Exudative epidermitis (EE), caused by Staphylococcus hyicus, represents an issue for swine production, particularly due to antimicrobial resistance. In this project, we isolated bacteriophages using S. hyicus as host and studied them as a potential alternative to antibiotic treatment in Quebec, Canada. Three phages, STAE-4, STAF-3, and STAM-1, were isolated from swine farm samples using a single S. hyicus strain (SC366) as the host. Transmission electron microscopy revealed that all three phages exhibited a siphovirus-like morphology, and RAPD-PCR profiling indicated that the phages were genetically distinct. Whole genome sequencing confirmed these differences and showed that the three phages were closely related to each other, and, more importantly, highly similar to phages previously described as infecting Staphylococcus chromogenes, a species closely related to S. hyicus. Host range analysis confirmed that the three phages preferentially infected the S. chromogenes strains included in the study, exhibiting minimal to no lytic activity against other strains of S. hyicus or Staphylococcus agnetis, another closely related species. The only exception was the host S. hyicus strain SC366, which was effectively infected by all three phages, albeit less efficiently than the most sensitive S. chromogenes strain (SC385). Adsorption tests further supported these observations, showing that phages bound to strain SC366 much more quickly than to SC385, despite the lower lytic activity observed. Taken together, these results highlight that while the phages retain some capacity to infect S. hyicus, their biological properties point to a stronger adaptation to S. chromogenes, indicating that they are not suitable candidates for controlling EE.

Exudative epidermitis (EE) in piglets, caused by the bacterium Staphylococcus hyicus, remains a major concern for the swine industry, particularly due to antimicrobial resistance. Bacteriophages represent a promising alternative treatment. This study aimed to isolate and characterize phages infecting S. hyicus from EE-affected swine farm environments in Quebec. Four novel phages (BQE1, BQE4, BQE7, BQL4) were isolated from water and manure samples. All produced clear plaques (<1 mm) and reached titers of 108-1010 PFU/mL. Electron microscopy revealed myovirus morphology. Their genomes (115.4-134.5 kb) are closely related to the already described Twort phage but distinct from other S. hyicus phages. No virulence or antimicrobial resistance genes were detected in their genome. Host range analysis across 58 staphylococcal strains showed broad activity against S. hyicus (77%-80% susceptibility), whereas the Twort phage was largely ineffective (4%). Adsorption and growth assays indicated efficient infection, with latent periods of ∼50 min and burst sizes of 10-13 PFU/cell. Phages remained stable at 4 °C and room temperature but were less stable at 31 °C. Overall, these Twort-like phages display strong lytic activity and favorable genomic and biological characteristics, supporting their potential as targeted alternatives to antibiotics for controlling EE in swine production.

AIMS:Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a fish disease highly aggressive for salmonids and responsible for significant economic losses in aquaculture worldwide. This study aimed to explore genomic and antimicrobial resistance traits of Western European A. salmonicida subsp. salmonicida strains and to develop an adapted infection model using larvae of the greater wax moth Galleria mellonella to assess the pathogenic potential of this psychrophilic subspecies. METHODS AND RESULTS:Three A. salmonicida subsp. salmonicida strains, isolated from salmonids displaying clinical signs of furunculosis, were tested against a panel of antibiotics and sequenced to characterize their genome. Virulence was evaluated in G. mellonella larvae using bacterial doses ranging from 101 to 106 CFU/larva. Two isolates exhibited multidrug resistance to antibiotics commonly used against furunculosis. Although closely related to the reference strain A449, genomic analyses revealed multiple plasmids known to encode antibiotic resistance genes. Virulence assays showed that this subspecies was lethal at doses as low as 101 CFU/larva, and that a fully functional Type III secretion system (T3SS) is not essential for the infection of G. mellonella, likely due to the presence of other virulence factors in T3SS-deficient strains. CONCLUSIONS:These findings enhance the genomic characterization of European A. salmonicida subsp. salmonicida and validate the use of G. mellonella larvae as a relevant alternative infection model for studying this psychrophilic subspecies.

