Distribution,Phylogeny and Evolution of Clinical and Environmental Vibrio vulnificus Antibiotic-Resistant Genes

Abstract

Vibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the evolution mechanism of antibiotic-resistant genes (ARGs) is still unclear. Twenty-two high-quality complete genomes of V. vulnificus were obtained and grouped into 16 clinical isolates and 6 environmental isolates. Genomic annotations found 23 ARG orthologous genes, among which 14 ARGs were shared by V. vulnificus and other Vibrio members. Furthermore, those ARGs were located in their chromosomes, rather than in the plasmids. Phylogenomic reconstruction based on single-copy orthologous protein sequences and ARG protein sequences revealed that clinical and environmental V. vulnificus isolates were in a scattered distribution. The calculation of non-synonymous and synonymous substitutions indicated that most of ARGs evolved under purifying selection with the Ka/Ks ratios lower than one, while h-ns, rsmA, and soxR in several clinical isolates evolved under the positive selection with Ka/Ks ratios >1. Our result indicated that V. vulnificus antibiotic-resistant armory was not only confined to clinical isolates, but to environmental ones as well and clinical isolates inclined to accumulate beneficial non-synonymous substitutions that could be retained to improve competitiveness.

Keywords

antibiotic-resistant genes clinical and environmental isolates comparative genomics phylogeny

Introduction

Vibrio vulnificus is an opportunistic pathogen for severe human infection,¹ and is one of the leading causes of non-Cholera, Vibrio-associated deaths globally.² Apart from clinical isolates, V. vulnificus inhabits a range of different environmental sources, including estuarine water, sediment and seafood produce.³ Moreover. V. vulnificus isolates have been classified into 3 biotypes based on biochemical traits.³ Biotype 1 as a human pathogen is the most common worldwide⁴ and biotype 2 is regarded as an eel pathogen,⁵ while biotype 3 is recorded as sharing biochemical properties of biotype 1 and 2.⁶ The advances of sequencing and bioinformatic technology facilitate researchers to obtain V. vulnificus genomes. Recent studies elucidate genetic and evolutionary mechanisms of infections and pathogenesis of V. vulnificus at the genomic level.^2,7,8

Antibiotic resistance in the V. vulnificus is a challenge that is associated with high morbidity and mortality.⁹ Particularly, the presence of antibiotic-resistant genes (ARGs) in environmental isolates can be a huge risk to the public health.¹⁰ However, the evolution mechanism of ARGs is still unclear. Moreover, the complete genomes can provide a more detailed gene information than the draft genomes.¹¹ In this study, the complete V. vulnificus genomes were obtained to demonstrate their ARGs distribution, phylogeny and evolutionary driving force, which could broaden our understandings of antibiotic-resistance in the V. vulnificus.

Materials and Methods

Collection of V. vulnificus and its relatives genomes

The complete genomes of V. vulnificus were obtained from the NCBI GenBank database.¹² In addition, other Vibrio type strain genomes were acquired from the gcType database¹³ and their accession numbers in the NCBI GenBank database were listed in Table 1. Moreover, Escherichia coli ATCC 11775^T was used as an outgroup in the phylogenomic analysis. Detailed information for the complete genomes in this study was shown in Table 1.

Table 1.

Genomic attributes of Vibrio genomes in this study.

