Overcoming Microbial Inhibition of S. Sonnei Through the Exploitation of Genomically Predicted Antibiotic Resistance Profiles for the Development of Food Enrichment Media.

Linking outbreaks of Shigella spp. to specific foods is challenging due to poor selectivity of current enrichment media. We have previously shown that enrichment media, tailored to the genomically-predicted antimicrobial resistance (AMR) of Shiga toxigenic E. coli strains, enhances their isolation from foods. This study investigates the application of this approach for Shigella isolation. The AMR gene profiles of 21,908 published S. sonnei genomes indicated a high prevalence of genes conferring resistance to streptomycin (aadA, aph(3″)-Ib, aph(6)-Id, 92.8%), sulfonamides (sul1, sul2, 74.8%), and/or trimethoprim (dfrA, 96.2%). Genomic analysis and antibiotic susceptibility testing conducted with a panel of 17 outbreak-associated S. sonnei strains confirmed the correlation of AMR gene detection with resistance phenotypes. Supplementation of Shigella Broth (SB) with up to 400 µg/mL of trimethoprim or sulfadiazine did not suppress the growth of sensitive strains, whereas 100 µg/mL of streptomycin increased the selectivity of this broth. All three antibiotics increased the selectivity of modified Tryptone Soya Broth (mTSB). Based on these results, supplemented media formulations were developed and assessed by measuring the relative growth of S. sonnei in cultures coinoculated with a strain of bacteriocin-producing E. coli that is inhibitory to Shigella growth. S. sonnei was not recovered from cocultures grown in SB or mTSB without antibiotics. In contrast, media supplemented with streptomycin at 50 and 100 µg/mL, trimethoprim at 25 and 50 µg/mL, and sulfadiazine at 100 µg/mL increased the relative proportion of S. sonnei in postenrichment cultures. The enhanced recovery of resistant S. sonnei strains achieved in this study indicates that, in cases where genomic data are available for clinical S. sonnei isolates, customization of selective enrichment media based on AMR gene detection could be a valuable tool for supporting the investigation of foodborne shigellosis outbreaks.

Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

Strain-specific Recovery of S. sonnei from Artificially Contaminated Baby Carrots: Enhancing Food-safety Investigations with a Customized Shigella Detection Method Based on Genomically predicted Antibiotic Resistance Traits.

Shigella spp. are Gram-negative gastrointestinal bacterial pathogens that cause bacillary dysentery or shigellosis in humans. Isolation of Shigella from outbreak-associated foods is often problematic due to the lack of selectivity of cultural enrichment broths. To facilitate Shigella recovery from foods, we have developed strain-specific enrichment media based on the genomically-predicted antimicrobial resistance (AMR) features of an outbreak-associated Shigella sonnei strain harboring resistance genes for streptomycin (STR) and trimethoprim (TMP). To assess performance of the method, baby carrots were artificially contaminated with the S. sonnei strain at low (2.4 CFU), medium (23.5 CFU), and high levels (235 CFU) along with 10-fold higher levels of a Shigella-inhibiting Escherichia coli strain. The target S. sonnei strain was successfully recovered from artificially-contaminated baby carrots when enriched in modified Tryptone Soya Broth (mTSB) supplemented with TMP, whereas Shigella was not recovered from Shigella broth (SB) or SB supplemented with STR. Quantitative PCR analysis indicated that supplementation of the enrichment broths with TMP or STR increased the relative proportion of S. sonnei in enrichment cultures, except at the lowest inoculation level for STR. Microbiome profiling of the baby carrot enrichment cultures conducted by 16S rRNA gene sequencing indicated that both SB-STR and mTSB-TMP repressed the growth of competing Enterobacteriaceae in the enrichment cultures, relative to SB without supplementation. Overall, improved Shigella recovery was achieved with the addition of the appropriate custom selective agent during cultural enrichments demonstrating that genomically informed custom selective enrichment of Shigella could be a valuable tool for supporting future foodborne shigellosis outbreak investigations.

