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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.

High-throughput genomic sequencing has helped to reveal the plasmidome of Aeromonas salmonicida. This literature review provides an overview of A. salmonicida's rich plasmidome by presenting all the plasmids identified so far, addressing their biological importance and the functional links between them. The plasmids of A. salmonicida, especially those bearing antibiotic resistance genes, can provide clues about interactions of this species with other pathogens (animals and humans), as is the case for pRAS3-3432 and Chlamydia suis or pSN254b and Salmonella enterica. In addition to antibiotic resistance, plasmids play an important role in the virulence of A. salmonicida, particularly for the subspecies salmonicida and the plasmid pAsa5, which carries genes for the type-three secretion system, a virulence factor essential for the bacterium. The A. salmonicida plasmidome also has many cryptic plasmids with no known biological function, but which can be used for the acquisition of new genetic elements. Striking examples are pAsa7 and pAsaXII that provide, respectively, resistance to chloramphenicol and formaldehyde and are derivatives of cryptic pAsa2.

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.

International audience; 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.

The bacterium Aeromonas salmonicida is known since long time as a major fish pathogen unable to grow at 37 °C. However, some cases of human infection by putative mesophilic A. salmonicida have been reported. The goal of the present study is to examine two clinical cases of human infection by A. salmonicida in Spain and to investigate the pathogenicity in mammals of selected mesophilic A. salmonicida strains. An evaluation of the pathogenicity in a mouse model of clinical and environmental A. salmonicida strains was performed. The genomes of the strains were sequenced and analyzed in order to find the virulence determinants of these strains. The experimental infection in mice showed a gradient in the virulence of these strains and that some of them can cause necrotizing fasciitis and tissue damage in the liver. In addition to demonstrating significant genomic diversity among the strains studied, bioinformatics analyses permitted also to shed light on crucial elements for the virulence of the strains, like the presence of a type III secretion system in the one that caused the highest mortality in the experimental infection. Clinicians and microbiologists should consider these results for the inclusion of A. salmonicida in diagnosis tests since it is now clear that some mesophilic strains are also pathogens for humans.

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.

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.

Aeromonas salmonicida is a Gram-negative bacterium, known as a fish pathogen since its discovery. Although the species was initially considered psychrophilic, a mesophilic subspecies (pectinolytica) and many other mesophilic strains still not attributed to subspecies have been described in the last two decades. These mesophilic strains were sampled from various sources, including humans, and some of them are known to be pathogenic. In this study, we describe a strain, JF2480, which was isolated from the spleen, and also found the kidney and liver of a dead pied avocet (Recurvirostra avosetta), a type of migratory bird inhabiting aquatic environments. A core genome phylogenomic analysis suggests that JF2480 is taxonomically distant from other known A.&nbsp, salmonicida subspecies. The genome sequence confirms that the strain possesses key virulence genes that are present in the typical A. salmonicida psychrophilic subspecies, with the exception of the genes encoding the type three secretion system (T3SS). Bacterial virulence assays conducted on the surrogate host Dictyostelium discoideum amoeba confirmed that the strain is virulent despite the lack of T3SS. Bacterial growth curves showed that strain JF2480 grow well at 40 &deg, C, the body temperature of the pied avocet, and even faster at 41 &deg, C, compared to other mesophilic strains. Discovery of this strain further demonstrates the extent of the phylogenomic tree of this species. This study also suggests that A. salmonicida can infect a wider array of hosts than previously suspected and that we need to rethink the way we perceive A. salmonicida&rsquo, s natural environment.

Phages infecting Aeromonas salmonicida subsp. salmonicida, the causative agent of the fish disease furunculosis, have been isolated for decades but very few of them have been characterized. Here, the host range of 12 virulent phages, including three isolated in the present study, was evaluated against a panel of 65 A. salmonicida isolates, including representatives of the psychrophilic subspecies salmonicida, smithia, masoucida, and the mesophilic subspecies pectinolytica. This bacterial set also included three isolates from India suspected of being members of a new subspecies. Our results allowed to elucidate a lytic dichotomy based on the lifestyle of A. salmonicida (mesophilic or psychrophilic) and more generally, on phage types (lysotypes) for the subspecies salmonicida. The genomic analyses of the 12 phages from this study with those available in GenBank led us to propose an A. salmonicida phage pan-virome. Our comparative genomic analyses also suggest that some phage genes were under positive selection and A. salmonicida phage genomes having a discrepancy in GC% compared to the host genome encode tRNA genes to likely overpass the bias in codon usage. Finally, we propose a new classification scheme for A. salmonicida phages.

