Aeromonas salmonicida isolates from Canada demonstrate wide distribution and clustering among mesophilic strains.

All the 36 known species to date of the genus Aeromonas are mesophilic except the species Aeromonas salmonicida, which includes both psychrophilic and mesophilic subspecies. For 20 years, more and more mesophilic A. salmonicida strains have been discovered. Only A. salmonicida subsp. pectinolytica has officially been classified as a mesophilic subspecies. Most mesophiles have been isolated in hot countries. We present, for the first time, the characterization of two new mesophilic isolates from Quebec (Canada). Phenotypic and genomic characterizations were carried out on these strains, isolated from dead fish from a fish farm. Isolates 19-K304 and 19-K308 are clearly mesophiles, virulent to the amoeba Dictyostelium discoideum, a surrogate host, and close to strain Y577, isolated in India. To our knowledge, this is the first time that mesophilic strains isolated from different countries are so similar. The major difference between the isolates is the presence of plasmid pY47-3, a cryptic plasmid that sometimes presents in mesophilic strains. More importantly, our extensive phylogenetic analysis reveals two well-defined clades of mesophilic strains with psychrophiles associated with one of these clades. This helps to have a better understanding of the evolution of this species and the apparition of psychrophilic subspecies.

Isolation of vB_AsaM_LPM4 reveals the dynamics of Prophage 3 in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida causes furunculosis, a major infection that affects fish farms worldwide. We isolated phage vB_AsaM_LPM4 (LPM4) from a diseased fish. Based on its DNA sequence, LPM4 is identical to the uncharacterized Prophage 3, a prophage present mostly in North American A. salmonicida subsp. salmonicida isolates that bear the genomic island AsaGEI2a. Prophage 3 and AsaGEI2a are inserted side by side in the bacterial chromosome. The LPM4/Prophage 3 sequence is similar to that of other prophages found in various members of the genus Aeromonas. LPM4 specifically infects A. salmonicida subsp. salmonicida strains that do not already bear Prophage 3. The presence of an A-layer on the surface of the bacteria is not necessary for the adsorption of phage LPM4 but seems to facilitate its infection process. We also successfully produced lysogenic strains that bear Prophage 3 using sensitive strains with different genetic backgrounds, suggesting that there is no interdependency between LPM4 and AsaGEIs. PCR analysis of the excision dynamics of Prophage 3 and AsaGEIs revealed that these genetic elements can spontaneously excise themselves from the bacterial chromosome independently of one another. Through the isolation and characterization of LPM4, this study reveals new facets of Prophage 3 and AsaGEIs.

Improvements of virulence factor phenotypic tests for Aeromonas salmonicida subsp. salmonicida, a major fish pathogen.

Aeromonas salmonicida subspecies salmonicida, a fish pathogen, expresses various virulence factors such as an A-layer, lipases and proteases during the infection process. Not all strains of this bacterium express the same virulence factors. It is important to be able to evaluate which factors are present when characterizing strains. The A-layer and secreted lipases and proteases are usually detected by agar-based tests that require long incubation (24 h and more) and may provide ambiguous results. In the present study, protocols have been optimized to determine the presence of these virulence factors using liquid tests. For A-layer detection, the optimized method stains the positive bacteria with Coomassie Brilliant Blue. The lipases are detected by a colorimetric biochemical reaction triggered by the degradation of p-nitrophenyl dodecanoate into a yellow product detectable by spectrophotometry, if the result is positive. Both of these tests show results in less than an hour. Finally, the protease activity is measured by clarification of a medium containing milk during an overnight bacterial growth. These new protocols provide opportunities for quicker characterization of A. salmonicida subsp. salmonicida strains and, particularly, provide more precise results.

Characterization of bacteriophage T7-Ah reveals its lytic activity against a subset of both mesophilic and psychrophilic Aeromonas salmonicida strains.

Aeromonas salmonicida strains cause problematic bacterial infections in the aquaculture industry worldwide. The genus Aeromonas includes both mesophilic and psychrophilic species. Bacteriophages that infect Aeromonas spp. strains are usually specific for mesophilic or psychrophilic species; only a few bacteriophages can infect both types of strains. In this study, we characterized the podophage T7-Ah, which was initially found to infect the Aeromonas salmonicida HER1209 strain. The burst size of T7-Ah against its original host is 72 new virions per infected cell, and its burst time is 30 minutes. It has been found that this phage can lyse both mesophilic and psychrophilic A. salmonicida strains, as well as one strain of Escherichia coli. Its genome comprises 40,153 bp of DNA and does not contain any recognizable toxin or antibiotic resistance genes. The adsorption rate of the phage on highly sensitive bacterial strains was variable and could not be related to the presence or absence of a functional A-layer on the surface of the bacterial strains. The lipopolysaccharide migration patterns of both resistant and sensitive bacterial strains were also studied and compared to investigate the nature of the potential receptor of this phage on the bacterial surface. This study sheds light on the surprising diversity of lifestyles of the bacterial strains sensitive to phage T7-Ah and opens the door to the potential use of this phage against A. salmonicida infections in aquaculture.