Analysis of Antimicrobial Resistance in Bacterial Pathogens Recovered from Food and Human Sources: Insights from 639,087 Bacterial Whole-Genome Sequences in the NCBI Pathogen Detection Database.

Understanding the role of foods in the emergence and spread of antimicrobial resistance necessitates the initial documentation of antibiotic resistance genes within bacterial species found in foods. Here, the NCBI Pathogen Detection database was used to query antimicrobial resistance gene prevalence in foodborne and human clinical bacterial isolates. Of the 1,843,630 sequence entries, 639,087 (34.7%) were assigned to foodborne or human clinical sources with 147,788 (23.14%) from food and 427,614 (76.88%) from humans. The majority of foodborne isolates were either Salmonella (47.88%), Campylobacter (23.03%), Escherichia (11.79%), or Listeria (11.3%), and the remaining 6% belonged to 20 other genera. Most foodborne isolates were from meat/poultry (95,251 or 64.45%), followed by multi-product mixed food sources (29,892 or 20.23%) and fish/seafood (6503 or 4.4%); however, the most prominent isolation source varied depending on the genus/species. Resistance gene carriage also varied depending on isolation source and genus/species. Of note, Klebsiella pneumoniae and Enterobacter spp. carried larger proportions of the quinolone resistance gene qnrS and some clinically relevant beta-lactam resistance genes in comparison to Salmonella and Escherichia coli. The prevalence of mec in S. aureus did not significantly differ between meat/poultry and multi-product sources relative to clinical sources, whereas this resistance was rare in isolates from dairy sources. The proportion of biocide resistance in Bacillus and Escherichia was significantly higher in clinical isolates compared to many foodborne sources but significantly lower in clinical Listeria compared to foodborne Listeria. This work exposes the gaps in current publicly available sequence data repositories, which are largely composed of clinical isolates and are biased towards specific highly abundant pathogenic species. We also highlight the importance of requiring and curating metadata on sequence submission to not only ensure correct information and data interpretation but also foster efficient analysis, sharing, and collaboration. To effectively monitor resistance carriage in food production, additional work on sequencing and characterizing AMR carriage in common commensal foodborne bacteria is critical.

Modeling the limits of detection for antimicrobial resistance genes in agri-food samples: a comparative analysis of bioinformatics tools.

BACKGROUND: Although the spread of antimicrobial resistance (AMR) through food and its production poses a significant concern, there is limited research on the prevalence of AMR bacteria in various agri-food products. Sequencing technologies are increasingly being used to track the spread of AMR genes (ARGs) in bacteria, and metagenomics has the potential to bypass some of the limitations of single isolate characterization by allowing simultaneous analysis of the agri-food product microbiome and associated resistome. However, metagenomics may still be hindered by methodological biases, presence of eukaryotic DNA, and difficulties in detecting low abundance targets within an attainable sequence coverage. The goal of this study was to assess whether limits of detection of ARGs in agri-food metagenomes were influenced by sample type and bioinformatic approaches. RESULTS: We simulated metagenomes containing different proportions of AMR pathogens and analysed them for taxonomic composition and ARGs using several common bioinformatic tools. Kraken2/Bracken estimates of species abundance were closest to expected values. However, analysis by both Kraken2/Bracken indicated presence of organisms not included in the synthetic metagenomes. Metaphlan3/Metaphlan4 analysis of community composition was more specific but with lower sensitivity than the Kraken2/Bracken analysis. Accurate detection of ARGs dropped drastically below 5X isolate genome coverage. However, it was sometimes possible to detect ARGs and closely related alleles at lower coverage levels if using a lower ARG-target coverage cutoff (< 80%). While KMA and CARD-RGI only predicted presence of expected ARG-targets or closely related gene-alleles, SRST2 (which allows read to map to multiple targets) falsely reported presence of distantly related ARGs at all isolate genome coverage levels. The presence of background microbiota in metagenomes influenced the accuracy of ARG detection by KMA, resulting in mcr-1 detection at 0.1X isolate coverage in the lettuce but not in the beef metagenome. CONCLUSIONS: This study demonstrates accurate detection of ARGs in synthetic metagenomes using various bioinformatic methods, provided that reads from the ARG-encoding organism exceed approximately 5X isolate coverage (i.e. 0.4% of a 40 million read metagenome). While lowering thresholds for target gene detection improved sensitivity, this led to the identification of alternative ARG-alleles, potentially confounding the identification of critical ARGs in the resistome. Further advancements in sequencing technologies providing increased coverage depth or extended read lengths may improve ARG detection in agri-food metagenomic samples, enabling use of this approach for tracking clinically important ARGs in agri-food samples.