Isolate	Accession number	Genetic materials	Genomic size (bp)	G + C content (%)	Completeness (%)	Contamination (%)
V. vulnificus YJ016	GCA_000009745.1	Two chromosomes, One plasmid	5260,086	46.7	100.0	0.1
V. vulnificus CMCP6	GCA_000039765.1	Two chromosomes	5126,696	46.7	100.0	0.2
V. vulnificus MO6-24/O	GCA_000186585.1	Two chromosomes	5007,768	46.9	100.0	0
V. vulnificus 93U204	GCA_000746665.1	Two chromosomes, One plasmid	5127,345	46.7	100.0	0.4
V. vulnificus FORC_009	GCA_001433435.1	Two chromosomes	5060,705	46.7	100.0	0.5
V. vulnificus FDAARGOS_119	GCA_001558515.2	Two chromosomes	4978,797	46.9	100.0	0.1
V. vulnificus FORC_016	GCA_001653775.1	Two chromosomes	5072,369	46.7	100.0	0.5
V. vulnificus FORC_017	GCA_001675245.1	Two chromosomes, One plasmid	5229,231	46.6	100.0	0.3
V. vulnificus FORC_036	GCA_002117205.1	Two chromosomes, One plasmid	6067,960	45.5	100.0	5.1
V. vulnificus FORC_037	GCA_002204915.1	Two chromosomes, One plasmid	5117,890	46.8	100.0	0.2
V. vulnificus CECT 4999	GCA_002215135.1	Two chromosomes, One plasmid	5163,135	46.5	100.0	0.1
V. vulnificus ATCC 27562^T	GCA_002224265.1	Two chromosomes	5007,160	46.7	100.0	0.3
V. vulnificus VV2014DJH	GCA_002850455.1	Two chromosomes	5074,562	46.8	99.7	0
V. vulnificus FORC_054	GCA_002863725.1	Two chromosomes, One plasmid	5120,766	46.7	100.0	0.4
V. vulnificus Env1	GCA_003047125.1	Two chromosomes	4954,048	46.7	99.9	0.6
V. vulnificus FORC_053	GCA_003522555.1	Three chromosomes	6019,009	45.4	100.0	5.4
V. vulnificus FORC_077	GCA_004319645.1	Two chromosomes	5018,260	46.9	100.0	0.3
V. vulnificus FDAARGOS_663	GCA_008693685.1	Two chromosomes	4974,815	46.7	100.0	0.3
V. vulnificus 2142-77	GCA_009665475.1	Two chromosomes	5079,985	46.8	99.9	0
V. vulnificus 2015AW-0208	GCA_009763305.1	Two chromosomes	5125,419	46.5	97.7	0.7
V. vulnificus 06-2410	GCA_009764095.1	Two chromosomes	4996,741	46.8	100.0	0.4
V. vulnificus 07-2444	GCA_009764115.1	Two chromosomes	5226,423	46.5	100.0	0.3
V. vulnificus Vv180806	GCA_014107515.1	Two chromosomes, One plasmid	5356,494	46.6	100.0	0.1
V. vulnificus 2497-87	GCA_014211935.1	Two chromosomes	5032,819	46.8	100.0	0.1
V. alfacsensis CAIM 1831^T	GCA_003544875.1	Two chromosomes, One plasmid	4910,231	44.2	99.4	0
V. alginolyticus ATCC 17749^T	GCA_000354175.2	Two chromosomes	5146,637	44.7	99.5	0.2
V. aphrogenes CA-1004T	GCA_002157735.2	Two chromosomes	3375,144	42.1	95.6	0.9
V. azureus LC2-005^T	GCA_002849855.1	Two chromosomes, Two plasmids	4833,901	42.3	99.0	0.2
V. campbellii ATCC 25920^T	GCA_002163755.1	Two chromosomes, One plasmid	5178,103	45.1	99.9	0.4
V. casei DSM 22364^T	GCA_002218025.2	Two chromosomes, Three plasmid	4140,771	40.7	96.3	1.0
V. cholerae CECT 514^T	GCA_013155105.1	Two chromosomes	4100,705	47.2	55.3	0.9
V. cidicii 2756-81^T	GCA_009763805.1	Two chromosomes	4750,222	47.9	97.2	0
V. fluvialis ATCC 33809^T	GCA_001558415.2	Two chromosomes	4827,733	49.9	99.9	0.9
V. hyugaensis 090810a^T	GCA_002906655.1	Two chromosomes	5612,082	45.0	100.0	0.3
V. natriegens ATCC 14048^T	GCA_001456255.1	Two chromosomes	5175,153	45.1	100.0	2.8
V. panuliri JCM 19500^T	GCA_009938205.1	Two chromosomes, One plasmid	4855,939	45.2	99.7	1.5
V. parahaemolyticus ATCC 17802^T	GCA_001558495.2	Two chromosomes	5152,461	45.3	100.0	0.1
V. ponticus DSM 16217^T	GCA_009938225.1	Two chromosomes, One plasmid	4796,932	44.8	98.3	2.0
V. rumoiensis FERM P-14531^T	GCA_002218045.2	Two chromosomes, Two plasmids	4207,152	42.3	95.6	0.5
V. tritonius JCM 16456^T	GCA_001547935.1	Two chromosomes	5221,926	43.9	98.1	3.2
V. tubiashii ATCC 19109	GCA_000772105.1	Two chromosomes, Four plasmids	5540,337	45.0	100.0	0.8
Escherichia coli ATCC 11775^T	GCA_003697165.1	One chromosome, One plasmid	5034,833	50.6	99.9	0.4

Genomic quality estimation and annotations

The quality of obtained genomes was estimated by CheckM v1.10.3 with the typical workflow, and the genome with the completeness of >90% and contamination of <5%. were regarded as a high-quality genome as recommended by Bowers et al¹⁴ Open reading frames (ORFs), rRNA and tRNA genes were annotated by using Prokka v.1.14.6¹⁵ with the command “–gram neg –kingdom Bacteria –gcode 11.” Non-redundant 16S rRNA genes were generated by CD-HIT program v4.8.1¹⁶ with the sequence identity of 99%. The classification of biotypes was analyzed based on the 16S rRNA gene phylogeny as described by Hoihuan et al⁴.

The ARGs were annotated against the Comprehensive Antibiotic Resistance Database (CARD) by using BLASTP with an e-value ⩽ 10,⁵ with an identity and query coverage thresholds of 50% and 50%.^17,18 In addition, those potential ARGs were double-checked by using the Resistance Gene Identifier (RGI) v.5.2.1¹⁷ with the command. The potential of horizontal gene transfer was carried out by using BLASTP against the NCBI RefSeq select proteins database based on the best hit taxon.

