Research Article | Ecological and Evolutionary Science

Continuous Genomic Surveillance Monitored the In Vivo Evolutionary Trajectories of Vibrio parahaemolyticus and Identified a New Virulent Genotype

Songzhe Fu, Qian Yang, Qingyao Wang, Bo Pang, Ruiting Lan, Dawei Wei, Baocheng Qu, Ying Liu
Naseer Sangwan, Editor
DOI: 10.1128/mSystems.01254-20

ABSTRACT

Our ability to predict evolutionary trajectories of pathogens is one of the promising leverages to fight against the pandemic disease, yet few studies have addressed this question in situ, due to the difficulty in monitoring the milestone evolutionary events for a given pathogen and in understanding the evolutionary strategies. In this study, we monitored the real-time evolution of Vibrio parahaemolyticus in response to successive antibiotic treatment in three shrimp farms in North China from 2011 to 2018 by whole-genome sequencing. Results showed that the stepwise emergence of resistance was associated with the antibiotic usage. Genomic analysis of resistant isolates showed that the acquisition of the resistant mobile genetic elements flanked by an insertion sequence (ISVal1) closely mirrored the antibiotics used in shrimp farms since 2014. Next, we also identified 50 insertion sites of ISVal1 in the chromosome, which facilitated the formation of pathogenicity islands (PAIs) and fitness islands in the following years. Further, horizontal transfers of a virulent trh-nik-ure genomic island (GI) and two GIs improving the fitness have been observed in two farms since 2016. In this case study, we proposed that the insertion sequence triggered four major evolutionary events during the outbreaks of shrimp disease in three farms, including horizontal transfer of transposon (HTT) (stage 1), the formation of resistance islands (stage 2) and the PAIs (stage 3), and horizontal transfer of the PAIs (stage 4). This study presented the first in vivo evolutionary trajectories for a given bacterial pathogen, which helps us to understand the emergence mechanisms of new genotypes.

IMPORTANCE Most human infectious diseases originate from animals. Thus, how to reduce or prevent pandemic zoonoses before they emerge in people is becoming a critical issue. Continuous genomic surveillance of the evolutionary trajectories of potential human pathogens on farms is a promising strategy to realize early warning. Here, we conducted an 8-year surveillance of Vibrio parahaemolyticus in three shrimp farms. The results showed that the use of antibiotics and horizontal transfer of transposons (HTT) drove the evolution of V. parahaemolyticus, which could be divided into four stages: HTT, formation of resistance islands, formation of pathogenicity islands (PAIs), and horizontal transfer of PAIs. This study presented the first in vivo monitoring of evolutionary trajectories for a given bacterial pathogen, providing valuable information for the prevention of pandemic zoonoses.

INTRODUCTION

Most human infectious diseases, especially recently emerging pathogens, originate from animals (1), and ongoing transmission of diseases such as coronavirus disease 2019 (COVID-19) presents a significant global health crisis (2). The recent emergence of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and COVID-19 has shown the vulnerability of humans to novel zoonotic pathogens and highlights that pathogen transmission from animals to humans can cause devastating consequences (3).

Although prediction of the emergence of novel pandemic agents often seems unachievable, efforts to reduce or prevent pandemic zoonoses before they emerge in people have also begun (4). For instance, Daszak et al. developed a model dividing cross-species transmission into three stages, animal infection without transmission to humans (stage 1), localized human infection (spillover; stage 2), and widespread human-to-human transmission (pandemic; stage 3) (5). Recent recognition that most emerging infectious disease events have wildlife origins highlights the need for a deep understanding of the evolutionary drivers for pathogens in stages 1 and 2 and the type of contact between animals and people that enables disease transmission (6). Opportunities for close contact between humans and wild animals are relatively rare compared to contact with domestic animals (7). Therefore, our ability to monitor the emergence and spread of pandemic diseases from farms to humans is critical to mitigate the exacerbation of the infectious disease crisis worldwide.

