John M. Fairbrother’s research while affiliated with Université de Montréal and other places

Bacteriophages have shown promise as an alternative to antibiotics for managing or preventing avian pathogenic Escherichia coli (APEC) in the egg industry. In a previous study, a phage cocktail comprising 3 virulent phages was shown to prevent APEC infections in laying hens. The aim of this study was to investigate whether orally and intramuscularly administered phages affect the intestinal barrier function of chickens, with a focus on cecal microbiome. In a 4-d trial, a total of 35 laying hens were randomly assigned to one of four treatment groups: 1) Medium control group (bacterial broth + phage buffer), 2) APEC only control (APEC + buffer); 3) IM group (Phage +APEC), or 4) DW group (phage in drinking water (4 d prior to APEC + APEC). Cecum samples were subjected to 16S rRNA gene sequencing targeting the V4 region to assess the effects of phages on cecal bacterial diversity. A 1-way ANOVA was used to evaluate alpha diversity with respect to treatment and time. Microbial community structure was analyzed using β-dispersion and permutational multivariate analysis of variance (PERMANOVA) to determine the effect of each treatment. Significant (P < 0.05) differentially abundant genera were identified with DESeq2 by fitting a negative binomial model to each treatment vs treatment comparison (“~Treatment”). Across the treatment and control groups, 13 phyla were identified. Firmicutes (53.81%), Bacteroidota (23.83%), Actinobacteriota (1.84%), and Proteobacteria (1.02%) were the most abundant phyla among the amplicon sequence variants (ASVs), with 18.39% being unclassified. Neither richness (P = 0.448) nor Shannon diversity (P = 0.687) exhibited significance in α- diversity metric between treatment and control groups. β-dispersion analysis showed no significant difference (P = 0.239) between treatment and control groups, indicating similar bacterial variability among groups. Moreover, the β-diversity representing microbiota structure was not affected by any phage treatment (P = 0.083). In each treatment group, 76 unique genera were identified, with Megamonas (15.80 %), Lactobacillus (8.76%), Faecalibacterium (7.70 %), Megasphaera (1.58%), Alloprevotella (1.54%), [Ruminococcus] torques group (1.29%), Prevotellaceae UCG-001 (1.14%), Bifidobacterium (1.04%), and Olsenella (0.48%) being the most abundant. Notably, Lactobacillus was more abundant (~ 10 to 20%) in medium control group compared with phage treated and APEC only treatment groups. Furthermore, gene expression profiles of various genera (pairwise) were analyzed using log2 fold change (log2FC), revealing differences (P < 0.05) between control and treatment groups for a total of 58 genera. It was concluded that bacteriophage selectively killed APEC population without comprising the population and structure of cecal microbiome in laying hens.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5–9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

Despite its importance in veterinary medicine, there is little information about antimicrobial resistance (AMR) and its transmission in dairy cattle. The aim of this work is to compare AMR phenotypes and genotypes in resistant Escherichia coli and to determine how the resistance genes spread among the E. coli population on dairy farms in Québec, Canada. From an existing culture collection of E. coli isolated from dairy manure, a convenient selection of the most resistant isolates (a high level of multidrug resistance or resistance to broad-spectrum β-lactams or fluoroquinolones) was analyzed (n = 118). An AMR phenotype profile was obtained for each isolate. Whole genome sequencing was used to determine the presence of resistance genes, point mutations, and mobile genetic elements. In addition, a subset of isolates from 86 farms was taken to investigate the phylogenetic relationship and geographic distribution of the isolates. The average agreement between AMR phenotypes and genotypes was 95%. A third-generation cephalosporin resistance gene (blaCTX-M-15), a resistance gene conferring reduced susceptibility to fluoroquinolones (qnrS1), and an insertion sequence (ISKpn19) were detected in the vicinity of each other on the genome. These genes were harbored in one triplet of clonal isolates from three farms located >100 km apart. Our study reveals the dissemination of resistant E. coli clones between dairy farms. Furthermore, these clones are resistant to broad-spectrum β-lactam and fluoroquinolone antimicrobials.

In animals, the most important pathotypes are intestinal pathogenic Escherichia coli : enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga toxin‐producing E. coli (STEC), and extraintestinal pathogenic E. coli . E. coli cause a variety of enteric and extraintestinal diseases in animals. Virulence factors of pathogenic E. coli are now grouped by functional categories, which include adhesins, toxins, autotransporters, secretion systems, invasins, metal transport/uptake systems, protectins, regulation, and fitness factors. ETEC enter the animal by the oral route and, when present in sufficient numbers, colonize the small intestine following attachment by fimbrial adhesins to receptors on the small intestinal epithelium or in the mucus coating the epithelium. The underlying principles of pathogenesis are the same for a wide range of clinical syndromes observed in animals with STEC disease.