Comparative genomic analysis and phylogenomic reconstruction

Based on the annotation, protein sequences translated from ORFs were compared by using OrthoFinder v.2.5.4¹⁹ with the default setting. Single-copy were chosen into the following phylogenomic reconstruction as described previously.²⁰ In brief, each were aligned by MAFFT v.7.490²¹ with the parameter “–auto.” Aligned protein sequences were refined by using trimAL v.10.2rev59²² with the parameter “-automated1” to remove spurious sequences or poorly aligned regions and then concatenated. The maximum-likelihood phylogenomic analysis were carried out by using IQ-TREE v.1.6.12²³ with the parameter “-st AA -m LG+F+R10 -bb 1000,” and the best amino acid substitution model was also inferred by the same software with the parameter “-st AA -m MFP.”

Evolutionary analysis of ARGs

Protein sequences of each group of ARGs were aligned as described in the phylogenomic analysis. And the best amino acid substitution model for those sequences were also estimated by using IQ-TREE v.1.6.12.²³

The aligned codon sequences were generated based on converting a multiple sequence alignment of proteins and the corresponding DNA by using PAL2NAL v.14²⁴ And then, the resulting codon alignments were subjected to the calculation of synonymous and non-synonymous substitution (Ka/Ks) rates by KaKs_Calculator package v.1.2²⁵ through model selection and model averaging. Furthermore, the codon usages of those ARGs were summarized by using the sequence manipulation suite.²⁶

Statistics and visualization

The regression analysis were performed by using the formulation lm implanted in R version.²⁷ The phylogenetic trees were visualized by using MEGA7 software²⁸ and edited by PowerPoint 2019 software (Microsoft Cooperation, Redmond, WA, USA). Other figures were generated by and PowerPoint 2019 software (Microsoft Cooperation, Redmond, WA, USA).

Results and Discussion

General genomic features and phylogenomic relationship of V. vulnificus

A collection of twenty-four V. vulnificus isolates and other 18 Vibrio type strains genomes were obtained in this study (Table 1). Genomic quality estimations indicated that all genomes were high-quality with the completeness of >90.0% and contamination of <5.0% (Table 1), except for V. vulnificus FORC_036 (completeness of 100.0% and contamination of 5.1%), V. vulnificus FORC_053 (completeness of 100.0% and contamination of 5.4%) and V. cholerae CECT 514^T (completeness of 55.3% and contamination of 0.8%). Based on isolation sources, twenty-two V. vulnificus isolates could be divided into 2 categories including clinical isolates(n = 16) isolated from hospital and patients,^1,29-32 and environmental isolates (n = 6) cultivated from estuarine, Anguilla anguilla, Konosirus punctatus, Mya arenaria, Oreochromis sp. etc.³³ The neighbor-joining phylogenetic analysis based on 16S rRNA gene sequences revealed that those strains were classified into 2 clades including biotype 1 clade (strains 2142-77, 93U204, CMCP6, FDAARGOS_119, FORC_009, FORC_016, FORC_017, FORC_077, MO6-24/O, Vv180806, VV2014DJH, and YJ016) and biotype 2 clade (strains 06-2410, 07-2444, 2015AW-0208, 2497-87, ATCC 27562, CECT 4999, Env1, FDAARGOS_663, FORC_037, and FORC_054), as shown in Supplemental Figure S1.

Genomic sizes of 22 V. vulnificus isolates varied from 4.95 to 5.36 Mbp, while their DNA G + C contents were in a narrow range with 46.5% to 46.9% (Table 1). Commonly, their genomes constituted of 2 chromosomes, while several isolates genomes contained one plasmid as an accessory genetic material that was both present in the clinical (n = 3) and environmental (n = 4) isolates. Genomic annotation revealed those V. vulnificus isolates encoded 4447 to 5739 genes in their genomes. Their gene counts were mostly positively correlated with their genomic size (r² = 0.96, P = 5.1e⁻¹⁵) except for the isolate 2015AW-0208, which harbored the most genes with the genomic size of 5.12 Mbp (Figure 1).

Figure 1.

Genomic size and gene counts of Vibrio vulnificus. Red and blue indicated clinical and environmental isolates, respectively. Circle and triangle represented absence and presence of the plasmid, respectively.