Continuous surveillance is essential for recognition of the evolutionary events involved in the cross-species transmission, spillover, and the spread of diseases, which can be used to predict the future disease emergence risks (8). The emergence of a pandemic pathogen transmitted from animals to humans usually involves a series of evolutionary events (9). A genomics-based pathogen surveillance system enables the real-time surveillance of such evolution (10).

For instance, a large outbreak of infections was reported in Germany in 2011, which was caused by Shiga toxin-producing Escherichia coli serotype O104:H4 (11). Genomic analysis revealed that this exceptionally virulent serotype acquired virulence factors from enteroaggregative E. coli, conferring mixed enteroaggregative and Shiga toxin-producing abilities and resulting in the emergence of a new virulent serotype (12).

In vivo monitoring of such evolutionary events becomes a long-standing issue. For example, Vibrio parahaemolyticus is a Gram-negative waterborne pathogen which has a large chromosome (chromosome 1) and a small chromosome (chromosome 2) (13). Recently, V. parahaemolyticus has become the leading cause of seafood-borne gastroenteritis (14). A recent phylogenetic analysis of pandemic V. parahaemolyticus isolates (emerged in 1996), and prepandemic isolates (isolated in the 1980s) suggested that the prepandemic strains initially acquired a genomic island containing thermostable direct hemolysin gene (tdh) and a toxRS new region (15); subsequent genomic analysis revealed that acquisition of seven novel genomic islands converted them into pandemic O3:K6 isolates within a decade (16).

Thus, such evolutionary events should be carefully monitored because once a pathogen has spilled over from farm animals to humans, human-to-human transmission of zoonoses can occur rapidly. However, few studies have addressed this issue in situ from farms to humans due to the difficulty of real-time surveillance of a given pathogen’s evolution and its precise microevolution in the long term. The recent next-generation sequencing revolution provided an unprecedented opportunity to explore the emergence of pandemic pathogens in situ (17). Whole-genome sequencing (WGS) has yielded novel insights into pathogen evolution, allowing inferences regarding population dynamics and geographic spread at both local and global levels (18). Acquisition of resistance by a given strain through horizontal transfer of mobile elements can also be monitored using this methodology. For instance, Du et al. sampled isolates during a 3-year large-scale outbreak of furunculosis in an Atlantic salmon farm and reconstructed the precise timeline of resistance island acquisition events in response to antibiotic pressure (19).

However, very little is currently known about the time needed for a given pathogen to transit from stage 1 to stage 2; that is, no in situ data are available to monitor the time required for an infectious agent in animals to become contagious to humans. In particular, the details of the microevolution and emergence of a pathogen from farms to humans remain largely unknown, as no long-term surveillance data from farms are available.

Consumption of undercooked seafood is one of the most essential infection routes for pathogenic V. parahaemolyticus-related infection (13). A recent study suggested that V. parahaemolyticus can also cause acute hepatopancreatic necrosis disease (AHPND) in shrimp due to the acquisition of a 70-kb plasmid encoding the binary toxin PirABvp (20). The pirABvp gene was flanked by two identical insertion sequences named ISVal1 and form a mobile genetic element (MGE) called Tn6264 (21). Our previous studies suggested that ISVal1 can excise from the plasmid and insert into the chromosomes both in vivo and in vitro (22). AHPND was first identified in South China and Vietnam in 2010 (20) and then reported in East China in 2011 and North China in 2013, which provided an extraordinary opportunity to observe the in vivo evolution of V. parahaemolyticus during the spread of this zoonosis. To record the spread of AHPND in North China, we monitored the real-time evolution of V. parahaemolyticus in three nearby shrimp-farming regions from 2011 to 2018 and accidentally found that ISVal1 mediated the evolution of V. parahaemolyticus.