Aeromonas salmonicida is a Gram-negative bacterium, known as a fish pathogen since its discovery. Although the species was initially considered psychrophilic, a mesophilic subspecies (pectinolytica) and many other mesophilic strains still not attributed to subspecies have been described in the last two decades. These mesophilic strains were sampled from various sources, including humans, and some of them are known to be pathogenic. In this study, we describe a strain, JF2480, which was isolated from the spleen, and also found the kidney and liver of a dead pied avocet (Recurvirostra avosetta), a type of migratory bird inhabiting aquatic environments. A core genome phylogenomic analysis suggests that JF2480 is taxonomically distant from other known A. salmonicida subspecies. The genome sequence confirms that the strain possesses key virulence genes that are present in the typical A. salmonicida psychrophilic subspecies, with the exception of the genes encoding the type three secretion system (T3SS). Bacterial virulence assays conducted on the surrogate host Dictyostelium discoideum amoeba confirmed that the strain is virulent despite the lack of T3SS. Bacterial growth curves showed that strain JF2480 grow well at 40 °C, the body temperature of the pied avocet, and even faster at 41 °C, compared to other mesophilic strains. Discovery of this strain further demonstrates the extent of the phylogenomic tree of this species. This study also suggests that A. salmonicida can infect a wider array of hosts than previously suspected and that we need to rethink the way we perceive A. salmonicida’s natural environment.

The bacterial species Aeromonas salmonicida officially has five subspecies. A large majority of the currently available sequences come from Aeromonas salmonicida subsp. salmonicida, which causes furunculosis in salmonids. We present the genomic sequences of four Aeromonas salmonicida subsp. achromogenes strains. This will help increase the robustness of genomic analyses for this subspecies.

International audience; The Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is an aquatic pathogen which causes furunculosis to salmonids, especially in fish farms. The emergence of strains of this bacterium exhibiting antibiotic resistance is increasing, limiting the effectiveness of antibiotherapy as a treatment against this worldwide disease. In the present study, we discovered an isolate of A. salmonicida subsp. salmonicida that harbors two novel plasmids variants carrying antibiotic resistance genes. The use of long-read sequencing (PacBio) allowed us to fully characterize those variants, named pAsa5-3432 and pRAS3-3432, which both differ from their classic counterpart through their content in mobile genetic elements. The plasmid pAsa5-3432 carries a new multidrug region composed of multiple mobile genetic elements, including a Class 1 integron similar to an integrated element of Salmonella enterica. With this new region, probably acquired through plasmid recombination, pAsa5-3432 is the first reported plasmid of this bacterium that bears both an essential virulence factor (the type three secretion system) and multiple antibiotic resistance genes. As for pRAS3-3432, compared to the classic pRAS3, it carries a new mobile element that has only been identified in Chlamydia suis. Hence, with the identification of those two novel plasmids harboring mobile genetic elements that are normally encountered in other bacterial species, the present study puts emphasis on the important impact of mobile genetic elements in the genomic plasticity of A. salmonicida subsp. salmonicida and suggests that this aquatic bacterium could be an important reservoir of antibiotic resistance genes that can be exchanged with other bacteria, including human and animal pathogens.

It is well known that antibiotics are playing leading roles in several areas such as human and animal health and farm production. Unfortunately, the use of antimicrobial compounds as a panacea is coming to an end. More and more antibiotic-resistant bacterial strains are listed in all spheres of activity where they were used. Aquaculture, which is a key industry in providing animal protein needs to an exponentially growing human population, is undergoing the attack of pathogens that are increasingly difficult to control due to antibiotic resistance. In this chapter, we will explore, from an aquaculture perspective, the discovery and use of antibiotics, explain the major mechanisms of antibiotic resistance and the adverse consequences of using broad-spectrum antibiotics, and finally discuss briefly on alternatives to traditional antimicrobial agents. We will also give a concrete example with the bacterium Aeromonas salmonicida subsp. salmonicida, which is a worrisome pathogen for the aquaculture industry and for which crucial discoveries have been recently made thanks to the advances in sequencing technologies.