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria and originate from their outer membrane. They can be extracted using ultracentrifugation or ultrafiltration using concentration columns, followed by purification with a density gradient. However, these methods may co-purify contaminants with similar physical properties. Several studies have identified GroEL, a chaperonin, as a major protein in OMV preparations. Using Aeromonas salmonicida subsp. salmonicida as a model, we detected GroEL by mass spectrometry and observed it in transmission electron microscopy images as separate from OMVs. As a cytoplasmic protein complex, GroEL is more likely a contaminant resulting from bacterial lysis during growth rather than an intrinsic OMV component. The model A. salmonicida subsp. salmonicida proved valuable in reaching this conclusion because it produces high levels of extracellular GroEL and low amounts of OMVs. This study emphasizes the need for caution when interpreting the presence of GroEL in OMV preparations and highlights the importance of rigorous purification methods to ensure OMV purity.

The crystal violet (CV) assay was the standard for quantifying biofilm biomass but is limited by its inability to replicate dynamic environmental conditions. In contrast, microfluidic flow cells provided a promising alternative, enabling real-time monitoring of biofilm development under controlled and continuously changing environments. This study evaluated the performance of different methods for assessing biofilm formation under various growth conditions, using Staphylococcus epidermidis as a model bacterium. S. epidermidis biofilms are particularly challenging in biomaterial-related infections, highlighting the need for accurate methods to assess biofilm dynamics in such contexts. Biofilm formation was evaluated using the CV assay (adapted from ISO 4768) and single-use polydimethylsiloxane (PDMS) microfluidic flow cells. These flow cells featured dual identical channels, which were individually operated by syringe pumps. In the microfluidic system, inoculum of varying concentrations and types were tested with a standard culture medium. This medium was either supplemented with 0.5 % glucose or diluted 1/5 to assess its effects on biofilm development. The CV assays showed similar biofilm growth in both standard and glucose-supplemented media. However, the microfluidic system revealed that glucose was detrimental to biofilm formation. The diluted medium condition impacted biofilm formation significantly with the CV assay but had minimal effect in microfluidics. Biofilm-like structures were consistently detected using microfluidics, even under diluted or low-inoculum conditions, whereas the CV assay failed to observe them. The microfluidic approach enabled real-time biofilm monitoring, offering greater sensitivity to medium composition and inoculum effects, while the CV assay reflects static conditions where environmental changes are mediated by biofilm itself. This study highlights the importance of adopting a dynamic approach to studying biofilm growth mechanisms. The microfluidic approach presented in this study was developed for blood bank applications to contribute to advancements in knowledge on transfusion safety in hospital settings.

The human pathogen Campylobacter jejuni can be packaged within multilamellar bodies (MLB), also called fecal pellets, produced by ciliates such as Tetrahymena pyriformis when these microorganisms are cocultivated. This packaging increases the survival of C. jejuni in oxygenic conditions and potentially protects it against other stressors. Traditional methods for detecting and quantifying these pellets, such as transmission electron microscopy (TEM) and fluorescence microscopy, are time-consuming and labor intensive. In this study, we devised an approach for utilizing flow cytometry to distinguish and quantify C. jejuni-containing pellets produced by both T. pyriformis and T. thermophila. Cocultures of each Tetrahymena species with four different C. jejuni strains, along with monoculture controls, were incubated for 24 h, stained with SYTO9, and analysed using flow cytometry. The results revealed ciliate species-specific and bacterial strain-specific differences in the number of pellets and their fluorescence intensity. TEM confirmed that this variability in fluorescence corresponds to differences in the number of bacteria per pellet. Our method provides a rapid and efficient means of quantifying bacteria-containing MLBs, which would facilitate the screening and comparison of a large quantity of C. jejuni strains and different conditions for studying the packaging of C. jejuni by ciliates.