Targeted metagenomics using bait-capture to detect antibiotic resistance genes in retail meat and seafood.

Metagenomics analysis of foods has the potential to provide comprehensive data on the presence and prevalence of antimicrobial resistance (AMR) genes in the microbiome of foods. However, AMR genes are generally present in low abundance compared to other bacterial genes in the food microbiome and consequently require multiple rounds of in-depth sequencing for detection. Here, a metagenomics approach, using bait-capture probes targeting antimicrobial resistance and plasmid genes, is used to characterize the resistome and plasmidome of retail beef, chicken, oyster, shrimp, and veal enrichment cultures (n = 15). Compared to total shotgun metagenomics, bait-capture required approximately 40-fold fewer sequence reads to detect twice the number of AMR gene classes, AMR gene families, and plasmid genes across all sample types. For the detection of critically important extended spectrum beta-lactamase (ESBL) genes the bait capture method had a higher overall positivity rate (44%) compared to shotgun metagenomics (26%), and a culture-based method (29%). Overall, the results support the use of bait-capture for the identification of low abundance genes such as AMR genes from food samples.

Correction: Genome Dynamics of Campylobacter jejuni in Response to Bacteriophage Predation.

[This corrects the article DOI: 10.1371/journal.ppat.0030119.].

Exploiting a targeted resistome sequencing approach in assessing antimicrobial resistance in retail foods.

BACKGROUND: With the escalating risk of antimicrobial resistance (AMR), there are limited analytical options available that can comprehensively assess the burden of AMR carried by clinical/environmental samples. Food can be a potential source of AMR bacteria for humans, but its significance in driving the clinical spread of AMR remains unclear, largely due to the lack of holistic-yet-sensitive tools for surveillance and evaluation. Metagenomics is a culture-independent approach well suited for uncovering genetic determinants of defined microbial traits, such as AMR, present within unknown bacterial communities. Despite its popularity, the conventional approach of non-selectively sequencing a sample's metagenome (namely, shotgun-metagenomics) has several technical drawbacks that lead to uncertainty about its effectiveness for AMR assessment; for instance, the low discovery rate of resistance-associated genes due to their naturally small genomic footprint within the vast metagenome. Here, we describe the development of a targeted resistome sequencing method and demonstrate its application in the characterization of the AMR gene profile of bacteria associated with several retail foods. RESULT: A targeted-metagenomic sequencing workflow using a customized bait-capture system targeting over 4,000 referenced AMR genes and 263 plasmid replicon sequences was validated against both mock and sample-derived bacterial community preparations. Compared to shotgun-metagenomics, the targeted method consistently provided for improved recovery of resistance gene targets with a much-improved target detection efficiency (> 300-fold). Targeted resistome analyses conducted on 36 retail-acquired food samples (fresh sprouts, n = 10; ground meat, n = 26) and their corresponding bacterial enrichment cultures (n = 36) reveals in-depth features regarding the identity and diversity of AMR genes, most of which were otherwise undetected by the whole-metagenome shotgun sequencing method. Furthermore, our findings suggest that foodborne Gammaproteobacteria could be the major reservoir of food-associated AMR genetic determinants, and that the resistome structure of the selected high-risk food commodities are, to a large extent, dictated by microbiome composition. CONCLUSIONS: For metagenomic sequencing-based surveillance of AMR, the target-capture method presented herein represents a more sensitive and efficient approach to evaluate the resistome profile of complex food or environmental samples. This study also further implicates retail foods as carriers of diverse resistance-conferring genes indicating a potential impact on the dissemination of AMR.