Clinical and environmental V. vulnificus isolates were in a scattered distribution as indicated by the maximum-likelihood phylogenomic tree, and they were also clustered into 2 clades classified as biotype 1 and 2 clade (Figure 2), which was identical with the phylogenetic reconstruction based on 16S rRNA gene sequences. Moreover, the absence/presence of plasmid(s) did not affect their phylogenetic relationship either (Figure 2). Our phylogenomic reconstruction is similar with those of others about V. vulnificus.^2,8,34 The recent phylogenomic reconstructions of V. vulnificus indicated that its isolates were divided into 4 or 5 clades, among which nearly 90% of isolates were clustered into 2 clades.^2,34 The biotype 1 clade appeared to contain a significantly higher proportion of clinical isolates, while biotype 2 clade contained both of clinical and environmental isolates, that was also reported by López-Pérez et al⁸ Despite the genomic divergence among clusters, a distinct pattern linking strain phylogeny, source of isolation, and virulent capabilities was not identified. This mixed distribution of clinical and environmental V. vulnificus isolates suggested that the genomic difference between 2 groups was subtle, which was also detected in other pathogens, such as Escherichia coli,³⁵ Legionella pneumophila,³⁶ Pseudomonas aeruginosa,³⁷ and Pseudomonas putida.³⁸ Moreover, this distribution indicated V. vulnificus antibiotic-resistant armory was not only confined to clinical isolates, but to environmental ones as well.

Figure 2.

The maximum-likelihood phylogenetic tree based on single-copy orthologous protein sequences. Red and blue indicate clinical and environmental isolates, respectively. Escherichia coli ATCC 11775^T was used as an outgroup.

Distribution of ARGs in the V. vulnificus

Twenty-three ARG orthologous proteins classified into 5 resistance mechanisms including antibiotic efflux, antibiotic inactivation, antibiotic target alteration, antibiotic target protection and antibiotic target replacement, were annotated in the genomes of V. vulnificus (Table 2). Besides, 2 ARGs annotated as ATP-dependent lipid A-core flippase MsbA and tetracycline efflux Na⁺/H⁺ antiporter family transporter Tet35 were compared into 2 different orthologous genes. Those ARGs were located in the V. vulnificus chromosomes, rather than in the plasmid, which was not common in other pathogens.^39,40 Furthermore, core and accessory ARGs showed highest sequence identities with V. vulnificus or other Vibrio species (Supplemental Table S1), indicating that the low horizontal gene transfer frequencies of those ARGs.⁴¹

Table 2.

Detailed information of antibiotic-resistant genes annotated in the V. vulnificus genomes.

Orthologous genes	Annotations	CARD accession	Resistance mechanism	Drug class	Reference
OG0000070	MCR-9.1	3004684	Antibiotic target alteration	Peptide antibiotic	Carroll et al⁴²
OG0000118 and OG0000942	MsbA	3003950	Antibiotic efflux	Nitroimidazole antibiotic	Singh et al⁴³
OG0000191	CatB9	3002681	Antibiotic inactivation	Phenicol antibiotic	Heidelberg et al⁴⁴
OG0000210	PmrE	3003577	Antibiotic target alteration	Peptide antibiotic	Lee et al⁴⁵
OG0000223	H-NS	3000676	Antibiotic efflux	Tetracycline antibiotic, penam, macrolide antibiotic, fluoroquinolone antibiotic, cephamycin, cephalosporin	Nishino and Yamaguchi⁴⁶
OG0000702	RsmA	3005069	Antibiotic efflux	Phenicol antibiotic, diaminopyrimidine antibiotic, fluoroquinolone antibiotic	Mulcahy et al⁴⁷
OG0001054	YajC	3005040	Antibiotic efflux	Tetracycline antibiotic, penam, phenicol antibiotic, rifamycin antibiotic, cephalosporin, glycylcycline, fluoroquinolone antibiotic, triclosan	Rundell et al⁴⁸
OG0001146	DfrA3	3003105	Antibiotic target replacement	Diaminopyrimidine antibiotic	Brolund et al⁴⁹
OG0001216	SoxR	3004107	Antibiotic efflux, Antibiotic target alteration	Tetracycline antibiotic, fluoroquinolone antibiotic, glycylcycline, cephalosporin, phenicol antibiotic, rifamycin antibiotic, penam, disinfecting agents and intercalating dyes, triclosan, acridine dye	Sakhtah et al⁵⁰
OG0001389	CRP	3000518	Antibiotic efflux	Macrolide antibiotic, penam, fluoroquinolone antibiotic	Nishino et al⁵¹
OG0001739 and OG0014842	Tet35	3000481	Antibiotic efflux	Tetracycline antibiotic	Teo et al⁵²
OG0002135	ArlR	3000838	Antibiotic efflux	Disinfecting agents and intercalating dyes, fluoroquinolone antibiotic, acridine dye	Fournier et al⁵³
OG0002643	QnrVC1	3002799	Antibiotic target protection	Fluoroquinolone antibiotic	Fonseca et al⁵⁴
OG0003164	MacB	3000535	Antibiotic efflux	Macrolide antibiotic	Xu et al⁵⁵
OG0003616	VarG	3004289	Antibiotic inactivation	Carbapenem	Lin et al⁵⁶
OG0006136	MexK	3003693	Antibiotic efflux	Macrolide antibiotic, tetracycline antibiotic, triclosan	Chuanchuen et al⁵⁷
OG0006781	FosC2	3002874	Antibiotic inactivation	Fosfomycin	Wachino et al⁵⁸
OG0014873	PmpM	3004077	Antibiotic efflux	Aminoglycoside antibiotic, fluoroquinolone antibiotic, benzalkonium chloride	He et al⁵⁹
OG0015064	TetT	3000193	Antibiotic target protection	Tetracycline antibiotic	Clermont et al⁶⁰
OG0015076	RpoB2	3000501	Antibiotic target alteration, antibiotic target replacement	Rifamycin antibiotic	Ishikawa et al⁶¹
OG0015104	TetWNW	3004442	Antibiotic target protection	Tetracycline antibiotic	Leclercq et al⁶²

