Plasmid composition in Aeromonas salmonicida subsp. salmonicida 01-B526 unravels unsuspected type three secretion system loss patterns.

BACKGROUND: Aeromonas salmonicida subsp. salmonicida is a ubiquitous psychrophilic waterborne bacterium and a fish pathogen. The numerous mobile elements, especially insertion sequences (IS), in its genome promote rearrangements that impact its phenotype. One of the main virulence factors of this bacterium, its type three secretion system (TTSS), is affected by these rearrangements. In Aeromonas salmonicida subsp. salmonicida most of the TTSS genes are encoded in a single locus on a large plasmid called pAsa5, and may be lost when the bacterium is cultivated at a higher temperature (25 °C), producing non-virulent mutants. In a previous study, pAsa5-rearranged strains that lacked the TTSS locus on pAsa5 were produced using parental strains, including 01-B526. Some of the generated deletions were explained by homologous recombination between ISs found on pAsa5, whereas the others remained unresolved. To investigate those rearrangements, short- and long-read high-throughput sequencing technologies were used on the A. salmonicida subsp. salmonicida 01-B526 whole genome. RESULTS: Whole genome sequencing of the 01-B526 strain revealed that its pAsa5 has an additional IS copy, an ISAS5, compared to the reference strain (A449) sequence, which allowed for a previously unknown rearrangement to occur. It also appeared that 01-B526 bears a second large plasmid, named pAsa9, which shares 40 kbp of highly similar sequences with pAsa5. Following these discoveries, previously unexplained deletions were elucidated by genotyping. Furthermore, in one of the derived strains a fusion of pAsa5 and pAsa9, involving the newly discovered ISAS5 copy, was observed. CONCLUSION: The loss of TTSS and hence virulence is explained by one consistent mechanism: IS-driven homologous recombination. The similarities between pAsa9 and pAsa5 also provide another example of genetic diversity driven by ISs.

The mosaic architecture of Aeromonas salmonicida subsp. salmonicida pAsa4 plasmid and its consequences on antibiotic resistance.

Aeromonas salmonicida subsp. 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 in A. salmonicida subsp. salmonicida and 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 in Escherichia 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-resistant A. salmonicida subsp. salmonicida bacteria.

Draft genome sequences of two Aeromonas salmonicida subsp. salmonicida isolates harboring plasmids conferring antibiotic resistance.

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 multidrug-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.

Variants of a genomic island in Aeromonas salmonicida subsp. salmonicida link isolates with their geographical origins.

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.

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.

Optimization of a plasmid electroporation protocol for Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a major fish pathogen. Molecular tools are required to study the virulence and genomic stability of this bacterium. An efficient electroporation-mediated transformation protocol for A. salmonicida subsp. salmonicida would make genetic studies faster and easier. In the present study, we designed the 4.1-kb pSDD1 plasmid as a tool for optimizing an electroporation protocol for A. salmonicida subsp. salmonicida. We systematically tested the electroporation conditions to develop a protocol that generates the maximum number of transformants. Under these optimal conditions (25 kV/cm, 200 Ω, 25 µF), we achieved an electroporation efficiency of up to 1×10(5) CFU/µg DNA. The electroporation protocol was also tested using another plasmid of 10.6-kb and three different strains of A. salmonicida subsp. salmonicida. The strains displayed significant differences in their electro-transformation competencies. Strain 01-B526 was the easiest to electroporate, especially with the pSDD1 plasmid. This plasmid was stably maintained in the 01-B526 transformants, as were the native plasmids, but could be easily cured by removing the selection conditions. This is the first efficient electroporation protocol reported for A. salmonicida subsp. salmonicida, and offers new possibilities for studying this bacterium.

Virulence, genomic features, and plasticity of Aeromonas salmonicida subsp. salmonicida, the causative agent of fish furunculosis.

The bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a systemic disease of fish in the salmonid family. Furunculosis is a ubiquitous disease that affects aquaculture operations worldwide and is characterized by high mortality and morbidity. A better understanding of the bacterium is required to find a cure. Thereby, this review centers on A. salmonicida subsp. salmonicida, its major virulence factors, and its genome. The classification and characteristics of A. salmonicida subsp. salmonicida, the virulence factors, such as the A-layer, extracellular molecules, and type three secretion system as well as the characteristics and plasticity of its genome are described.

Insertion sequence AS5 (IS AS5 ) is involved in the genomic plasticity of Aeromonas salmonicida.

The genome of the fish pathogen Aeromonas salmonicida subsp salmonicida harbors a large number of insertion sequences (ISs), many of which are located on plasmids. In the present study, we analyzed the small plasmid profile of A. salmonicida strains to identify evidences of plasmid alterations. Ten out of 78 strains analyzed displayed an unconventional plasmid profile. However the HER1104 strain was unique, having a positive PCR signal for pAsal1 plasmid despite not carrying this plasmid. Instead, HER1104 was bearing a plasmid at higher molecular weight than pAsal1. We characterized this new larger plasmid, which we called pAsal1B since it is a derivative of pAsal1 containing one more complete IS (ISAS5) than the parental plasmid. An additional 96 bp relic of ISAS5 was also present in pAsal1B. These results propose that ISAS5 is another active mobile genetic element in A. salmonicida subsp salmonicida and provided further proof of the genomic plasticity of this bacterium.

Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system.

The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida.

IS-mediated loss of virulence by Aeromonas salmonicida: A tangible piece of an evolutionary puzzle.

Insertion sequences (IS) are abundant in the bacterial fish pathogen Aeromonas salmonicida genome. IS are involved in rearrangement events that lead to the loss of virulence. In previous work, we studied a plasmid rearrangement that causes the deletion of the type three secretion system in A. salmonicida, resulting in a loss of virulence. We showed that the rearrangement is caused by the recombination of two IS (ISAS11) on an unstable plasmid (pAsa5). However, many rearrangements cannot be explained by our experimental approach and are thought to be the result of more complex or incomplete rearrangement events, as suggested by other plasmid loss profiles observed in various A. salmonicida strains. In this commentary, we examine the genetic instability of A. salmonicida indicating that its genome is rapidly evolving.

An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system.

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida.

Draft genome sequence of the virulent strain 01-B526 of the fish pathogen Aeromonas salmonicida.

Aeromonas salmonicida is an important fish pathogen, mainly of salmonids. This bacterium causes a disease named furunculosis, which is particularly detrimental for the aquaculture industry. Here, we present the draft genome sequence of A. salmonicida 01-B526, a strain isolated from a brook trout that is more virulent than A. salmonicida reference strain A449, for which a genome sequence is available.