Strains of Aeromonas salmonicida subsp. salmonicida, a major pathogen of salmonids, typically do not grow at temperatures above 30 °C. The effects of thermal stress on this bacterium have been extensively studied. Recently, we demonstrated that repeated exposure to cyclical thermal stress, reaching up to 37 °C, can induce permanent thermoadaptation in certain strains of this bacterium. Many aspects of this adaptation process remain poorly understood. We generated 88 thermoadapted strains of A. salmonicida subsp. salmonicida capable of sustained growth at 33 °C or higher demonstrating that prolonged heat exposure can shift a substantial proportion of psychrophilic strains toward a more mesophilic-like behavior. Although growth at 35 °C was still weaker than in naturally mesophilic A. salmonicida strains, some thermoadapted strains were able to grow up to 37 °C. North American strains harboring the genomic island AsaGEI1a, a known biomarker, exhibited a significantly reduced capacity for thermoadaptation, suggesting a possible genetic constraint, although genomic analyses indicate that AsaGEI1a itself is not directly responsible for this limitation. Genotyping and phenotypic analyses revealed that thermoadaptation is frequently associated with the loss of Type III secretion system and the A-layer, two key virulence factors. Only 7% of the thermoadapted strains retained both features. Overall, our findings suggest that thermoadaptation may represent a potential mechanism influencing the persistence of some psychrophilic A. salmonicida subsp. salmonicida strains in warming aquatic environments under climate change.

The impact of dietary treatments on the phage community of porcine intestinal microbiota is not well understood. An antibiotic (tylvalosin), a probiotic (Pediococcus acidilactici), and a combination of these were given to six cannulated pigs in a double crossover design study. Samples of ileal digesta and feces were collected and whole genome shotgun sequencing was performed. The variations in phage and bacterial communities were compared for each treatment and sample type. The bacteriophages present in the gut microbiome exhibited greater variations in both α- and β-diversity between sample types (digesta, feces) than between treatments. β-diversity and differential abundance showed that the effect of the combined antibiotic and probiotic treatment was the same as with the antibiotic alone. However, the effects of the probiotic and antibiotic treatments were statistically significantly different in the fecal samples. β-diversity was different in those two treatments, and differential abundance analysis identified multiple phages as markers for each treatment. No significant variations in relative abundance were found in phage lifestyle (i.e., virulent, temperate) between treatments.

BACKGROUND:Antibiotic resistance is a major issue affecting all spheres of human activity, including agriculture. One significant example is the Avian Pathogenic Escherichia coli (APEC), a bacterium that infects poultry and leads to substantial economic losses in the farming industry. As antibiotics lose efficacity, bacteriophages (phages) -viruses that specifically target bacteria-are emerging as a promising alternative to antibiotics for treating and preventing bacterial infections. However, bacteria can develop resistance to phages through various mechanisms. Studying the coevolution between a phage and its host bacterium is important to gain insight into the phage's potential as a therapeutic agent. This study investigates the evolutionary responses of an APEC strain and a laboratory E. coli strain to a commercial phage originally isolated from APEC. RESULTS:In most cases, phage resistance resulted in a significant increase in mucoidy. Genomic analysis revealed that this resistance consistently correlated with amino acid changes, particularly in proteins involved in colanic acid production, such as YrfF. Further investigation of a mutation found in the YrfF protein demonstrated that this mutation altered the protein's structure and its interaction with the membrane. Transcriptomic analysis confirmed that the genes involved in colanic acid production were significantly overexpressed. Although the strains possessed a CRISPR-Cas system, it did not contribute to phage resistance. CONCLUSIONS:This study suggests that specific amino acid changes in key proteins may be a mechanism employed by E. coli, including APEC, to defend against phage infections.

Staphylococcus hyicus is the etiological agent of exudative epidermitis (EE) in suckling and weaned piglets, which is an endemic disease of concern for animal health and welfare on swine farms. Staphylococcus hyicus is not widely studied, and there is no recent scientific literature on Canadian strains. In a preliminary study, we presented an Eastern Canadian S. hyicus strain isolated from an EE case and bearing a plasmidic tet(L) gene conferring resistance to tetracyclines, one of the most commonly used antibiotics in Canadian swine farms. The current study characterized the tet(L)-bearing plasmids present in S. hyicus isolates from pigs in Eastern Canadian to better understand the potential vectors for this gene dissemination. Eleven S. hyicus isolates harboring tet(L) as the only tetracycline resistance gene were sequenced using Oxford Nanopore Technology and analyzed. Eight different tet(L)-bearing plasmids were identified that share similarities with Staphylococcus aureus sequences available in the National Center for Biotechnology Information. Only one of the plasmids was present in more than one isolate and two isolates carried tet(L) on their chromosome. This suggests a high diversity of tet(L)-bearing plasmids in S. hyicus and the possible need to analyze swine farm samples over time to understand the dynamics of tet(L) in S. hyicus.