Among those ARGs, 61% (14/23) of them shared by V. vulnificus and other Vibrio type strains were chloramphenicol acetyltransferase CatB9, translational regulators CRP and RsmA, dihydrofolate reductase Dfr-A3, fosfomycin resistance phosphotransferase FosC2, histone-like nucleoid structuring protein H-NS, ABC-type macrolide antibiotic exporter MacB, mobile colistin resistance phosphoethanolamine transferase MCR-9, ATP-dependent lipid A-core flippase MsbA, polymyxin resistance phosphoethanolamine transferase PmrE, quinolone resistance protein QnrVC1, redox-sensitive transcriptional activator SoxR, tetracycline efflux Na⁺/H⁺ antiporter family transporter Tet35 and AcrAB-TolC multidrug efflux pump YajC (Figure 3). Furthermore, the metallo-beta-lactamase VarG were exclusively in all of V. vulnificus isolates, other than the isolate VV2014DJH. Other exclusive genes encoding the response regulator ArlR, multidrug efflux RND transporter permease subunit MexK, ATP-dependent lipid A-core flippase MsbA, H⁺-coupled multidrug efflux pump PmpM, rifampin-resistant beta-subunit of RNA polymerase RpoB2, tetracycline efflux Na⁺/H⁺ antiporter family transporter Tet35, tetracycline-resistant ribosomal protection proteins TetT and TetWNW were mostly in the isoalte 2015AW-0208, which showed genomic differences compared with other V. vulnificus isolates.

Figure 3.

Distribution of antibiotic-resistant genes in the V. vulnificus genomes.

Antibiotic resistance determinations revealed that V. vulnificus could resist various antibiotics including fluoroquinolones, β-lactam combination agent, lipopeptide, macrolide, nitrofurans, penicillin and phenicols,^63-66 which were consistent with those ARGs found in their genomes (Table 2). Furthermore, the genomes of environmental isolates 93U204, ATCC 27562^T, CECT 4999, Env1, FORC_037, and FORC_054 encoded 13 or 14 ARG orthologous genes, which were similar with the ARGs profile of clinical isolates (Figure 3). Therefore, environmental V. vulnificus isolates may serve as reservoirs for transmission of their antibiotic resistance.

Evolution of ARGs in the V. vulnificus

Except for FosC2 only annotated in the V. vulnificus VV2014DJH and V. campbellii ATCC 25920^T, other 13 shared ARG orthologous proteins and VarG were processed into phylogenetic analysis. V. vulnificus isolates were clustered into an independent clade, which was separated from other Vibrio strains, of phylogenetic trees based on most ARG orthologous proteins including CatB9, CRP, Dfr-A3, H-NS, MacB, MCR-9, MsbA, QnrVC1, SoxR, Tet35, and YajC (Supplemental Figure S1). However, V. vulnificus isolates were in a scattered distribution in the phylogenetic trees of PmrE and RsmA, that were classified into antibiotic target alteration or antibiotic efflux resistance mechanisms. Clinical and environmental isolates in those scattered distribution phylogenetic trees were still mixed (Supplemental Figure S1), demonstrating that environmental ones had a similar evolutionary history with clinical ones which were also observed in other pathogens.^67,68

The calculation of non-synonymous and synonymous substitutions indicated that most of ARGs evolved under purifying selection with the Ka/Ks ratios lower than one⁶⁹ (Figure 4). While several pairwise gene sequences of ARGs had the Ka/Ks ratios higher than 1 indicating that those genes evolved under positive selection⁷⁰ (Figure 4), especially for those antibiotic efflux ARG proteins H-NS, RsmA, and SoxR among which the group of Ka/Ks > 1 accounted for 6.5% to 10.0%. And those high Ka/Ks ratios were mostly confined to isolates 2015AW-0208, FDAARGOS_119, FDAARGOS_663, VV2014DJH, and YJ016, which were all isolated from clinical sources. Compared with environmental sources, clinical settings, where most antibiotics are prescribed, are hypothesized to serve as a major hotspot,⁷¹ that forced several beneficial non-synonymous substitutions could be retained to improve competitiveness.

Figure 4.

Ka/Ks ratios of each orthologous ARG in the V. vulnificus.

