORTANCE: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen.

Assessment of commercial chromogenic solid media for the detection of non-O157 Shiga toxin-producing Escherichia coli (STEC).

Detection of Shiga toxin-producing Escherichia coli (STEC) has evolved significantly since the introduction of sorbitol-MacConkey agar. This study compares four chromogenic media (CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157, and Colorex® O157) in their identification of non-O157 STEC. When 161 non-O157 STEC were directly inoculated onto each medium, detection rates on CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157 and Colorex® O157 were 90%, 70%, 3.7% and 6.8%, respectively. Tellurite minimal inhibitory concentrations (MICs) correlated with growth on CHROMagar™ STEC as 20 of 22 isolates with poor or no growth had MICs ≤1µg/mL. Stool spiking experiments revealed that CHROMagar™ STEC had the highest recovery of the six most common non-O157 STEC, ranging from 30% (in mucoid stool) to 98% (in watery stool). When using clinical stool samples, CHROMagar™ STEC had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.6%, 87%, 13.9%, and 99.6%, respectively.

Phenotypic and Genotypic Characteristics of Shiga Toxin-Producing Escherichia coli Isolated from Surface Waters and Sediments in a Canadian Urban-Agricultural Landscape.

A hydrophobic grid membrane filtration-Shiga toxin immunoblot method was used to examine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in four watersheds located in the Lower Mainland of British Columbia, Canada, a region characterized by rapid urbanization and intensive agricultural activity. STEC were recovered from 21.6, 23.2, 19.5, and 9.2% of surface water samples collected monthly from five sites in each watershed over a period of 1 year. Overall prevalence was subject to seasonal variation however, ranging between 13.3% during fall months and 34.3% during winter months. STEC were also recovered from 23.8% of sediment samples collected in one randomly selected site. One hundred distinct STEC isolates distributed among 29 definitive and 4 ambiguous or indeterminate serotypes were recovered from water and sediments, including isolates from Canadian "priority" serogroups O157 (3), O26 (4), O103 (5), and O111 (7). Forty seven isolates were further characterized by analysis of whole genome sequences to detect Shiga toxin gene (stx 1 and stx 2), intimin gene (eaeA) allelic variants and acquired virulence factors. These analyses collectively showed that surface waters from the region support highly diverse STEC populations that include strains with virulence factors commonly associated with human pathotypes. The present work served to characterize the microbiological hazard implied by STEC to support future assessments of risks to public health arising from non-agricultural and agricultural uses of surface water resources in the region.

Biofilm Formation, Virulence Gene Profiles, and Antimicrobial Resistance of Nine Serogroups of Non-O157 Shiga Toxin-Producing Escherichia coli.

The objectives of this study were to characterize the phenotype and genotype of 36 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from humans, ovines, or bovines, including the top 6 (O26, O45, O103, O111, O121, and O145) and three other serogroups implicated in serious illness (O91, O113, and O128). Biofilms were formed by all strains with intermediate to strong biofilm producers (n = 24) more common at 22°C than at 37°C (p < 0.001) and 48 and 72 h (p < 0.001) than 24 h of incubation time. Biofilm-forming potential differed by serogroup and origin with O113 and human strains exhibiting the highest potential (p < 0.001). Biofilm-associated genes, csgA/csgD/crl/fimH (100%), flu (94%), rpoS (92%), ehaA(α) (89%), and cah (72%), were most prevalent, while mlrA (22%) and ehaA(ß) (14%) were least prevalent, although there was no clear compliment of genes associated with strains exhibiting the greatest biofilm-forming capacity. Among 12 virulence genes screened, iha and ehxA were present in 92% of the strains. The occurrence of stx1 in the top 6 serogroups (8/12, 67%) did not differ (p = 0.8) from other serogroups (17/24, 71%), but stx2 was less likely (confidence interval [CI] = 0.14-1.12; p = 0.04) to be in the former (9/24, 38%) than the latter (9/12, 75%). Excluding serogroups, O91 and O121, at least one strain per serogroup was resistant to between three and six antimicrobials. Streptomycin (31%), sulfisoxazole (31%), and tetracycline (25%) resistance was most common and was 35-50% less likely (p < 0.05) in human than animal strains. All non-O157 STEC strains were able to form biofilms on an abiotic surface, with some exhibiting resistance to multiple antimicrobials. Potential as a reservoir of antimicrobial resistance genes may be another hazard of biofilms in food-processing plants. As a result, future strategies to control these pathogens may include measures to prevent biofilms.