The genus Flavobacterium constitutes a vast pool of microorganisms living in multiple environmental niches including fish pathogens and species in the fish microbiome. Veterinary reports have identified flavobacteria in fish from Quebec's fish farms, confirming their association with infections. However, these reports have not conducted in-depth characterization, and the diversity of nonpathogenic flavobacteria in Quebec remains unknown. This study is the first step in assessing the diversity of Flavobacterium in Quebec's fish farms, without focusing solely on pathogenic strains. Seventeen isolates were collected from different fish farms, from either the water or fish. Microbial species identification was performed using PCR genotyping of the gyrB gene, whole-genome sequencing, and phylogenetic analysis. Antimicrobial susceptibility tests for tetracycline and florfenicol, the two most commonly used antibiotics in Quebec aquaculture, along with predictive tools, were employed to assess resistance. This study revealed potential new species among the isolates. No known pathogenic species were detected, and all 17 isolates clustered within CIIIb or CIIIc, recently described phylogenetic clades of Flavobacterium found in various environments, and the majority showed resistance to antibiotics. This study highlights the expanding diversity of Flavobacterium, particularly among species associated with fish, and underscores the need for further research in Quebec.

Platelet concentrates (PCs) are stored at 20-24˚C in a biologically favorable environment that may support bacterial growth. Staphylococcus epidermidis, a typical contaminant, can form biofilms in PCs, complicating detection and increasing the risk of transfusion-transmitted bacterial infections. The material composition and surface texture of PC storage bags may influence biofilm formation. The impact of different PC storage bag materials on S. epidermidis biofilm formation was evaluated using the ISO 4768:2023(E) crystal violet (CV) assay. Four surface conditions were tested: polyvinyl chloride (PVC) plasticized with n-butyryl-tri(n-hexyl)-citrate (BTHC) - both smooth and rough sides, PVC plasticized with tri-(2-ethylhexyl)-trimellitate (TEHTM) and ethylene-vinyl acetate (EVA). Coupons and bags made from each material were used in the experiments. Biofilm-positive S. epidermidis was cultured in tryptic soy broth (TSB), PCs and plasma and added on plastic coupons under static conditions or directly in the bags with agitation. Bacterial enumeration and CV assay were performed on days 2, 5, and 7. In TSB, EVA coupons significantly formed more biofilm than the smooth side of PVC-BTHC or TEHTM over seven days. In PCs, more biofilm formed on the rough side of PVC-BTHC coupons than the smooth side, with no other differences between plastics, suggesting similar biofilm amount across PC bag materials in the presence of platelets. No biofilm was detected on coupons in plasma. Under continuous agitation and reduced oxygen levels, only the rough side of PVC-BTHC showed significant biofilm formation in TSB in PC storage bags over seven days. These findings highlight the need for standardized biofilm testing and suggest that some plastics are more conducive to biofilm formation under static conditions. However, during blood bank storage (i.e., continuous agitation and reduced oxygen levels), biofilm formation is limited, regardless of the platelet bag material, thereby reducing the risk of undetected bacterial contamination.