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 &deg, 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 &deg, 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.

In Aeromonas salmonicida subsp. salmonicida, a bacterium that causes fish disease, there are two types of small plasmids (

The Gram-negative bacterium Aeromonas salmonicida contains five subspecies: salmonicida, smithia, achromogenes, masoucida and pectinolytica. Pectinolytica is a mesophilic subspecies with the ability to thrive at a wide range of temperatures, including 37°C, while the four other subspecies are psychrophilic, restricted to lower temperatures. The psychrophilic subspecies are known to infect a wide range of fishes. However, there is no evidence of pathogenicity for the mesophilic subspecies pectinolytica. Study of the differences between the mesophilic and psychrophilic subspecies is hampered by the lack of completely sequenced and closed genomes from the mesophilic subspecies. A previous study reported that insertion sequences, which can induce genomic rearrangements at temperatures around 25°C, could be one of the determinants explaining the differences in lifestyle (mesophilic or psychrophilic) between the subspecies. In this study, the genome of mesophilic strain A527 of A. salmonicida was sequenced, closed and analyzed to investigate the mesophilic-psychrophilic discrepancy. This reference genome supports the hypothesis that insertion sequences are major determinants of the lifestyle differences between the A. salmonicida subspecies. Moreover, the phylogenetic analysis performed to position strain A527 within the taxonomy raises an issue regarding the intraspecies structure of A. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen. Analysis of its genomic characteristics is required to determine the worldwide distribution of the various populations of this bacterium. Genomic alignments between the 01-B526 pathogenic strain and the A449 reference strain have revealed a 51-kb chromosomal insertion in 01-B526. This insertion (AsaGEI1a) has been identified as a new genomic island (GEI) bearing prophage genes. PCR assays were used to detect this GEI in a collection of 139 A. salmonicida subsp. salmonicida isolates. Three forms of this GEI (AsaGEI1a, AsaGEI1b, AsaGEI2a) are now known based on this analysis and the sequencing of the genomes of seven additional isolates. A new prophage (prophage 3) associated with AsaGEI2a was also discovered. Each GEI appeared to be strongly associated with a specific geographic region. AsaGEI1a and AsaGEI2a were exclusively found in North American isolates, except for one European isolate bearing AsaGEI2a. The majority of the isolates bearing AsaGEI1b or no GEI were from Europe. Prophage 3 has also a particular geographic distribution and was found only in North American isolates. We demonstrated that A. salmonicida subsp. salmonicida possesses unsuspected elements of genomic heterogeneity that could be used as indicators to determine the geographic origins of isolates of this bacterium.

Aeromonas salmonicida strains are roughly classified into two categories, typical and atypical strains. The latter mainly regroup isolates that present unusual phenotypes or hosts, comparatively to the typical strains that belong to the salmonicida subspecies. This study focuses on an uncharacterized atypical strain, M18076-11, isolated from lumpfish (Cyclopterus lumpus) and not part of the four recognized Aeromonas salmonicida subspecies. This isolate presents an unreported phenotype in the A. salmonicida species: the formation of large granular aggregates. Granules are formed of a heterogeneous mix of live and dead cells, with live cells composing the majority of the population. Even if no mechanism was determined to cause cellular aggregation, small globular structures at the cell surface were observed, which might affect granular formation. Pan-genome phylogenetic analysis indicated that this strain groups alongside the masoucida subspecies. However, phenotypic tests showed that these strains have diverging phenotypes, suggesting that M18076-11 might belong to a new subspecies. Also, a pAsal1-like plasmid, which was only reported in strains of the subspecies salmonicida, was discovered in M18076-11. This study sheds light on unsuspected diversity in A. salmonicida subspecies and stresses the need of thorough identification when a new strain is encountered, as unique traits might be discovered.