RESULTS

Progression of shrimp disease and the development of antibiotic resistance of V. parahaemolyticus in Tianjin.We conducted continuous pathogen monitoring in three nearby shrimp farms in Tianjin, Tangshan, and Huanghua cities, China, to observe the microevolution of V. parahaemolyticus in vivo (Fig. S1). In Tianjin, shrimps were cultured in a recirculating aquaculture system (RAS) which consisted of ponds and a wastewater treatment unit, whereas shrimps were cultivated in open ponds in Tangshan and Huanghua without wastewater treatment units. From 2011 to 2018, diseased shrimps were sampled from each outbreak-related pond annually to isolate the V. parahaemolyticus. The V. parahaemolyticus isolates were then subjected to multilocus sequence typing (MLST) to confirm the sequence type (ST) (23). The presence of virulence factors, including pirABvp, T3SS1, T3SS2, and tdh/trh, were also screened by PCR (24–26).

FIG S1

The location of sampled shrimp farms in Tianjin, Tangshan and Huanghua. The sampling sites were mapped by the ArcGIS Desktop 10.2 software. Download FIG S1, PDF file, 1.0 MB.
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From 2011 to 2012, no V. parahaemolyticus was isolated and no massive shrimp disease was recorded in three farming regions. In 2013, an occasional outbreak of shrimp disease was first identified in Tianjin; oxytetracycline was only occasionally used for treatment. Only one ST1743 strain and one ST150 strain were identified in the shrimps in 2013, of which ST150 strain VP17 was a pirABvp-positive strain (Table S1). This is the first record of a pirABvp-positive strain on this farm. Accordingly, except for the resistance to amoxicillin, no antibiotic resistance out of nine tested antibiotics was found in 2013 isolates (Table S2). In July 2014, the outbreak was identified in 30% of the ponds. Three V. parahaemolyticus strains were isolated and were divided into ST424, ST1743, and ST919, respectively (Fig. 1A). All of them were pirABvp-positive. As the outbreak spread to more ponds, oxytetracycline and florfenicol were then widely used to manage the outbreak beginning in 2014. Meanwhile, to eliminate the introduced V. parahaemolyticus, the seawater used for evaporation compensation was treated with ozone for 30 min before it entered the pond.

FIG 1
FIG 1

(A to C) The dynamics of V. parahaemolyticus sequence types (STs) from 2011 to 2018 obtained from three-shrimp farming regions in Tianjin (A), Tangshan (B), and Huanghua (C), China.

TABLE S1

Virulence and plasmid profiles of genomes used in this study. Download Table S1, XLSX file, 0.02 MB.
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TABLE S2

Antibiotic resistance profile of sequence strains in three shrimp farms. Download Table S2, XLSX file, 0.02 MB.
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However, the combined use of antibiotics did not stop the outbreaks, which affected 50% of the ponds in July 2015. Fifteen strains were isolated from the shrimp ponds, which were subtyped into ST1, ST1743, ST424, ST415, ST809, ST1749, ST122, and ST917a. Resistance to florfenicol was detected in ST1, ST122, and ST809 strains in 2015.

Due to the emergence of florfenicol-resistant strains in 2015, antibiotic treatment was shifted to sulfamethoxazole, enrofloxacin, and streptomycin. However, the situation still became worse in July 2016 as outbreaks spread to more than 80% of the ponds; 14 V. parahaemolyticus strains were identified in the shrimp, including 4 pirABvp-positive STs (ST1743, ST424, ST150, ST917a) and three pirABvp-negative STs (ST1, ST122, and ST1797). Resistance to amoxicillin, sulfamethoxazole, streptomycin, and florfenicol was identified in ST1797, ST1, and ST122 strains. In April 2017, a larger outbreak occurred and spread out to almost all ponds, resulting in the isolation of 39 V. parahaemolyticus strains that can be subtyped into ST1, ST122, ST809, ST1749, and ST424. Corresponding multiple-drug resistances were found in ST1, ST122, and ST809 strains. For the virulence genes, T3SS1 was found in all isolates, while T3SS2 was only presented in ST1 isolates which were trh-positive. Interestingly, trh was also amplified in one ST424 strain by PCR.

