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

Abstract

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.

Figure 1. Putative evolutionary scenario of the multidrug region of pAsa8.

Only the elements involved are shown: the gene umuC of ancestral pAsa8, the genes tnpR and the one encoding for the chemotaxis protein (CP) located on the transposon Tn1721, the insertion sequence IS5 and finally the integron In104-like. Cassettes aadA2 and qacEΔ1 are located within integron 1 of In104 (dashed arrow), and also within integron 2 of In104-like found on pAsa8 (solid line arrow) (isolate M16474-11).

Figure 2. Comparisons of the multi-drug region of pAsa8 with the transposon Tn1721 and the integron In104 found in SGI.

Figure 3

Multiplex PCR targeting genes coding for resistance to chloramphenicol/florfenicol (A), sulfonamides (B), and tetracyclines (C,D). Electrophoresis gels with amplicons generated using DNA isolated from positive controls (A449 + M15879-11 in (A,C), M15879-11 in B, 2009-144K3 + M16474-11 in D) and from a negative control (01-B526). A specific control for A. salmonicida subsp. salmonicida using a primer pair that amplified an element of the prophage 1 was included in each multiplex PCR reaction. The amplicon of this product can be seen at the bottom of each gel (control). (A) The target cat (448 bp) and floR genes (632 bp) and (B) the sul2 (449 bp) and sul1 (550 bp) genes were detected when present. (C) Amplicons for tet(E) (351 bp) and tetA (526 pb). (D) Amplicons for tet(H) (326 bp), tet(G) (460 bp), and tet(C) (629 bp). Water was used as a negative control.

Figure 4. Map of the Quebec showing the regions to which the various isolates were assigned.

Northwest (NW, orange), northeast (NE, purple), southwest (SW, pink), and southeast (SE, green). Most of the fish farms analyzed were less than 200 km from the St. Lawrence River, which crosses the province from west to east. The map has been drawn using Adobe Photoshop CS4 version 11.0.2 (www.adobe.com).