Mélanie V. Trudel

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.

Detection of Variants of the pRAS3, pAB5S9, and pSN254 Plasmids in Aeromonas salmonicida subsp. salmonicida: Multidrug Resistance, Interspecies Exchanges, and Plasmid Reshaping

ABSTRACT 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.

Diversity of antibiotic-resistance genes in Canadian isolates of Aeromonas salmonicida subsp. salmonicida : dominance of pSN254b and discovery of pAsa8

The bacterium Aeromonas salmonicida subsp. salmonicida is a common pathogen in fish farms worldwide. Since the antibiotic resistance of this bacterial species is on the increase, it is important to have a broader view on this issue. In the present study, we tested the presence of known plasmids conferring multi-drug resistance as well as antibiotic resistance genes by a PCR approach in 100 Canadian A. salmonicida subsp. salmonicida isolates. Our study highlighted the dominance of the conjugative pSN254b plasmid, which confers multi-drug resistance. We also identified a new multi-drug plasmid named pAsa8, which has been characterized by a combination of sequencing technologies (Illumina and Oxford nanopore). This new plasmid harbors a complex class 1 integron similar to the one of the Salmonella genomic island 1 (SGI1) found in Salmonella enterica and Proteus mirabilis. Consequently, in addition to providing an update on the A. salmonicida subsp. salmonicida isolates that are resistant to antibiotics, our data suggest that this bacterium is potentially an important reservoir of drug resistance genes and should consequently be monitored more extensively. In addition, we describe a screening method that has the potential to become a diagnostic tool that is complementary to other methods currently in use.

Insertion sequence AS5 (ISAS5) 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.

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.

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.

Diversity and Homogeneity among Small Plasmids of Aeromonas salmonicida subsp. salmonicida Linked with Geographical Origin.

Furunculosis, which is caused by Aeromonas salmonicida subsp. salmonicida, is a major salmonid disease in fish farms worldwide. Several plasmids found in this bacterium confer phenotypes such drug resistance and virulence. Small plasmids (pAsa1, pAsa2, pAsa3, and pAsal1) related to ColE1- and ColE2-type replicons are usually present in its normal plasmidome. In the present study, with the objective to investigate if these plasmids display particularities related to the origin of the isolates bearing them, a total of 153 isolates, including 78 new and 75 previously described, were analyzed for the presence of small plasmids by PCR and DNA restriction fragment profiling. A geographical dichotomy between Canadian and European isolates for their propensity to do not have pAsa3 or pAsal1 was found. In addition, the genotyping analysis led to the identification of two European isolates harboring an unusual pAsal1. An investigation by next-generation sequencing (NGS) of these two isolates shed light on two pAsal1 variants (pAsal1C and pAsal1D). As with pAsal1B, another pAsal1 variant previously described, these two new variants bore a second insertion sequence (ISAS5) in addition to the usual ISAS11. The characterization of these variants suggested that they could predominate over the wild-type pAsal1 in stressful conditions such as growth at temperatures of 25°C and above. To obtain a comprehensive portrait of the mutational pressure on small plasmids, 26 isolates whose DNA had been sequenced by NGS were investigated. pAsa3 and pAsal1 were more prone to mutations than pAsa1 and pAsa2, especially in the mobA gene, which encodes a relaxase and a primase. Lastly, the average copy number of each plasmid per cell was assessed using raw sequencing data. A clear trend with respect to the relative proportion per cell of each plasmid was identified. Our large-scale study revealed a geographical dichotomy in small plasmid repertoire in addition to a clear trend for pAsa3 and pAsal1 to be more frequently altered. Moreover, we present the discovery of two new variants of pAsal1: pAsal1C and pAsal1D.

AsaGEI2b: a new variant of a genomic island identified in the Aeromonas salmonicida subsp. salmonicida JF3224 strain isolated from a wild fish in Switzerland.

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.

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.