Genomically Informed Custom Selective Enrichment of Shiga Toxigenic Escherichia coli (STEC) Outbreak Strains in Foods Using Antibiotics.

Shiga toxigenic Escherichia coli (STEC) have been implicated in major foodborne outbreaks worldwide. The STEC family of pathogens is biochemically diverse, and current microbiological methods for detecting STEC are limited by the lack of a universal selective enrichment approach and prone to interference by high levels of background microbiota associated with certain types of foods. A novel approach has been developed for the recovery of foodborne illness outbreak strains during outbreak investigations based on the analysis of whole genome sequence data of implicated clinical isolates to determine antimicrobial resistance (AMR) genes. The presence of certain AMR genes in STEC has been correlated with the ability to grow in the presence of a specific antibiotic, which can be used to supplement enrichment broths to improve the recovery of a target strain. The enhanced recovery of STEC strains with different AMR profiles from various food types (beef, sprouts, leafy greens, and raw milk cheese) containing high levels of background microbiota was demonstrated using AMR predictions for nine different antibiotics. This genomically informed custom selective enrichment approach increases the availability of analytical options and improves the reliability of food microbiological analyses in confirming food vehicles implicated in outbreak events and defining the scope of product contamination to support risk assessment and risk management actions.

Virulence potential of antimicrobial-resistant extraintestinal pathogenic Escherichia coli from retail poultry meat in a Caenorhabditis elegans model.

Healthy poultry can be a reservoir for extraintestinal pathogenic Escherichia coli (ExPEC), some of which could be multidrug resistant to antimicrobials. These ExPEC strains could contaminate the environment and/or food chain representing thus, food safety and human health risk. However, few studies have shown the virulence of poultry-source antimicrobial-resistant (AMR) ExPEC in humans. This study characterized AMR ExPEC and investigated the virulence potential of some of their isolates in a Caenorhabditis elegans infection model. A total of 46 E. coli isolates from poultry (chicken, n = 29; turkey, n = 12) retail meats and chicken feces (n = 4), or humans (n = 1) were sequenced and identified as ExPEC. Except eight, all remaining 38 ExPEC isolates were resistant to at least one antibiotic and carried corresponding antimicrobial resistance genes (ARGs). About 27 of the 46 ExPEC isolates were multidrug-resistant (≥3 antibiotic classes). Seven ExPEC isolates from chicken or turkey meats were of serotype O25:H4 and sequence type (ST) 131 which clustered with an isolate from a human urinary tract infection (UTI) case having the same serotype and ST. The C. elegans challenge model using eight of studied ExPEC isolates harboring various ARGs and virulence genes (VGs) showed that regardless of their ARG or VG numbers in tested poultry meat and feces, ExPEC significantly reduced the life span of the nematode (P < 0.05) similarly to a human UTI isolate. This study indicated the pathogenic potential of AMR ExPEC from retail poultry meat or feces, but more studies are warranted to establish their virulence in poultry and human. Furthermore, relationships between specific resistance profiles and/or VGs in these E. coli isolates for their pathogenicity deserve investigations.

Phage and Antibiotic Combinations Reduce Staphylococcus aureus in Static and Dynamic Biofilms Grown on an Implant Material.

Staphylococcus aureus causes the majority of implant-related infections. These infections present as biofilms, in which bacteria adhere to the surface of foreign materials and form robust communities that are resilient to the human immune system and antibiotic drugs. The heavy use of broad-spectrum antibiotics against these pathogens disturbs the host's microbiome and contributes to the growing problem of antibiotic-resistant infections. The use of bacteriophages as antibacterial agents is a potential alternative therapy. In this study, bioluminescent strains of S. aureus were grown to form 48-h biofilms on polyether ether ketone (PEEK), a material used to manufacture orthopaedic implants, in either static or dynamic growth conditions. Biofilms were treated with vancomycin, staphylococcal phage, or a combination of the two. We showed that vancomycin and staph phages were able to independently reduce the total bacterial load. Most phage-antibiotic combinations produced greater log reductions in surviving bacteria compared to single-agent treatments, suggesting antimicrobial synergism. In addition to demonstrating the efficacy of combining vancomycin and staph phage, our results demonstrate the importance of growth conditions in phage-antibiotic combination studies. Dynamic biofilms were found to have a substantial impact on apparent treatment efficacy, as they were more resilient to combination treatments than static biofilms.

Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast.

BACKGROUND: Sequence-based methods for the detection of bacteria such as 16S rRNA amplicon sequencing and metagenomics can provide a comprehensive view of the bacterial microbiome of food. These methods rely on the detection of gene sequences to indicate the presence of viable bacteria. This indirect form of detection can be prone to experimental artefacts. Sample handling and processing are key sources of variation that require standard approaches. Extracting sufficient quantities of high quality DNA from food matrices is challenging because target bacterial species are usually minor components of the microbiota and foods contain an array of compounds that are inhibitory to downstream DNA applications. Here, three DNA extraction methods are compared for their ability to extract high quality bacterial DNA from retail chicken breast rinses, with or without enrichment. Method performance was assessed by comparing ease of use, DNA yield, DNA quality, PCR amplicon yield, and the detection of bacterial taxa by 16S rRNA amplicon sequencing. RESULTS: All three DNA extraction methods yielded DNA of sufficient quantity and quality to perform quantitative PCR and 16S rRNA amplicon sequencing. The extraction methods differed in ease of use, with the two commercial kits (PowerFood, PowerSoil) offering considerable time and cost savings over a hybrid method that used laboratory reagents for lysis and commercial column based kits for further purification. Bacterial richness as determined by 16S rRNA amplicon sequencing was similar across the three DNA extraction methods. However, differences were noted in the relative abundance of bacterial taxa, with significantly higher abundance of Gram-positive genera detected in the DNA samples prepared using the PowerFood DNA extraction kit. CONCLUSION: The choice of DNA extraction method can affect the detection of bacterial taxa by 16S rRNA amplicon sequencing in chicken meat rinses. Investigators should be aware of this procedural bias and select methods that are fit for the purposes of their investigation.

Microbial Antagonism in Food-Enrichment Culture: Inhibition of Shiga Toxin-Producing Escherichia coli and Shigella Species.

Bacterial pathogens, such as Shiga toxin-producing Escherichia coli (STEC) and Shigella spp., are important causes of foodborne illness internationally. Recovery of these organisms from foods is critical for food safety investigations to support attribution of illnesses to specific food commodities; however, isolation of bacterial cultures can be challenging. Methods for the isolation of STEC and Shigella spp. from foods typically require enrichment to amplify target organisms to detectable levels. Yet, during enrichment, target organisms can be outcompeted by other bacteria in food matrices due to faster growth rates, or through production of antimicrobial agents such as bacteriocins or bacteriophages. The purpose of this study was to evaluate the occurrence of Shigella and STEC inhibitors produced by food microbiota. The production of antimicrobial compounds in cell-free extracts from 200 bacterial strains and 332 food-enrichment broths was assessed. Cell-free extracts produced by 23 (11.5%) of the strains tested inhibited growth of at least one of the five Shigella and seven STEC indicator strains used in this study. Of the 332 enrichment broths tested, cell-free extracts from 25 (7.5%) samples inhibited growth of at least one of the indicator strains tested. Inhibition was most commonly associated with E. coli recovered from meat products. Most of the inhibiting compounds were determined to be proteinaceous (34 of the 48 positive samples, 71%; including 17 strains, 17 foods) based on inactivation by proteolytic enzymes, indicating presence of bacteriocins. The cell-free extracts from 13 samples (27%, eight strains, five foods) were determined to contain bacteriophages based on the observation of plaques in diluted extracts and/or resistance to proteolytic enzymes. These results indicate that the production of inhibitors by food microbiota may be an important challenge for the recovery of foodborne pathogens, particularly for Shigella sonnei. The performance of enrichment media for recovery of Shigella and STEC could be improved by mitigating the impact of inhibitors produced by food microbiota during the enrichment process.