Cryptosporidium and Giardia are protozoan parasites responsible for gastrointestinal illnesses in humans and in animal species. The main way these parasites are transmitted is by ingestion of their (oo)cysts in drinking water. Monitoring (oo)cysts in water sources is beneficial to evaluate the quality of raw water supplying treatment plants. Currently, the only standardized protocol to enumerate these parasites from water samples is United States Environmental Protection Agency (USEPA) Method 1623.1. With this method, we monitored three major water sources in Quebec over a year to assess temporal and geographical variations of these parasite (oo)cysts. These three water sources have independent watersheds despite being in the same region. We found a general pattern for Giardia, with high concentrations of cysts during cold and transition periods, and significantly lower concentrations during the warm period. Cryptosporidium's concentration was more variable throughout the year. Statistical correlations (Pearson's correlation coefficients) were established between the concentration of each parasite and various environmental parameters. The three study sites each showed unique factors correlating with the presence of both protozoa, supporting the idea that each water source must be seen as a unique entity with its own particular characteristics and therefore, must be monitored independently. Although some environmental parameters could be interesting proxies to the parasitic load, no parameter was strongly correlated throughout the whole sampling year and none of the parameters could be used as a single proxy for all three studies sources.

Phage replication can be studied using various approaches, including measuring the optical density (OD) of a bacterial culture in a liquid medium in the presence of phages. A few quantitative methods are available to measure and compare the efficiency of phages by using a single index based on the analysis of OD curves. However, these methods are not always applicable to non-canonical OD curves. Using the concept of center of area (centroid), we developed a metric called Centroid Index (CI), sensitive to the trend of the growth curves (OD distribution) including bacterial regrowth, which is not considered by the methods already available. We also provide a user-friendly software to facilitate the calculation of CI. This method offers an alternative and more precise way to determine phage efficiency by considering the OD variations over time, which may help in the selection of phages for biocontrol applications.

Aeromonas salmonicida is studied using Dictyostelium discoideum as a model host, with predation resistance measured as a key parameter. Aeromonas salmonicida mesophilic isolates exhibit inconclusive results with the amoebic model. This study focuses on new mesophilic isolates (S24-S38, S26-S10, and S28-S20) from Alberta, Canada, and introduces an improved predation test method. Phylogenetic analysis reveals two subgroups, with S24-S38 and S26-S10 clustering with the subspecies pectinolytica from Argentina, and S28-S20 with strains from India (Y567) and Spain (AJ83), showcasing surprising mesophilic strain diversity across geographic locations. Predation tests were carried out with various mesophilic and psychrophilic strains of A. salmonicida, including Alberta isolates. The amoeba cell lines used were DH1-10 and AX2. Although the mesophilic isolates were very resistant to predation by the amoeba DH1-10, some lost this resistance to the AX2 strain, which appeared more voracious in the conditions tested. In addition, when diluting the culture medium used in a predation test with AX2, a loss of the capacity to predation resistance was observed for all the mesophilic isolates, including the highly resistant S28-S20 isolate. This study provides insights into the predation resistance of A. salmonicida isolates and offers avenues for better characterizing mesophilic isolates.

The genus Flavobacterium comprises a diversity of species, including fish pathogens. Multiple techniques have been used to identify isolates of this genus, such as phenotyping, polymerase chain reaction genotyping, and in silico whole-genome taxonomy. In this study, we demonstrate that whole-genome-based taxonomy, using average nucleotide identity and molecular phylogeny, is the most accurate approach for Flavobacterium species. We obtained various isolated strains from official collections; these strains had been previously characterized by a third party using various identification methodologies. We analyzed isolates by PCR genotyping using previously published primers targeting gyrB and gyrA genes, which are supposedly specific to the genus Flavobacterium and Flavobac- terium psychrophilum, respectively. After genomic analysis, nearly half of the isolates had their identities re-evaluated: around a quarter of them were re-assigned to other genera and two isolates are new species of flavobacteria. In retrospect, the phenotyping method was the least accurate. While gyrB genotyping was accurate with the isolates included in this study, bioinformatics analysis suggests that only 70% of the Flavobacterium species could be appropriately identified using this approach. We propose that whole-genome taxonomy should be used for accurate Flavobacterium identification, and we encourage bacterial collections to review the identification of isolates identified by phenotyping.