Beyond the A-layer: adsorption of lipopolysaccharides and characterization of bacteriophage-insensitive mutants of Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage-insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A-layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A-layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. salmonicida cells.

Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium cells mutant for the Arf GAP ACAP-A.

Dictyostelium discoideum ACAP-A is an Arf GTPase-activating protein (GAP) involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here, we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, the efficiency of fusion between post-lysosomes and the plasma membrane was reduced, resulting in the accumulation of post-lysosomes. Moreover, internalized fluid-phase markers showed extended intracellular transit times, and the transfer kinetics of phagocyted particles from lysosomes to post-lysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA(-) cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Taken together, these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfA-dependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.

Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis.

Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence of C. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized that C. jejuni could be packaged under aerobic conditions by the amoeba Acanthamoeba castellanii or the ciliate Tetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellanii did not produce MLBs containing C. jejuni In contrast, when incubated with T. pyriformis,C. jejuni was ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics of C. jejuni culturability showed that packaging increased the survival of C. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests that T. pyriformis may increase the risk of persistence of C. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging.

Detection of variants of the pRAS3, pAB5S9, and pSN254 plasmids in Aeromonas salmonicida subsp. salmonicida: multidrug resistance, interspecies exchanges, and plasmid reshaping.

The ubiquitous water-borne Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a worldwide disease in fish farms. Plasmids carrying antibiotic resistance genes have already been described for this bacterium. The aim of the present study was to identify and characterize additional multidrug resistance plasmids in A. salmonicida subsp. salmonicida. We sequenced the plasmids present in two multiple antibiotic-resistant isolates using high-throughput technologies. We also investigated 19 other isolates with various multidrug resistance profiles by genotyping PCR and assessed their resistance to tetracycline. We identified variants of the pAB5S9 and pSN254 plasmids that carry several antibiotic resistance genes and that have been previously reported in bacteria other than A. salmonicida subsp. salmonicida, which suggests a high level of interspecies exchange. Genotyping analyses and the antibiotic resistance profiles of the 19 other isolates support the idea that multiple versions of pAB5S9 and pSN254 exist in A. salmonicida subsp. salmonicida. We also identified variants of the pRAS3 plasmid. The present study revealed that A. salmonicida subsp. salmonicida harbors a wide variety of plasmids, which suggests that this ubiquitous bacterium may contribute to the spread of antibiotic resistance genes in the environment.

Lipid composition of multilamellar bodies secreted by Dictyostelium discoideum reveals their amoebal origin.

When they are fed with bacteria, Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs), which are composed of membranous material. It has been proposed that MLBs are a waste disposal system that allows D. discoideum to eliminate undigested bacterial remains. However, the real function of MLBs remains unknown. Determination of the biochemical composition of MLBs, especially lipids, represents a way to gain information about the role of these structures. To allow these analyses, a protocol involving various centrifugation procedures has been developed to purify secreted MLBs from amoeba-bacterium cocultures. The purity of the MLB preparation was confirmed by transmission electron microscopy and by immunofluorescence using H36, an antibody that binds to MLBs. The lipid and fatty acid compositions of pure MLBs were then analyzed by high-performance thin-layer chromatography (HPTLC) and gas chromatography (GC), respectively, and compared to those of amoebae as well as bacteria used as a food source. While the bacteria were devoid of phosphatidylcholine (PC) and phosphatidylinositol (PI), these two polar lipid species were major classes of lipids in MLBs and amoebae. Similarly, the fatty acid composition of MLBs and amoebae was characterized by the presence of polyunsaturated fatty acids, while cyclic fatty acids were found only in bacteria. These results strongly suggest that the lipids constituting the MLBs originate from the amoebal metabolism rather than from undigested bacterial membranes. This opens the possibility that MLBs, instead of being a waste disposal system, have unsuspected roles in D. discoideum physiology.

MQM1, a bacteriophage infecting strains of Aeromonas salmonicida subspecies salmonicida carrying Prophage 3.

Aeromonas salmonicida subsp. salmonicida is a Gam-negative bacterium responsible for furunculosis in fish. Because this aquatic bacterial pathogen has a rich reservoir of antibiotic-resistant genes, it is essential to investigate antibacterial alternatives, including the use of phages. Yet, we have previously demonstrated the inefficiency of a phage cocktail designed against A. salmonicida subsp. salmonicida strains due to a phage resistance phenotype associated to a prophage, namely Prophage 3. To bypass this resistance, one of the solutions is to isolate novel phages capable of infecting Prophage 3-bearing strains. Here we report on the isolation and characterization of the new virulent phage vB_AsaP_MQM1 (or MQM1), which is highly specific to A. salmonicida subsp. salmonicida strains. Phage MQM1 inhibited the growth of 01-B516, a strain carrying Prophage 3, including when combined to the previous phage cocktail. MQM1 infected 26 out of the 30 (87%) Prophage 3-bearing strains tested. Its linear dsDNA genome contains 63,343 bp, with a GC content of 50.2%. MQM1 genome can encode 88 proteins and 8 tRNAs, while no integrase or transposase-encoding genes were found. This podophage has an icosahedral capsid and a non-contractile short tail. We suggest that MQM1 may be a good addition to future phage cocktails against furunculosis to resolve the Prophage 3-resistance issue.