Conclusions

With regard to uptake of antibiotic resistance factors, marine environments with highly variable ecological niches provide an unrivaled gene pool with a diversity that considerably exceeds that of the human and marine animal microbiota. Indeed, the most remarkable feature of marine microbiome is its enormous diversity, providing numerous genes that potentially could be acquired and used by pathogens to counteract the effect of antibiotics.⁷² Moreover, metals and antibiotic pollutions co-selecting for antibiotic-resistant strains via cross-resistance or co-resistance should also be taken into consideration seriously to retard the rapid evolutionary expansion and spread of antibiotic resistance factors.⁷³ Clinical and environmental V. vulnificus isolates were in a scatter distribution based on the genomic size and constitutes as well as the phylogenomic relationship. Genomic annotation and comparative genomic analysis also indicated that this mixed distribution of ARGs in clinical and environmental isolates. Unexpectedly, those ARGs were located in their chromosomes, rather than in the plasmids of them, suggesting that those genes were conserved in the V. vulnificus. The calculation of non-synonymous and synonymous substitutions indicated that most of ARGs evolved under purifying selection with the Ka/Ks ratios lower than one, while h-ns, rsmA and soxR in several clinical isolates evolved under the positive selection with Ka/Ks ratios >1. Therefore, V. vulnificus antibiotic-resistant armory was not only confined to clinical isolates, but to environmental ones as well and clinical isolates inclined to accumulate beneficial non-synonymous substitutions that could be retained to improve their competitiveness.

Supplemental Material

sj-docx-1-evb-10.1177_11769343221134400 – Supplemental material for Distribution, Phylogeny and Evolution of Clinical and Environmental Vibrio vulnificus Antibiotic-Resistant Genes

Supplemental material, sj-docx-1-evb-10.1177_11769343221134400 for Distribution, Phylogeny and Evolution of Clinical and Environmental Vibrio vulnificus Antibiotic-Resistant Genes by Nan Geng, Guojin Sun, Wen-Jia Liu, Bin-Cheng Gao, Cong Sun, Cundong Xu, Ertian Hua and Lin Xu in Evolutionary Bioinformatics

Footnotes

Funding:

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the Natural Science Foundation of Zhejiang Province (LQ19C010006), the Key Technology Research and Development Program of Zhejiang (2021C03019, 2021C03196), the Zhejiang Basic Public Welfare Research Project (LGF19E090001), and the National Science and Technology Fundamental Resources Investigation Program of China (2019FY100700, 2021FY100900).

Declaration of Conflicting Interests:

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ORCID iD

Lin Xu

Supplemental Material

Supplemental material for this article is available online.

References

Chen

C-Y

K-M

Chang

Y-C

, et al Comparative genome analysis of Vibrio vulnificus, a marine pathogen. Genome Res. 2003;13:2577-2587.

López-Pérez

Jayakumar

Grant

Zaragoza-Solas

Cabello-Yeves

Almagro-Moreno

Ecological diversification reveals routes of pathogen emergence in endemic Vibrio vulnificus populations. Proc Natl Acad Sci U S A. 2021;118:e2103470118.

Baker-Austin

Oliver

JD.

Vibrio vulnificus: new insights into a deadly opportunistic pathogen. Environ Microbiol. 2018;20:423-430.

Hoihuan

Soonson

Bunlipatanon

, et al Molecular genotyping and phenotyping of Vibrio vulnificus isolated from diseased, brown-marbled grouper (Epinephelus fuscoguttatus) in Thailand with preliminary vaccine efficacy analysis. Aquaculture. 2021;545:737188.

Tison

Nishibuchi

Greenwood

Seidler

RJ.

Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl Environ Microbiol. 1982;44:640-646.

Bisharat

Agmon

Finkelstein

, et al Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Lancet. 1999;354:1421-1424.

Liang

KYH

Orata

Islam

, et al A Vibrio cholerae core genome multilocus sequence typing scheme to facilitate the epidemiological study of cholera. J Bacteriol. 2020;202:e00086-20.

López-Pérez

Jayakumar

Haro-Moreno

, et al Evolutionary model of cluster divergence of the emergent marine pathogen Vibrio vulnificus: from genotype to ecotype. mBio. 2019;10:e02852-18.

Frieri

Kumar

Boutin

Antibiotic resistance. J Infect Public Health. 2017;10:369-378.

10.

Zhu

Y-G

Johnson

, et al Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc Natl Acad Sci U S A. 2013;110:3435-3440.

11.

Land

Hauser

Jun

, et al Insights from 20 years of bacterial genome sequencing. Funct Integr Genomics. 2015;15:141-161.

12.

Benson

Cavanaugh

Clark

, et al GenBank. Nucleic Acids Res. 2012;41:D36-D42.

13.

Shi

Sun

Fan

, et al gcType: a high-quality type strain genome database for microbial phylogenetic and functional research. Nucleic Acids Res. 2021;49: D694-D705.

14.