To tackle antimicrobial resistance (AMR), one of the major health threats of this century, the World Health Organization (WHO) endorsed a global action plan in 2015. This plan calls countries to develop national actions to address AMR. The province of Québec, Canada, adopted a new regulation on the 25th of February 2019, to limit the use in food animals of antimicrobials of very high importance in human medicine. We aimed to establish the impact of this regulation by comparing the AMR situation in dairy cattle in Québec ~2 years before and 2 years after its introduction. We sampled calves, cows, and the manure pit in 87 farms. Generic and putative ESBL/AmpC E. coli were tested for susceptibility to 20 antimicrobials. Logistic regression was used to investigate whether the probability of antimicrobial resistance differed between isolates obtained from the pre and post regulation periods by sample type (calves, cows, manure pit) and in general. To identify AMR genes dissemination mechanisms, we sequenced the whole genome of 15 generic isolates. In the generic collection, at the herd level, the proportion of multidrug resistant (MDR) isolates, decreased significantly from 83 to 71% (p = 0.05). Folate inhibitor and aminoglycoside resistances demonstrated a significant decrease. However, when analyzed by sample type (calves, cows, manure pit), we did not observe a significant AMR decrease in any of these categories. In the ESBL/AmpC collection, we did not detect any significant difference between the two periods. Also, the general resistance gene profile was similar pre and post regulation. We identified both clonal and plasmidic dissemination of resistance genes. In conclusion, as early as 2 years post regulation implementation, we observed a significant decrease in MDR in the dairy industry in Quebec in the generic E. coli collection with folate inhibitor and aminoglycoside resistances showing the most significant decrease. No other significant decreases were yet observed.

Antimicrobial resistance (AMR) is an important burden for public health and veterinary medicine. For Québec (Canada) dairy farms, the prevalence of AMR is mostly described using passive surveillance, which may be misleading. In addition, the presence of extended spectrum β-lactamase (ESBL)/AmpC producing Escherichia coli is unknown. This observational cross-sectional study used random dairy farms (n = 101) to investigate AMR and extended spectrum β-lactamase (ESBL)/AmpC producing Escherichia coli. Twenty antimicrobials were tested on E. coli isolates (n = 593) recovered from fecal samples (n = 599) from calves, cows, and the manure pit. Isolates were mostly susceptible (3% AMR or less) to the highest priority critically important antimicrobials in humans. The highest levels of AMR were to tetracycline (26%), sulfisozaxole (23%) and streptomycin (19%). The resistance genes responsible for these resistances were, respectively: tet(A), tet(B), sul1, sul2, sul3, aph(3”)-Ib (strA), aph(6)-Id (strB), aadA1, aadA2, and aadA5. ESBL analysis revealed two predominant phenotypes: AmpC (51%) and ESBL (46%) where blaCMY−2 and blaCTX−M (blaCTX−M−1, blaCTX−M−15, and blaCTX−M−55) were the genes responsible for these phenotypes, respectively. During this study, 85% of farms had at least one ESBL/AmpC producing E. coli. Isolates from calves were more frequently resistant than those from cows or manure pits. Although prevalence of AMR was low for critically important antimicrobials, there was a high prevalence of ESBL/AmpC-producing E. coli on Quebec dairy farms, particularly in calves. Those data will help determine a baseline for AMR to evaluate impact of initiatives aimed at reducing AMR.

Aims: Describe the temporal trends in E. coli pathotypes and antimicrobial resistance detected in isolates from diseased-pig cases submitted to the EcL from 2008 to 2016, in Quebec, Canada, and investigate the presence of spatio-temporal and phylogenetic clusters. Methods and results: Detection of 12 genes coding for virulence factors in pathogenic E. coli in pigs by PCR and antimicrobial resistance standard disk diffusion assay were performed. Demographic and clinical data were entered in the Animal Pathogenic and Zoonotic Escherichia coli (APZEC) database. ETEC:F4 was the most prevalent pathovirotype among the 3773 cases submitted. The LT:STb:F4 virotype was predominant until 2014, then was overtaken by the LT:STb:STa:F4 virotype. More than 90% of the ETEC:F4 isolates were multidrug resistant. A spatio-temporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 04/2015 and 09/2016. Pulsed-field gel electrophoresis analysis of 137 ETEC:F4 isolates revealed the presence of a cluster composed mainly of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin. Conclusions: The APZEC database was useful to highlight temporal trends in E. coli pathotypes. A high-risk ETEC:F4 clone might disseminate in the pig population in Quebec since 2015. Significance and impact of the study: Surveillance is crucial to identify new clones and develop control strategies.