Aeromonas salmonicidasubsp.salmonicida, the causative agent of furunculosis in salmonids, is an issue especially because many isolates of this bacterium display antibiotic resistances, which limit treatments against the disease. Recent results suggested the possible existence of alternative forms of pAsa4, a large plasmid found inA. salmonicidasubsp.salmonicidaand bearing multiple antibiotic resistance genes. The present study reveals the existence of two newly detected pAsa4 variants, pAsa4b and pAsa4c. We present the extensive characterization of the genomic architecture, the mobile genetic elements and the antimicrobial resistance genes of these plasmids in addition to the reference pAsa4 from the strain A449. The analysis showed differences between the three architectures with consequences on the content of resistance genes. The genomic plasticity of the three pAsa4 variants could be partially explained by the action of mobile genetic elements like insertion sequences. Eight additional isolates from Canada and Europe that bore similar antibiotic resistance patterns as pAsa4-bearing strains were genotyped and specific pAsa4 variants could be attributed to phenotypic profiles. pAsa4 and pAsa4c were found in Europe, while pAsa4b was found in Canada. In accordance with their content in conjugative transfer genes, only pAsa4b and pAsa4c can be transferred by conjugation inEscherichia coli. The plasticity of pAsa4 variants related to the acquisition of antibiotic resistance indicates that these plasmids may pose a threat in terms of the dissemination of antimicrobial-resistantA. salmonicidasubsp.salmonicidabacteria.

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonids. We recently identified a group of genomic islands (AsaGEI) in this bacterium. AsaGEI2a, one of these genomic islands, has almost exclusively been identified in isolates from North America. To date, Aeromonas salmonicida subsp. salmonicida JF3224, a strain isolated from a wild brown trout (Salmo trutta) caught in Switzerland, was the only European isolate that appeared to bear AsaGEI2a. We analyzed the genome of JF3224 and showed that the genomic island in JF3224 is a new variant of AsaGEI, which we have called AsaGEI2b. While AsaGEI2b shares the same integrase gene and insertion site as AsaGEI2a, it is very different in terms of many other features. Additional genomic investigations combined with PCR genotyping revealed that JF3224 is sensitive to growth at 25°C, leading to insertion sequence-dependent rearrangement of the locus on the pAsa5 plasmid that encodes a type three secretion system, which is essential for the virulence of the bacterium. The analysis of the JF3224 genome confirmed that AsaGEIs are accurate indicators of the geographic origins of A. salmonicida subsp. salmonicida isolates and is another example of the susceptibility of the pAsa5 plasmid to DNA rearrangements.

The bacterium Aeromonas salmonicida is the etiological agent of furunculosis, a widespread fish disease causing important economic losses to the fish farming industry. Antibiotic treatments in fish farms may be challenging given the existence of multi-drug resistant isolates of this bacterium. Here, we report the draft genome sequences of the 2004-05MF26 and 2009-144K3 isolates, which harbor plasmids conferring antibiotic resistance. Both isolates also carry the large plasmid pAsa5, which is known to encode a type three secretion system (TTSS) and the pAsal1 plasmid which has the aopP gene producing a TTSS effector. These two isolates are good representatives of the plasmid diversity in A. salmonicida subsp. salmonicida .

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.

Background Aeromonads make up a group of Gram-negative bacteria that includes human and fish pathogens. The Aeromonas salmonicida species has the peculiarity of including five known subspecies. However, few studies of the genomes of A. salmonicida subspecies have been reported to date. Results We sequenced the genomes of additional A. salmonicida isolates, including three from India, using next-generation sequencing in order to gain a better understanding of the genomic and phylogenetic links between A. salmonicida subspecies. Their relative phylogenetic positions were confirmed by a core genome phylogeny based on 1645 gene sequences. The Indian isolates, which formed a sub-group together with A. salmonicida subsp. pectinolytica, were able to grow at either at 18 °C and 37 °C, unlike the A. salmonicida psychrophilic isolates that did not grow at 37 °C. Amino acid frequencies, GC content, tRNA composition, loss and gain of genes during evolution, pseudogenes as well as genes under positive selection and the mobilome were studied to explain this intraspecies dichotomy. Conclusion Insertion sequences appeared to be an important driving force that locked the psychrophilic strains into their particular lifestyle in order to conserve their genomic integrity. This observation, based on comparative genomics, is in agreement with previous results showing that insertion sequence mobility induced by heat in A. salmonicida subspecies causes genomic plasticity, resulting in a deleterious effect on the virulence of the bacterium. We provide a proof-of-concept that selfish DNAs play a major role in the evolution of bacterial species by modeling genomes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2381-3) contains supplementary material, which is available to authorized users.