Bowers

Kyrpides

Stepanauskas

, et al Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea. Nat Biotechnol. 2017;35:725-731.

15.

Seemann

Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068-2069.

16.

Godzik

Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006;22:1658-1659.

17.

Alcock

Raphenya

Lau

TTY

, et al CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res. 2020;48:D517-D525.

18.

Altschul

Gish

Miller

Myers

Lipman

DJ.

Basic local alignment search tool. J Mol Biol. 1990;215:403-410.

19.

Emms

Kelly

OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20:238.

20.

Sun

Fang

Oren

XW.

Genomic-based taxonomic classification of the family Erythrobacteraceae. Int J Syst Evol Microbiol. 2020;70:4470-4495.

21.

Katoh

Standley

DM.

MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772-780.

22.

Capella-Gutiérrez

Silla-Martínez

Gabaldón

trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972-1973.

23.

Nguyen

L-T

Schmidt

von Haeseler

Minh

BQ.

IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32:268-274.

24.

Suyama

Torrents

Bork

PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 2006;34:W609-W612.

25.

Zhang

Zhao

X-Q

Wang

Wong

G-K

KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genom Proteom Bioinform. 2006;4:259-263.

26.

Stothard

The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques. 2000;28:1102, 1104.

27.

Ihaka

Gentleman

R: a language for data analysis and graphics. J Comput Graph Stat. 1996;5:299-314.

28.

Kumar

Stecher

Tamura

MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870-1874.

29.

Kim

Jeong

, et al Integrative genome-scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery. Mol Syst Biol. 2011;7:460.

30.

Kim

Lee

Kim

, et al Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun. 2003;71:5461-5471.

31.

Park

Cho

Y-J

Chun

, et al Complete genome sequence of Vibrio vulnificus MO6-24/O. J Bacteriol. 2011;193:2062-2063.

32.

Chung

Kim

Y-T

Kim

, et al Complete genome sequence of Vibrio vulnificus FORC_017 isolated from a patient with a hemorrhagic rash after consuming raw dotted gizzard shad. Gut Pathog. 2016;8:22.

33.

Chen

C-Y

Kuo

C-H.

Complete genome sequence of Vibrio vulnificus 93U204, a bacterium isolated from diseased tilapia in Taiwan. Genome Announc. 2014;2:e01005-e01014.

34.

Roig

González-Candelas

Sanjuán

, et al Phylogeny of Vibrio vulnificus from the analysis of the core-genome: implications for intra-species taxonomy. Front Microbiol. 2017;8:2613.

35.

Han

Liu

Han

, et al Genetic and phylogenetic characterization of Shiga toxin-producing Escherichia coli and enteropathogenic E. coli from livestock in Jiangsu by using whole-genome sequencing. J Appl Microbiol. 2022;132: 3925-3936.

36.

Coscollá

González-Candelas

Comparison of clinical and environmental samples of Legionella pneumophila at the nucleotide sequence level. Infect Genet Evol. 2009;9:882-888.

37.

Nair

Joseph

Krishna

, et al

A comparative study of coastal and clinical isolates of

Pseudomonas aeruginosa. Braz J Microbiol. 2015;46:725-734.

38.

Yonezuka

Shimodaira

Tabata

, et al Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group. J Gen Appl Microbiol. 2017;63:1-10.

39.

Bennett

PM.

Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. 2008;153 Suppl 1:S347-S357.

40.

San Millan

. Evolution of plasmid-mediated antibiotic resistance in the clinical context. Trends Microbiol. 2018;26:978-985.

41.

Bonham

Wolfe

Dutton

RJ.

Extensive horizontal gene transfer in cheese-associated bacteria. eLife. 2017;6:e22144.

42.

Carroll

Gaballa

Guldimann

Sullivan

Henderson

Wiedmann

Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype typhimurium isolate. mBio. 2019;10:e00853-19.

43.

Singh

Velamakanni

Deery

Howard

Wei

van Veen

HW.

ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled. Nat Commun. 2016;7:12387.

44.

Heidelberg

Eisen

Nelson

, et al

DNA sequence of both chromosomes of the cholera pathogen

Vibrio cholerae. Nature. 2000;406:477-483.

45.

Lee

Hsu

F-F

Turk

Groisman

EA.

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica. J Bacteriol. 2004;186:4124-4133.

46.

Nishino

Yamaguchi

Role of histone-like protein H-NS in multidrug resistance of

Escherichia coli. J Bacteriol. 2004;186:1423-1429.

47.

Mulcahy

O’Callaghan

O’Grady

Adams

O’Gara

. The posttranscriptional regulator RsmA plays a role in the interaction between Pseudomonas aeruginosa and human airway epithelial cells by positively regulating the type III secretion system. Infect Immun. 2006;74:3012-3015.

48.

Rundell

Commodore

Goodman

Kazmierczak

BI.

