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Plasmids that carry antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen with severe consequences in salmonid farming. Here, we describe a 67 kb plasmid found in the A. salmonicida subsp. salmonicida Strain SHY15-2939 from Quebec, Canada. This new plasmid, named pAsa-2939 and identified by high throughput sequencing, displays features never found before in this bacterial species. It contains a transposon related to the Tn21 family, but with an unusual organization. This transposon bears a catB3 gene (chloramphenicol resistance) that has not been detected yet in A. salmonicida subsp. salmonicida. The plasmid is transferable by conjugation into Aeromonas hydrophila, but not into Escherichia coli. Based on PCR analysis and genomic sequencing (Illumina and PacBio), we determined that the transposon is unstable in A. salmonicida subsp. salmonicida Strain SHY15-2939, but it is stable in A. hydrophila trans-conjugants, which explains the chloramphenicol resistance variability observed in SHY15-2939. These results suggest that this bacterium is likely not the most appropriate host for this plasmid. The presence of pAsa-2939 in A. salmonicida subsp. salmonicida also strengthens the reservoir role of this bacterium for antibiotic resistance genes, even those that resist antibiotics not used in aquaculture in Québec, such as chloramphenicol.

Aeromonas salmonicida subsp. salmonicida is a pathogenic bacterium responsible for furunculosis in salmonids. Following an outbreak of furunculosis, the infection can be treated with antibiotics, but it is common to observe ineffective treatment due to antibiotic resistance. This bacterium has a wide variety of plasmids responsible for this resistance. Among them, pRAS3 carries a tetracycline resistance gene. Several variants of this plasmid have been discovered over the years (pRAS3-3432 and pRAS3.1 to 3.4). During the present study, two new variants of the plasmid pRAS3 were identified (pRAS3.5 and pRAS3-3759) in strains of A. salmonicida subsp. salmonicida. Plasmid pRAS3-3759, which has been found in many strains from the same region over the past three years, has an additional genetic element identical to one found in pRAS3-3432. This genetic element was also found in Chlamydia suis, a swine pathogen. In this study, we analyzed the bacteria’s resistance to tetracycline, the number of copies of the plasmids, and the growth of the strains that carry five of the pRAS3 variants (pRAS3.3 to 3.5, pRAS3-3432, and pRAS3-3759). The results show no particular trend despite the differences between the plasmids, except for the resistance to tetracycline when analyzed in an isogenic background. Blast analysis also revealed the presence of pRAS3 plasmids in other bacterial species, which suggests that this plasmid family has widely spread. This study once again highlights the ability of A. salmonicida subsp. salmonicida to adapt to furunculosis antibiotic treatments, and the still-growing family of pRAS3 plasmids.

Antimicrobial resistance (AMR) is continuing to grow across the world. Though often thought of as a mostly public health issue, AMR is also a major agricultural and environmental problem. As such, many researchers refer to it as the preeminent One Health issue. Aerial transport of antimicrobial-resistant bacteria via bioaerosols is still poorly understood. Recent work has highlighted the presence of antibiotic resistance genes in bioaerosols. Emissions of AMR bacteria and genes have been detected from various sources, including wastewater treatment plants, hospitals, and agricultural practices; however, their impacts on the broader environment are poorly understood. Contextualizing the roles of bioaerosols in the dissemination of AMR necessitates a multidisciplinary approach. Environmental factors, industrial and medical practices, as well as ecological principles influence the aerial dissemination of resistant bacteria. This article introduces an ongoing project assessing the presence and fate of AMR in bioaerosols across Canada. Its various sub-studies include the assessment of the emissions of antibiotic resistance genes from many agricultural practices, their long-distance transport, new integrative methods of assessment, and the creation of dissemination models over short and long distances. Results from sub-studies are beginning to be published. Consequently, this paper explains the background behind the development of the various sub-studies and highlight their shared aspects.

Aquaculture is a rapidly growing food production sector. Fish farmers are experiencing increasing problems with antibiotic resistance when fighting against pathogenic bacteria such as Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis. Phage therapy may provide an alternative, but effective use must be determined. Here, we studied the inhibition of A. salmonicida subsp. salmonicida strains by five phages (HER98 [44RR2.8t.2], HER110 [65.2], SW69-9, L9-6 and Riv-10) used individually or as combinations of two to five phages. A particular combination of four phages (HER98 [44RR2.8t.2], SW69-9, Riv-10, and HER110 [65.2]) was found to be the most effective when used at an initial multiplicity of infection (MOI) of 1 against the A. salmonicida subsp. salmonicida strain 01-B526. The same phage cocktail is effective against other strains except those bearing a prophage (named Prophage 3), which is present in 2/3 of the strains from the province of Quebec. To confirm the impact of this prophage, we tested the effectiveness of the same cocktail on strains that were either cured or lysogenized with Prophage 3. While the parental strains were sensitive to the phage cocktail, the lysogenized ones were much less sensitive. These data indicate that the prophage content of A. salmonicida subsp. salmonicida can affect the efficacy of a cocktail of virulent phages for phage therapy purposes.

The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity.

Protozoa are natural predators of bacteria, but some bacteria can evade digestion once phagocytosed. Some of these resistant bacteria can be packaged in the fecal pellets produced by protozoa, protecting them from physical stresses and biocides. Depending on the bacteria and protozoa involved in the packaging process, pellets can have different morphologies. In the present descriptive study, we evaluated the packaging process with 20 bacteria that have never been tested before for packaging by ciliates. These bacteria have various characteristics (shape, size, Gram staining). All of them appear to be included in pellets produced by the ciliates Tetrahymena pyriformis and/or T. thermophila in at least one condition tested. We then focused on the packaging morphology of four of these bacteria. Our results demonstrated that, as shown previously for Mycobacterium smegmatis, the packaging of Microbacterium oxydans, Micrococcus luteus, and Cupriavidus sp. was formed of a single layer of material. The packaging of Cellulosimicrobiumfunkei was made of indistinguishable material. A different pellet morphology was obtained for each of the four bacterial strains studied. The ingestion of small bacteria resulted in rounder, denser, and more regular pellets. These results support the idea that bacteria packaging is a relatively widespread phenomenon.