Although antimicrobial resistance is an increasing threat in equine medicine, molecular and epidemiological data remain limited in North America. We assessed the prevalence of, and risk factors for, shedding multidrug-resistant (MDR) and extended-spectrum -lactamase (ESBL) and/or AmpC β-lactamase-producing E. coli in healthy horses in Quebec, Canada. We collected fecal samples in 225 healthy adult horses from 32 premises. A questionnaire on facility management and horse medical history was completed for each horse. Indicator (without enrichment) and specific (following enrichment with ceftriaxone) E. coli were isolated and tested for antimicrobial susceptibility. The presence of ESBL/AmpC genes was determined by PCR. The prevalence of isolates that were non-susceptible to antimicrobials and to antimicrobial classes were estimated at the horse and the premises level. Multivariable logistic regression was used to assess potential risk factors for MDR and ESBL/AmpC isolates. The shedding of MDR E. coli was detected in 46.3% of horses. Non-susceptibility was most commonly observed to ampicillin, amoxicillin/clavulanic acid or streptomycin. ESBL/AmpC producing isolates were detected in 7.3% of horses. The most commonly identified ESBL/AmpC gene was blaCTX-M-1, although we also identified blaCMY-2. The number of staff and equestrian event participation were identified as risk factors for shedding MDR isolates. The prevalence of healthy horses harboring MDR or ESBL/AmpC genes isolates in their intestinal microbiota is noteworthy. We identified risk factors which could help to develop guidelines to preclude their spread.

The objective was to identify the genetic determinants and supports of expanded-spectrum cephalosporin (ESC) resistance in commensal Escherichia coli from healthy horses in France in 2015. Faecal samples from 744 adult horses were screened for ESC-resistant E. coli isolates. The ESBL/AmpC resistance genes were identified using PCR and sequencing. ESC phenotypes were horizontally transferred by conjugation or transformation. Plasmids carrying ESBL/AmpC genes were typed by PCR-based replicon typing, restriction fragment length polymorphism, and plasmid MLST. The ESC-resistant E. coli isolates were typed by XbaI macrorestriction analysis. Sixteen stables out of 41 harboured at least one horse carrying ESC-resistant E. coli. The proportion of individually tested horses carrying ESC-resistant E. coli was 8.5% (28/328). Fifty non-redundant ESC-resistant E. coli isolates showing a great diversity of XbaI macrorestriction profiles, belonged mainly to phylogroup B1, and were negative for major E. coli virulence genes suggesting that they are commensal isolates. ESBL blaCTX-M genes were dominant (blaCTX-M-1, n=34; blaCTX-M-2, n=8; blaCTX-M-14, n=2) and located on conjugative plasmids belonging to various incompatibility groups (IncHI1, IncI1, IncN, IncY, or non-typeable). Among these, the multidrug-resistance IncHI1-pST9 plasmids were dominant and simultaneously harboured the blaCTX-M-1/2 genes and an operon enabling the metabolism of short-chain fructo-oligosaccharides (scFOS). In conclusion, commensal E. coli of French horses displayed a significant distribution of IncHI1-pST9 plasmids carrying both the blaCTX-M-1/2 gene and the fos metabolism operon. This finding highlights the risk of co-selection of multidrug-resistance IncHI1 plasmids carrying ESBL gene possibly mediated by the use of scFOS as prebiotic in horses.

Antimicrobial resistance is a global problem. The antimicrobial ceftiofur has been used worldwide for disease prevention in poultry production, resulting in a greatly increased resistance to this antimicrobial important in poultry and human medicine. Our study examined the impact of ceftiofur cessation and its replacement with the antimicrobial combination lincomycin-spectinomycin, a common practice in the industry. Our study demonstrated a decrease in ceftiofur resistance after the cessation of ceftiofur use, although the resistance genes remain ubiquitous in all phases of poultry production, showing that poultry remains a reservoir for ceftiofur resistance and requiring continued vigilance. We also observed a decrease in multidrug resistance involving different antimicrobial classes after cessation of ceftiofur but an increase following use of lincomycin-spectinomycin, indicating that this antimicrobial use should be questioned. Reduced resistance to ceftiofur in poultry may translate to better treatment efficacy, decreased morbidity/mortality, and enhanced food safety for humans.