Characterization of Atypical Shiga Toxin Gene Sequences and Description of Stx2j, a New Subtype.

Shiga toxin (Stx) is the definitive virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx variants are currently organized into a taxonomic system of three Stx1 (a, c, and d) and seven Stx2 (a, b, c, d, e, f, and g) subtypes. In this study, seven STEC isolates from food and clinical samples possessing stx2 sequences that do not fit current Shiga toxin taxonomy were identified. Genome assemblies of the STEC strains were created from Oxford Nanopore and Illumina sequence data. The presence of atypical stx2 sequences was confirmed by Sanger sequencing, as were Stx2 expression and cytotoxicity. A strain of O157:H7 was found to possess stx1a and a truncated stx2a, which were originally misidentified as an atypical stx2. Two strains possessed unreported variants of Stx2a (O8:H28) and Stx2b (O146:H21). In four of the strains, we found three Stx subtypes that are not included in the current taxonomy. Stx2h (O170:H18) was identified in a Canadian sprout isolate; this subtype has only previously been reported in STEC from Tibetan Marmots. Stx2o (O85:H1) was identified in a clinical isolate. Finally, Stx2j (O158:H23 and O33:H14) was found in lettuce and clinical isolates. The results of this study expand the number of known Stx subtypes, the range of STEC serotypes, and isolation sources in which they may be found. The presence of the Stx2j and Stx2o in clinical isolates of STEC indicates that strains carrying these variants are potential human pathogens.

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.

Randomized, Double-Blind Placebo-Controlled Trial to Assess the Effect of Probiotics on Irritable Bowel Syndrome in Veterans With Gulf War Illness.

Background: Many veterans who served in Operation Desert Storm (August 1990 to March 1991) experienced a complex of symptoms of unknown etiology called Gulf War illness (GWI), which significantly impacts the health and quality of life (QOL) and may have contributed to irritable bowel syndrome (IBS). Methods: We performed a prospective, double-blind placebocontrolled study to determine the efficacy of the multistrain De Simone Formulation probiotic containing 8 strains of bacteria on symptoms of IBS and GWI. Veterans of Operation Desert Storm who had IBS and ≥ 2 nonintestinal symptoms of GWI were included. The primary study endpoint was change in bowel symptom score. The secondary endpoints were mean change in symptoms, QOL, and extra-intestinal and posttraumatic stress disorder (PTSD) symptoms. Results: A total of 101 Gulf War veterans with IBS and GWI were screened at the Veteran Affairs Medical Center in Salt Lake City, Utah. The study was completed by 53 veterans; 47 (89%) were male with a mean (SD) age of 55 (8) years. The probiotic did not improve IBS symptoms or other extra-intestinal symptoms common to IBS and GWI. Conclusions: Our study did not demonstrate statistically significant improvement in IBS symptoms or QOL after treatment with the probiotic. We also did not find any improvement in symptoms of GWI or PTSD.

Combining analytical epidemiology and genomic surveillance to identify risk factors associated with the spread of antimicrobial resistance in Salmonella enterica subsp. enterica serovar Heidelberg.

Antimicrobial resistance (AMR) has become a critical threat to public health worldwide. The use of antimicrobials in food and livestock agriculture, including the production of poultry, is thought to contribute to the dissemination of antibiotic resistant bacteria (ARB) and the genes and plasmids that confer the resistant phenotype (ARG). However, the relative contribution of each of these processes to the emergence of resistant pathogens in poultry production and their potential role in the transmission of resistant pathogens in human infections, requires a deeper understanding of the dynamics of ARB and ARG in food production and the factors involved in the increased risk of transmission.

Systematic Evaluation of Whole-Genome Sequencing Based Prediction of Antimicrobial Resistance in Campylobacter jejuni and C. coli.

