Comparative sequence analysis of a multidrug-resistant plasmid from Aeromonas hydrophila

Abstract

Aeromonas hydrophila is a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated from A. hydrophila obtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1_Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn21 type transposon. This transposon contains the drug resistance genes qacH, bla(OXA-10), aadA1, and sul1 in a class 1 integron; tetA and tetR in transposon Tn1721; and catA2 and a duplicate sul1 in a locus showing 100% similarity to IncU plasmids isolated from fish. The bla(OXA-10) and aadA1 genes showed 100% similarity to those from the Acinetobacter baumannii AYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred.

Fig 1

Circular representation of pR148. The assembly and annotation information are shown. Circles from inside to outside: GC content (blue, GC⁺; red, GC⁻), reverse CDS, forward CDS, nucleotide position (relative to the repA gene, which is represented as an arrowhead), region in pR148 homologous to E. coli pNDM-1_Dok01, region homologous to E. coli pNDM-1_Dok01, region homologous to S. enterica (Newport) pSN254, inserts found in pNDM-1_Dok01, inserts found in pNDM102337, and inserts found in pSN254. Genes are color coded according to function. The locus for the type 4 secretion system (T4SS), the mercuric resistance operon, Tn1721, and the class 1 integron are enclosed in blue boxes.

Fig 2

BLAST homology search results. The homology of the whole nucleotide sequence of pR148 was generated against the nr/nt database using BLASTN with a 2 to 3 match/mismatch scores, gap scores of 5 for existence and 2 for extension, and an expected threshold of 1,000 on 17 February 2012. Sequences showing ≥50% query coverage were chosen for initial comparative analysis. The black lines transverse to the alignment indicate regions of least similarity, while gaps indicate deletion regions (regions that contain nucleotide sequences in pR148 but not in the subject sequence).

Fig 3

MAUVE alignment of local colinear blocks (LCBs) of pR148, pNDM-1_Dok01, pNDM102337, and pSN254. The topmost panel is a linear representation of pR148 for reference, arranged from top to bottom representing reverse CDS, forward CDS, and nucleotide position relative to the repA gene. Genes are color coded according to function. The representation of each sequence from MAUVE contains, from top to bottom, the LCBs, forward CDs, reverse CDs, and annotated features. Lines are drawn to connect the similar LCBs, and a colored similarity plot is shown for each genome, the height of which is proportional to the level of sequence identity in that region. Mauve-colored segments are conserved among all sequences, while other colors indicate segments which are conserved only among some of the sequences.

Fig 4

Representation of the accessory and 4th indel and accessory region of pR148 compared to the flanking backbone sequences from the representative plasmids pNDM-1_Dok01, pNDM102337, and pSN254. The light blue background represents regions showing alignment scores of ≥200 in BLASTN. The scale on the top represents nucleotide positions in pR148. Genes are color coded according to function.

Fig 5

Phylogenetic alignment and source map of closely related plasmids. The phylogenetic tree of parallelized sequences was aligned using ClustalW and was generated using the neighbor-joining method with 1,000 bootstrap replications.

Fig 6

Network representation of the closely related plasmids. A network representation for pR148 was produced using Cytoscape by merging the available network for mobile genetic elements (MGEs) generated from the ACLAME database. Each node represents a genome, and nodes are connected if they share homologous DNA. The edges are weighted, meaning genomes sharing more families have a higher significance value and are closer on the display. (A) pR148 resides in a cluster of several MGEs that are closely related to one another. (B) The MGEs extracted from the ACLAME database that were also chosen for comparative analysis show that all of these MGEs are closely related to one another but pR148 has fewer shared elements. Circles, plasmids; lines, edges representing the significance value.