Draft genome sequences of four Staphylococcus hyicus strains, SC302, SC304, SC306, and SC310, isolated from swine from Eastern Canada.

The bacterium Staphylococcus hyicus causes porcine exudative epidermitis in piglets, which represents both health and welfare concerns. Few genome sequences of this pathogen are published. We provide four additional ones to help future genomic analysis of S. hyicus. These are genomes of strains isolated from Canadian swine.

Host Dependent-Transposon for a Plasmid Found in Aeromonas salmonicida subsp. salmonicida That Bears a catB3 Gene for Chloramphenicol Resistance.

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 intraspecies divergence revealed by the various strategies displayed when grazed by Tetrahymena pyriformis.

Worldwide, Aeromonas salmonicida is a major bacterial pathogen of fish in both marine and freshwater environments. Despite psychrophilic growth being common for this species, the number of characterized mesophilic strains is increasing. Thus, this species may serve as a model for the study of intraspecies lifestyle diversity. Although bacteria are preyed upon by protozoan predators, their interaction inside or outside the phagocytic pathway of the predator can provide several advantages to the bacteria. To correlate intraspecies diversity with predation outcome, we studied the fate of psychrophilic and mesophilic strains of A. salmonicida cocultured with the ciliate Tetrahymena pyriformis. A total of three types of outcome were observed: digestion, resistance to phagocytosis, and pathogenicity. The psychrophilic strains are fully digested by the ciliate. In contrast, the mesophilic A. salmonicida subsp. pectinolytica strain is pathogenic to the ciliate. All the other mesophilic strains display mechanisms to resist phagocytosis and/or digestion, which allow them to survive ciliate predation. In some cases, passage through the phagocytic pathway resulted in a few mesophilic A. salmonicida being packaged inside fecal pellets. This study sheds light on the great phenotypic diversity observed in the complex range of mechanisms used by A. salmonicida to confront a predator.

Expansion of the pRAS3 Plasmid Family in Aeromonas salmonicida subsp. salmonicida and Growing Evidence of Interspecies Connections for These Plasmids.

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 in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes.

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.

[Dictyostelium discoideum: a model for the study of bacterial virulence]. / Dictyostelium discoideum: un modèle pour l'étude de la virulence bactérienne.

The amoeba Dictyostelium discoideum, a bacterial predator, has emerged as a valuable tool for studying bacterial virulence. All its features make this unicellular eukaryote a versatile model organism. It can be used to study virulence factors of pathogenic bacteria as well as host elements involved in resistance to pathogens. The virulence of more than 20 bacterial species pathogenic for humans or animals has been studied using D. discoideum so far. These bacteria are either extracellular or intracellular pathogens. This review presents an overview of the question, with special emphasis on the reasons why D. discoideum is a suitable host model to study bacterial virulence, as well as on the type of information on host­pathogen relationship this amoeba can provide.

Phage Cocktail Development against Aeromonas salmonicida subsp. salmonicida Strains Is Compromised by a Prophage.

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.

Draft Genome Sequence of an Aeromonas salmonicida subsp. salmonicida Strain from the Canadian Pacific Coast Bearing a Variant of pRAS1.

The genome sequencing of Aeromonas salmonicida subspecies salmonicida strain 2004-072 revealed a plasmid bearing a region carrying antibiotic resistance genes very similar to the one found in the plasmid pRAS1, an IncU family plasmid. This new plasmid was named pRAS1b.

AsaGEI2d: a new variant of a genomic island identified in a group of Aeromonas salmonicida subsp. salmonicida isolated from France, which bears the pAsa7 plasmid.

Genomic islands (Aeromonas salmonicida genomic islands, AsaGEIs) are found worldwide in many isolates of Aeromonas salmonicida subsp. salmonicida, a fish pathogen. To date, five variants of AsaGEI (1a, 1b, 2a, 2b and 2c) have been described. Here, we investigate a sixth AsaGEI, which was identified in France between 2016 and 2019 in 20 A. salmonicida subsp. salmonicida isolates recovered from sick salmon all at the same location. This new AsaGEI shares the same insertion site in the chromosome as the other AsaGEI2s as they all have a homologous integrase gene. This new AsaGEI was thus named AsaGEI2d, and has five unique genes compared to the other AsaGEIs. The isolates carrying AsaGEI2d also bear the plasmid pAsa7, which was initially found in an isolate from Switzerland. This plasmid provides resistance to chloramphenicol thanks to a cat gene. This study reveals more about the diversity of the AsaGEIs.

Evidence that Bacteria Packaging by Tetrahymena Is a Widespread Phenomenon.

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.