The increasing prevalence of antimicrobial resistance (AMR) in Campylobacter spp. is a global concern. This study evaluated the use of whole-genome sequencing (WGS) to predict AMR in Campylobacter jejuni and C. coli. A panel of 271 isolates recovered from Canadian poultry was used to compare AMR genotype to antimicrobial susceptibility testing (AST) results (azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin). The presence of antibiotic resistance genes (ARGs) was determined for each isolate using five computational approaches to evaluate the effect of: ARG screening software, input data (i.e., raw reads, draft genome assemblies), genome coverage and genome assembly software. Overall, concordance between the genotype and phenotype was influenced by the computational pipelines, level of genome coverage and the type of ARG but not by input data. For example, three of the pipelines showed a 99% agreement between detection of a tet(O) gene and tetracycline resistance, whereas agreement between the detection of tet(O) and TET resistance was 98 and 93% for two pipelines. Overall, higher levels of genome coverage were needed to reliably detect some ARGs; for example, at 15X coverage a tet(O) gene was detected in >70% of the genomes, compared to <60% of the genomes for bla(OXA). No genes associated with florfenicol or gentamicin resistance were found in the set of strains included in this study, consistent with AST results. Macrolide and fluoroquinolone resistance was associated 100% with mutations in the 23S rRNA (A2075G) and gyrA (T86I) genes, respectively. A lower association between a A2075G 23S rRNA gene mutation and resistance to clindamycin and telithromycin (92.8 and 78.6%, respectively) was found. While WGS is an effective approach to predicting AMR in Campylobacter, this study demonstrated the impact that computational pipelines, genome coverage and the genes can have on the reliable identification of an AMR genotype.

Shiga Toxin-Producing Escherichia coli (STEC) and STEC-Associated Virulence Genes in Raw Ground Pork in Canada.

ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses.

Antimicrobial Resistance Phenotypes and Genotypes of Escherichia coli Isolates from Broiler Chickens Fed Encapsulated Cinnamaldehyde and Citral.

ABSTRACT: This study was conducted to investigate the effects of in-feed encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination on antimicrobial resistance (AMR) phenotypes and genotypes of Escherichia coli isolates recovered from feces of 6-, 16-, 23-, and 27-day-old broiler chickens. The five dietary treatments including the basal diet (negative control [NC]) and the basal diet supplemented with 55 ppm of bacitracin (BAC), 100 ppm of encapsulated CIN, 100 ppm of encapsulated CIT, or 100 ppm each of encapsulated CIN and encapsulated CIT (CIN+CIT). Antimicrobial susceptibility testing of 240 E. coli isolates revealed that the most common resistance was to ß-lactams, aminoglycosides, sulfonamides, and tetracycline; however, the prevalence of AMR decreased (P < 0.05) as birds aged. The prevalence of resistance to amoxicillin-clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, gentamicin, and sulfonamide was lower (P < 0.05) in isolates from the CIN or CIN+CIT groups than in isolates from the NC or BAC groups. Whole genome sequencing of 227 of the 240 isolates revealed 26 AMR genes and 19 plasmids, but the prevalence of some AMR genes and the number of plasmids were lower (P < 0.05) in E. coli isolated from CIN or CIN+CIT birds than in isolates from NC or BAC birds. The most prevalent resistance genes were tet(A) (108 isolates), aac(3)-VIa (91 isolates), aadA1 (86 isolates), blaCMY-2 (78 isolates), sul1 (77 isolates), aph(3)-Ib (58 isolates), aph(6)-Id (58 isolates), and sul2 (24 isolates). The numbers of most virulence genes carried by isolates increased (P < 0.05) in chickens from 6 to 27 days of age. The prevalence of E. coli O21:H16 isolates was lower (P < 0.05) in CIN and CIN+CIT, and the colibacillosis-associated multilocus sequence type (ST117) was most prevalent in isolates from 23-day-old chickens. A phylogenetic tree of whole genome sequences revealed a close relationship between 25 of the 227 isolates and human or broiler extraintestinal pathogenic E. coli strains. These findings indicate that AMR and virulence genotypes of E. coli could be modulated by providing encapsulated CIN or CIN+CIT feed supplements, but further investigation is needed to determine the mechanisms of the effects of these supplements.