A screen for antibiotic resistance determinants reveals a fitness cost of the flagellum in Pseudomonas aeruginosa. J Bacteriol. 2020;202:e00682-19.

49.

Brolund

Sundqvist

Kahlmeter

Grape

Molecular characterisation of trimethoprim resistance in Escherichia coli and Klebsiella pneumoniae during a two year intervention on trimethoprim use. PLoS One. 2010;5:e9233.

50.

Sakhtah

Koyama

Zhang

, et al The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development. Proc Natl Acad Sci U S A. 2016;113: E3538-E3547.

51.

Nishino

Senda

Yamaguchi

CRP regulator modulates multidrug resistance of Escherichia coli by repressing the mdtEF multidrug efflux genes. J Antibiot. 2008;61:120-127.

52.

Teo

Tan

Poh

CL.

Genetic determinants of tetracycline resistance in Vibrio harveyi. Antimicrob Agents Chemother. 2002;46:1038-1045.

53.

Fournier

Aras

Hooper

DC.

Expression of the multidrug resistance transporter NorA from Staphylococcus aureus is modified by a two-component regulatory system. J Bacteriol. 2000;182:664-671.

54.

Fonseca

Dos Santos Freitas

Vieira

Vicente

AC.

New qnr gene cassettes associated with superintegron repeats in Vibrio cholerae O1. Emerg Infect Dis. 2008;14:1129-1131.

55.

Sim

S-H

Nam

, et al Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter. Biochemistry. 2009;48:5218-5225.

56.

Lin

HTV

Massam-Wu

Lin

C-P

, et al The Vibrio cholerae var regulon encodes a metallo-β-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor. PLoS One. 2017;12:e0184255.

57.

Chuanchuen

Narasaki

Schweizer

HP.

The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan. J Bacteriol. 2002;184:5036-5044.

58.

Wachino

Yamane

Suzuki

Kimura

Arakawa

Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes. Antimicrob Agents Chemother. 2010;54:3061-3064.

59.

Kuroda

Mima

Morita

Mizushima

Tsuchiya

An H⁽⁺⁾-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from

Pseudomonas aeruginosa. J Bacteriol. 2004;186:262-265.

60.

Clermont

Chesneau

De Cespédès

Horaud

New tetracycline resistance determinants coding for ribosomal protection in streptococci and nucleotide sequence of tet(T) isolated from Streptococcus pyogenes A498. Antimicrob Agents Chemother. 1997;41:112-116.

61.

Ishikawa

Chiba

Kurita

Satoh

Contribution of rpoB2 RNA polymerase beta subunit gene to rifampin resistance in Nocardia species. Antimicrob Agents Chemother. 2006;50:1342-1346.

62.

Leclercq

Wang

Zhu

, et al Diversity of the tetracycline mobilome within a Chinese pig manure sample. Appl Environ Microbiol. 2016;82:6454-6462.

63.

Gxalo

Digban

Igere

Olapade

Okoh

Nwodo

UU.

Virulence and antibiotic resistance characteristics of Vibrio isolates from rustic environmental freshwaters. Front Cell Infect Microbiol. 2021;11:732001.

64.

M Kurdi Al-Dulaimi

Abd Mutalib

Abd Ghani

Mohd Zaini

Ariffin

. Multiple antibiotic resistance (MAR), plasmid profiles, and dna polymorphisms among Vibrio vulnificus isolates. Antibiotics. 2019;8:68.

65.

Heng

S-P

Letchumanan

Deng

C-Y

, et al Vibrio vulnificus: an environmental and clinical burden. Front Microbiol. 2017;8:997.

66.

Elmahdi

DaSilva

Parveen

Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: a review. Food Microbiol. 2016;57: 128-134.

67.

Yang

Higgins

Rehman

, et al Genomic diversity, virulence, and antimicrobial resistance of Klebsiella pneumoniae strains from cows and humans. Appl Environ Microbiol. 2019;85:e02654-18.

68.

Carvalho

Martínez-Murcia

Esteves

Correia

Saavedra

MJ.

Phylogenetic diversity, antibiotic resistance and virulence traits of Aeromonas spp. from untreated waters for human consumption. Int J Food Microbiol. 2012; 159:230-239.

69.

Hurst

LD.

The Ka/ks ratio: diagnosing the form of sequence evolution. Trends Genet. 2002;18:486-487.

70.

Vitti

Grossman

Sabeti

PC.

Detecting natural selection in genomic data. Annu Rev Genet. 2013;47:97-120.

71.

Kunhikannan

Thomas

Franks

Mahadevaiah

Kumar

Petrovski

Environmental hotspots for antibiotic resistance genes. Microbiol Open. 2021; 10:e1197.

72.

Larsson

DGJ

Flach

C-F.

Antibiotic resistance in the environment. Nat Rev Microbiol. 2022;20:257-269.

73.

Pal

Asiani

Arya

, et al Metal resistance and its association with antibiotic resistance. Adv Microb Physiol. 2017;70:261-313.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

1.50 MB