Plasmids carrying antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen. In this study, we describe three new plasmids found in strains of A. salmonicida subsp. salmonicida from Québec, Canada: pAsa-2358, pAsa-2900, and pAsa-2900b. The last two plasmids have a tet(D) resistance gene against tetracycline, which has not been previously found in A. salmonicida. The only difference between these two plasmids is the presence of a florfenicol resistance gene (floR) surrounded by two copies of the same insertion sequence, indicating that pAsa-2900 and pAsa-2900b are plasmid variants. All three plasmids represent an issue for aquaculture because they bear resistance genes against antibiotics used to treat infected fish: floR and tet(A) for pAsa-2358; floR, sul2, and tet(D) for pAsa-2900; and sul2 and tet(D) for pAsa-2900b. Conjugation assays confirmed that the three plasmids are capable of conjugation with one A. salmonicida mesophilic strain, one A. hydrophila strain, and Escherichia coli DH5α; except for pAsa-2358 that is not able to conjugate into E. coli DH5α. Based on the results of antibiotic resistance assays, the tet(D) gene provides a greater resistance to tetracycline than the tet(A) gene found in many A. salmonicida subsp. salmonicida plasmids. This is the case whether the plasmids are in their parental A. salmonicida subsp. salmonicida strains or in Aeromonas sp. transconjugates. The presence of these three plasmids in A. salmonicida subsp. salmonicida strengthens the role of this bacterium as a reservoir for antibiotic resistance genes.

Streptomycin thallous acetate actidione medium is typically used to isolate Brochothrix thermosphacta bacteria from food. Using this medium, three bacterial strains were isolated from the environment. Genomic sequences demonstrated that these bacteria are of the genera Lysinibacillus and Paenibacillus and are of biotechnological interest.

Flavobacterium is a genus of microorganisms living in a variety of hosts and habitats across the globe. Some species are found in fish organs, and only a few, such as Flavobacterium psychrophilum and Flavobacterium columnare, cause severe disease and losses in fish farms. The evolution of flavobacteria that are pathogenic to fish is unknown, and the protein changes accountable for the selection of their colonization to fish have yet to be determined. A phylogenetic tree was constructed with the complete genomic sequences of 208 species of the Flavobacterium genus using 861 softcore genes. This phylogenetic analysis revealed clade CII comprising nine species, including five pathogenic species, and containing the most species that colonize fish. Thirteen specific amino acid changes were found to be conserved across 11 proteins within the CII clade compared with other clades, and these proteins were enriched in functions related to replication, recombination, and repair. Several of these proteins are known to be involved in pathogenicity and fitness adaptation in other bacteria. Some of the observed amino acid changes can be explained by preferential selection for certain codons and tRNA frequency. These results could help explain how species belonging to the CII clade adapt to fish environments.

Background/Objectives: Antibiotic resistance gene (ARG) spread is driven by horizontal gene transfer (HGT). Ciliated protozoa may contribute to this process, as their predation has been shown to facilitate HGT in certain bacteria. Here, this phenomenon was further investigated using A. salmonicida subsp. salmonicida. This fish pathogen bears an extensive and dynamic plasmidome, suggesting a high potential for HGT. Methods: A. salmonicida strains carrying one of three conjugative plasmids bearing ARGs (pSN254b, pRAS1b or pAsa4b) were cocultured with a recipient, either A. salmonicida, E. coli or A. hydrophila. Conjugation rates were assessed in the presence and absence of the ciliate Tetrahymena borealis. PCR genotyping confirmed the acquisition of the conjugative plasmids and was used to verify the mobilization of other plasmids. Results: The basal rate of conjugation observed was high. Under the conditions studied, ciliate predation did not appear to influence the conjugation rate, except at higher proportions of ciliates, which typically hampered conjugation. Microscopy revealed that most bacteria were digested in these conditions. PCR screening demonstrated that small mobilizable plasmids from A. salmonicida (pAsa1, pAsa2, pAsa3, and pAsal1) were acquired by the recipients along with the conjugative plasmids, with a slight effect of the ciliates in some donor/recipient cell combination. Conclusions: These results highlight how A. salmonicida can conjugate efficiently with different species and how complex its relationship with ciliates is.