Host Dependent-Transposon for a Plasmid Found in Aeromonas salmonicida subsp. salmonicida That Bears a catB3 Gene for Chloramphenicol Resistance

Abstract

Plasmids that carry antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen with severe consequences in salmonid farming. Here, we describe a 67 kb plasmid found in the A. salmonicida subsp. salmonicida Strain SHY15-2939 from Quebec, Canada. This new plasmid, named pAsa-2939 and identified by high throughput sequencing, displays features never found before in this bacterial species. It contains a transposon related to the Tn21 family, but with an unusual organization. This transposon bears a catB3 gene (chloramphenicol resistance) that has not been detected yet in A. salmonicida subsp. salmonicida. The plasmid is transferable by conjugation into Aeromonas hydrophila, but not into Escherichia coli. Based on PCR analysis and genomic sequencing (Illumina and PacBio), we determined that the transposon is unstable in A. salmonicida subsp. salmonicida Strain SHY15-2939, but it is stable in A. hydrophila trans-conjugants, which explains the chloramphenicol resistance variability observed in SHY15-2939. These results suggest that this bacterium is likely not the most appropriate host for this plasmid. The presence of pAsa-2939 in A. salmonicida subsp. salmonicida also strengthens the reservoir role of this bacterium for antibiotic resistance genes, even those that resist antibiotics not used in aquaculture in Québec, such as chloramphenicol.

Keywords: Aeromonas salmonicida subsp. salmonicida; antibiotic resistance gene; chloramphenicol; pAsa-2939; plasmid; transposon.

Figure 1

Gene map of the pAsa-2939 plasmid. This map was produced with the DNA plotter software. The red genes code for antibiotic resistance proteins, the blue genes for hypothetical proteins, and the purple genes for proteins with other known functions. The black arc indicates the transposon (Tn21-like). The green arc corresponds to conjugation genes and the inner circle corresponds to the GC skew. The primers used for PCR genotyping are also shown on the map with arrowheads.

Figure 2

Nucleotide alignment of pAsa-2939 transposon with Tn21 and Tn1721. Comparison of Tn1721 (accession X61367) and the transposon from pAsa-2939 (Tn21-like) and Tn21 (accession AF071413). The gray areas represent similar or identical segments in the sequences of the plasmids compared. The ARGs are shown in red, the mobile elements are in blue and the orange arrows represent other genes. The figure was made using Easyfig software.

Figure 3

Transposon instability in SHY15-2939 confirmed by PCR analysis. (A) Analysis of different lysates from A. salmonicida subsp. salmonicida SHY15-2939 grown on different medium using different primers. This confirms the excision of the transposon in a part of the bacterial population (in orange) while the majority of the bacteria keep an intact plasmid (in blue). (B) Schematic representation of pAsa-2939 found in A. salmonicida subsp. salmonicida SHY15-2939. The region in green corresponds to the gene involve in the conjugation of the plasmid and the Tn21-like is represented by the region in red. The primers are shown in blue or orange depending on the combination used for the PCR. The Tn21_integrase_R1 primer was used in both PCR reaction. (C) Confirmation of the specificity of the Tn21_Merc_F1 and Tn21_integrase R1 primers (orange combination) for transposon excision. Strains tested are in this order from lane 1 to 6: 01-B526, 01-B516, A449, SHY16-3432, SHY15-2939 and water as a negative control. (D) The PCR result from 7 trans-conjugants of A. hydrophila with the pAsa-2939 plasmid (line 1 to 7) using the primers Tn21_Merc_F1 and Tn21_integrase R1 (orange combination). The eighth well corresponds to the positive control which is A. salmonicida ssp. salmonicida SHY15-2939. (E) A PCR amplification of the primers Tn21_integrase_F1 and Tn21_integrase_R1 (blue combination) for A. hydrophila with pAsa-2939 on the same samples than in D.

Figure 4

Transposon instability in SHY15-2939 confirmed by bioinformatics analysis. Comparison between a read from PacBio and the sequence of pAsa-2939 without the transposon (theoretical) and the pAsa-2939 plasmid including the transposon. The gray area represents similarities between the two plasmid versions. The red arrows represent the ARGs and the orange arrows represent other genes. The white parallelogram represents the region used to detect PacBio reads corresponding to the junction consequent to transposon excision.