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

Abstract

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.

Keywords: Aeromonas salmonicida; furunculosis; insertion sequence; pAsal1; plasmid stability.

Figure 1

Maps of the ColE2-type replicon plasmids pAsa1, pAsa3, and pAsal1. The gray zones represent high identity regions of the sequences. The dark and light blue arrows represent coding sequences (CDSs) and the RNA I regulator, respectively. The red rectangle represents the ISAS11 of pAsal1. HP means “hypothetical protein.” The complete map of pAsal1 is given in Figure S1.

Figure 2

Pulse field gel electrophoresis of digested small plasmids. The purified small plasmids of isolates JF2506 and JF2507 were digested with EcoRI and were separated on 1% agarose gel by PFGE. HER1104 was also added since the bands were similar to those reported for the pAsal1 variant pAsal1B. Strain 01-B526 was used as a control of a normal plasmidome of small plasmids.

Figure 3

Comparison of pAsal1 (JF2267), pAsal1B (HER1104), pAsa1C (JF2506), and pAsal1D (JF2507). The dark and light blue arrows represent coding sequences (CDSs) and the RNA I regulator, respectively. The purple and red rectangles represent ISAS5 and ISAS11, respectively, while the green rectangles represent pseudogenes. The gray zones represent high identity regions of the sequences. The orange zone indicates the difference between pAsal1C and pAsal1D. The CDSs related to transposases and a 3′ part of the truncated mobA were not added for purposes of clarity. The complete map of pAsal1 is given in Figure S1.

Figure 4

Boxplot analysis of the plasmid copy number ratios relative to pAsa1. This analysis included small plasmids for all the isolates for which the DNA was sequenced and which are listed in Table S3.