Investigation des éléments génomiques impliqués dans la perte de virulence d’Aeromonas salmonicida ssp. salmonicida suite à un stress thermique

Aeromonas salmonicida ssp. salmonicida est l’agent étiologique de la furonculose chez les salmonidés, spécialement les poissons d’élevage. Les pertes économiques pour l’aquaculture au Québec liées à cette maladie sont considérables. En raison des traitements par antibiotiques, on observe une émergence de souches d’A. salmonicida ssp. salmonicida résistantes aux antibiotiques. C’est pourquoi différentes alternatives, dont la vaccination, sont étudiées afin de prévenir la furonculose. Généralement, les vaccins utilisés injectent des bactéries mortes. En revanche cette méthode peut être laborieuse et inefficace. Ce projet s’intéresse au développement de souches vivantes atténuées qui pourraient être utilisées comme vaccin dans les piscicultures. Des études antérieures ont démontré que la virulence de la bactérie pouvait être atténuée lorsqu’exposée à un stress. Cela est expliqué par la perte des gènes du système de sécrétion de type trois (SSTT), qui ont un rôle essentiel dans la virulence de la bactérie. Cependant, le mécanisme reste encore inconnu et conséquemment, ce projet avait comme principal objectif d’identifier et caractériser les différents éléments génomiques pouvant contribuer à l’instabilité du SSTT. Tout d’abord, plusieurs souches d’A. salmonicida ssp. salmonicida ont été cultivées à 25 °C, ce qui est supérieur à leur température optimale de 18 °C. Les résultats ont révélé que certaines souches pouvaient perdre leur SSTT, mais que d’autres étaient réfractaires à la délétion des gènes malgré leur exposition à un stress. Une analyse génomique de ces différentes souches a permis d’identifier la présence d’un regroupement de gènes qui semble jouer un rôle dans l’instabilité du SSTT. Ces gènes sont retrouvés en majorité chez les souches sensibles et ils sont absents pour les souches réfractaires. Ce regroupement de gènes sera étudié en vue de créer des souches atténuées à partir de souches réfractaires. Ces souches seront ensuite testées sur les poissons pour déterminer l’efficacité de cette approche vaccinale contre la furonculose.Aeromonas salmonicida ssp. salmonicida is the causative agent of furunculosis in salmonids, especially in farmed fish. The economic losses for aquaculture in Quebec related to this disease are considerable. As a result of antibiotic treatments, there is the emergence of antibiotic-resistant strains of Aeromonas salmonicida ssp. salmonicida. This is why different alternatives, including vaccination, are being studied to prevent furunculosis. Historically, the vaccines used are composed of dead bacteria. However, this method can be laborious and inefficient. This project is interested in the development of live attenuated strains that could be used as a vaccine in fish farms. Previous studies have shown that the virulence of the bacteria can be reduced when exposed to stress. This is explained by the loss of the genes of the type three secretion system (TTSS), which is essential in the virulence of the bacteria. However, the mechanism is still not fully understood and consequently the main objective of this project was to identify and characterize the different genomic elements that can contribute to the instability of TTSS. First, several strains of A. salmonicida ssp. salmonicida were grown at 25 ° C, which is above their optimum temperature of 18 ° C. The results revealed that some strains could lose their TTSS, but others were resistant to gene deletion despite their exposure to stress. Genomic analysis of these different strains identified the presence of a cluster of genes that appears to play a role in the instability of TTSS. These genes are found mostly in sensitive strains and they are absent in refractory strains. This cluster of genes will be studied to create attenuated strains from refractory strains. These strains will then be tested on fish to determine the effectiveness of these strains as a vaccine approach against furunculosis

Expansion of the tetracycline resistome in Aeromonas salmonicida with a tet(D) gene found in plasmids pAsa-2900 and pAsa-2900b

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

Systematic Analysis of the Stress-Induced Genomic Instability of Type Three Secretion System in Aeromonas salmonicida subsp. salmonicida

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

Interaction of pAsa5 and pAsa8 Plasmids in <i>Aeromonas salmonicida</i> subsp. <i>salmonicida</i>

The plasmid known as pAsa5 is present in Aeromonas salmonicida subsp. salmonicida, a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium’s virulence. Recombination events are known to occur in pAsa5, resulting in the loss of certain segments or the acquisition of additional genetic elements. For example, the transposon carried by the large pAsa8 plasmid was found to be inserted into the pAsa5 plasmid in the SHY16-3432 strain, enabling the addition of antibiotic resistance genes to this plasmid, which does not normally possess any. In this study, we present the isolation of additional strains carrying pAsa8. Further analyses of these strains revealed that a fusion between pAsa5 and the complete version of pAsa8 is possible. The pAsa8 transposon insertion in pAsa5 seen in the SHY16-3432 strain appears to be an aberrant event compared to the fusion of the two full-length plasmids. A 22-nucleotide sequence, present in both plasmids, serves as the site for the fusion of the two plasmids. Moreover, it is possible to introduce pAsa8 through conjugation into naive strains of A. salmonicida subsp. salmonicida and once the plasmid is within a new strain, the fusion with pAsa5 is detectable. This study reveals a previously unexplored aspect of pAsa5 plasmid biology, highlighting an additional risk for the spread of antibiotic resistance genes in A. salmonicida subsp. salmonicida.</i

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

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 Qu&eacute;bec, such as chloramphenicol

Host Dependent-Transposon for a Plasmid Found in <i>Aeromonas salmonicida</i> subsp. <i>salmonicida</i> That Bears a <i>catB3</i> 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