Fecal prevalence, serotype distribution and antimicrobial resistance of Salmonellae in dairy cattle in central Ethiopia.

BACKGROUND: Salmonellae are major worldwide zoonotic pathogens infecting a wide range of vertebrate species including humans. Consumption of contaminated dairy products and contact with dairy cattle represent a common source of non-typhoidal Salmonella infection in humans. Despite a large number of small-scale dairy farms in Addis Ababa and its surrounding districts, little is known about the status of Salmonella in these farms. RESULTS: Salmonella was recovered from the feces of at least one animal in 7.6% (10/132) of the dairy farms. Out of 1203 fecal samples examined, 30 were positive for Salmonella resulting in a weighted animal level prevalence of 2.3%. Detection of diarrhea in an animal and in a farm was significantly associated with animal level (p = 0.012) and herd level (p < 0.001) prevalence of Salmonella. Animal level prevalence of Salmonella was significantly associated with age (p = 0.023) and study location; it was highest among those under 6 months of age and in farms from Adaa district and Addis Ababa (p < 0.001). Nine different serotypes were identified using standard serological agglutination tests. The most frequently recovered serotypes were Salmonella Typhimurium (23.3%), S. Saintpaul (20%), S. Kentucky (16.7%) and S. Virchow (16.7%). All isolates were resistant or intermediately resistant to at least one of the 18 drugs tested. Twenty-six (86.7%), 19 (63.3 %), 18 (60%), 16 (53.3%) of the isolates were resistant to streptomycin, nitrofurantoin, sulfisoxazole and tetracycline , respectively. Resistance to 2 drugs was detected in 27 (90%) of the isolates. Resistance to 3 or more drugs was detected in 21 (70%) of the isolates, while resistance to 7 or more drugs was detected in 11 (36.7%) of the isolates. The rate of occurrence of multi-drug resistance (MDR) in Salmonella strains isolated from dairy farms in Addis Ababa was significantly higher than those isolated from farms outside of Addis Ababa (p = 0.009). MDR was more common in S. Kentucky, S. Virchow and S. Saintpaul. CONCLUSION: Isolation of Salmonella serotypes commonly known for causing human salmonellosis that are associated with an MDR phenotype in dairy farms in close proximity with human population is a major public health concern. These findings imply the need for a strict pathogen reduction strategy.

Complete Genome and Plasmid Sequences of Three Canadian Isolates of Salmonella enterica subsp. enterica Serovar Heidelberg from Human and Food Sources.

Isolates of Salmonella enterica subsp. enterica serovar Heidelberg are often associated with poultry products and may cause severe human illness. Here, we report the fully assembled genome and plasmid sequences of three S. Heidelberg strains with phage types 9, 29, and 41.

Complete Genome and Plasmid Sequences of Three Canadian Strains of Salmonella enterica subsp. enterica Serovar Enteritidis Belonging to Phage Types 8, 13, and 13a.

Salmonella enterica subsp. enterica serovar Enteritidis is a prominent cause of human salmonellosis frequently linked to poultry products. In Canada, S. Enteritidis phage types 8, 13, and 13a predominate among both clinical and poultry isolates. Here, we report the complete genome and plasmid sequences of poultry isolates of these three phage types.

Feces of feedlot cattle contain a diversity of bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli.

This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.