Vibrio parahaemolyticus from Migratory Birds in China Carries an Extra Copy of tRNA-Gly and Plasmid-Mediated Quinolone Resistance Gene qnrD

Abstract

Vibrio parahaemolyticus is a marine bacterium coming from estuarine environments, where the migratory birds can easily be colonized by V. parahaemolyticus. Migratory birds may be important reservoirs of V. parahaemolyticus by growth and re-entry into the environment. To further explore the spreading mechanism of V. parahaemolyticus among marine life, human beings, and migratory birds, we aimed to investigate the characteristics of the genetic diversity, antimicrobial resistance, virulence genes, and a potentially informative gene marker of V. parahaemolyticus isolated from migratory birds in China. This study recovered 124 (14.55%) V. parahaemolyticus isolates from 852 fecal and environmental (water) samples. All of the 124 strains were classified into 85 known sequence types (STs), of which ST-2738 was most frequently identified. Analysis of the population structure using whole-genome variation of the 124 isolates illustrated that they grouped into 27 different clonal groups (CGs) belonging to the previously defined geographical populations VppX and VppAsia. Even though these genomes have high diversity, an extra copy of tRNA-Gly was presented in all migratory bird-carried V. parahaemolyticus isolates, which could be used as a potentially informative marker of the V. parahaemolyticus strains derived from birds. Antibiotic sensitivity experiments revealed that 47 (37.10%) isolates were resistant to ampicillin. Five isolates harbored the plasmid-mediated quinolone resistance (PMQR) gene qnrD, which has not previously been identified in this species. The investigation of antibiotic resistance provides the basic knowledge to further evaluate the risk of enrichment and reintroduction of pathogenic V. parahaemolyticus strains in migratory birds. IMPORTANCE The presence of V. parahaemolyticus in migratory birds' fecal samples implies that the human pathogenic V. parahaemolyticus strains may also potentially infect birds and thus pose a risk for zoonotic infection and food safety associated with re-entry into the environment. Our study firstly highlights the extra copy of tRNA as a potentially informative marker for identifying the bird-carried V. parahaemolyticus strains. Also, we firstly identify the plasmid-mediated quinolone resistance (PMQR) gene qnrD in V. parahaemolyticus. To further evaluate the risk of enrichment and reintroduction of pathogenic strains carried by migratory birds, we suggest conducting estuarine environmental surveillance to monitor the antibiotic resistance and virulence factors of bird-carried V. parahaemolyticus isolates.

Keywords: MLST; Vibrio parahaemolyticus; antimicrobial resistance; bacterial genome-wide association studies; genomic analysis; population structure; virulence factors.

FIG 1

This map was drawn by ArcGIS online (https://www.arcgis.com). The geographical locations of the sampling sites highlighted using red dots. (A) Overseas Chinese Town Wetland Park (OCTWP), Shenzhen, Guangdong; (B) Futian Red Forest Reserve (FRFR), Shenzhen, Guangdong; (C) Zhaoqing, Guangdong; (D) Beihai, Guangxi; (E) Fangchenggang, Guangxi; (F) Nanning, Guangxi; (G) Dongxing, Guangxi; (H) Zhongning, Ningxia; (I) Zhanjiang, Guangdong; (L) Leizhou, Guangdong; (M) Chifeng, Inner Mongolia autonomous region. All of the isolates in this study are from sites A, B, I, and L.

FIG 2

Neighbor-joining tree and population structure of V. parahaemolyticus. (A) A core SNP-based phylogenetic tree of the 593 genomes. The colors on the clades represent population groups (VppUS1, VppUS2, VppX, and VppAsia). The purple dots at the tips of the branches show the 28 clonal groups (CGs). The location, source, and collection time of the 124 genomes and the 70 non-bird-carried V. parahaemolyticus genomes with an extra copy of tRNA-Gly in this study are shown by strips. (B) The population structure of the 325 genomes after grouping the strains (pairwise SNPs < 23,500). The 325 genomes represent the 588 genomes. The column represents donor strain, and the row represents recipient strain. The color of the cell shows the number of sequences that the donor transfers to the recipient. The black line indicates the lineage boundary.

FIG 3

The illustration of the extra copy of tRNA-Gly in bird-carried V. parahaemolyticus strains. VP-Bird represents the bird-carried V. parahaemolyticus, and Non-VP-Bird represents the non-bird-carried V. parahaemolyticus. VPt08x and VPt09x in the top row show the positions of the tRNA. The numbers in Gly-xTR-y in the very left column show the number of copies of tRNA-Gly (x) and the concatenation-style of tRNA-Gly and tRNA-Met (y).

FIG 4

Percentages of the 46 V. parahaemolyticus isolates resistant to ampicillin in relation to isolation source (A) and origin (B); antibiotic resistance genes (C) identified in the samples, and MIC value (D) of samples. OCTWP, Overseas Chinese Town Wetland Park; FRFR, Futian Red Forest Reserve.

FIG 5

Plasmid pColM within the five V. parahaemolyticus isolates (SH10-3, SH13-1, SH14-2, SH15-2, SH16-2) aligned to reference P. mirabilis plasmid pM510 (KJ190020.1). Gene locations of the hypothetical proteins and PMQR gene qnrD in these plasmids are indicated in the outer ring.