Due to the severe outbreaks in shrimp culture, all of the shrimp ponds were cleaned up and replaced with fresh rearing water in July 2018. Microbiological analysis of 60 shrimp samples from 4 ponds only found three ST1748 isolates.

Progression of shrimp disease in Tangshan and Huanghua.Just 1 year after AHPND was identified in Tianjin, suspected symptoms of AHPND were also identified in shrimp farms from Tangshan and Huanghua in 2014. Since then, V. parahaemolyticus has been continuously isolated from shrimp. Initially, the majority of them belonged to ST424, ST1743, and ST415 during 2014 to 2015 (Fig. 1B and C). Since 2015, several other genotypes have also been identified, including ST150, ST864, ST1800, ST1166, ST1073, and ST12. Interestingly, Tianjin, Tangshan, and Huanghua shared similar genotypes of V. parahaemolyticus (including ST424, ST1743, ST150, and ST415) from 2013 to 2015, probably because the close locations facilitate the exchange of V. parahaemolyticus. Apart from T3SS1, virulence genes were not found in the Tangshan and Huanghua data sets.

As the outbreak spread to more ponds since 2014, oxytetracycline and florfenicol were both used in the Tangshan and Huanghua ponds. Moreover, combined usage of sulfamethoxazole, streptomycin, and florfenicol was also applied to the ponds in 2016. However, all treatments failed to control the outbreaks. Farming activities ceased in Huanghua and Tangshan in 2016 and 2017, respectively.

Antibiotic resistance was conferred by mobile gene elements (MGEs) with IS.The above-described observation provides a clear example to show the emergence of resistance linked to the use of antibiotics. These observations raised the speculation that whether the stepwise acquisition of MGEs was mediated by ISVal1, which was previously found to be involved with the formation of resistance islands (22). Next, we conducted PCR to detect the presence of ISVal1 in the isolates collected from three farms, 26 of which were ISVal1-positive. To understand whether ISVal1 shaped the genomic plasticity as found previously (22), all ISVal1-positive isolates and the isolates from other representative STs in each year were selected for WGS.

Except for the intrinsic resistance to beta-lactam, no antibiotic resistance genes (ARGs) were found in 2013 and 2014 isolates. A genomic analysis of the isolates from 2015 showed that the emergence of resistance to florfenicol coincided with the acquisition of a plasmid harboring floR and ISVal1 in an ST1 isolate, VP120 (Fig. 2A). This plasmid was also found in 2013 strain VP17 but without ISVal1. In 2016, the plasmid in an ST1 strain further acquired an additional composite transposon flanked by ISVal1, which contains resistance genes sul1, aac(6′)-Ib-cr, and ARR-3. Accordingly, multiple-drug resistance emerged in an ST1 strain in 2016 (Table S2).

FIG 2
FIG 2

(A to C) Schematic diagrams showing the comparative genome analysis of ST1 (A), ST122 (B), and plasmid pLH80-1 (C) and the stepwise formation of resistant MGEs; the maximum-likelihood tree of V. parahaemolyticus is vertically on the top of rings; the antibiotic resistance genes of each isolates are shown under the tree.

In addition, a stepwise acquisition of ARGs was also found in ST122 isolates. In 2015, ST122 isolate VP157 only harbored four ARGs (blaCARB-17, blaCARB-20, sul2, and tet [59]). In 2016, one ST122 isolate acquired another resistance island (flanked by ISVal1) containing floR, sul2, aph(6)-Id, and aph(3′')-Ib (Fig. 2B). In 2017, the above-described ARGs was still present in one ST122 and one ST809 isolate but without ISVal1. Additionally, one ST1797 isolate harbored qnrS5 in 2016, suggesting the role of ISVal1 in the response to the antibiotic treatment and the acquisition of ARGs (Table S2).

Likewise, acquisition of resistance islands was also observed in the Tangshan and Huanghua data sets. ST1800 strains did not harbor any transferable ARGs in 2015. However, a resistance island conferring resistance to tetracycline was identified in a plasmid of ST1800 isolates in 2016 (Fig. 2C). Additionally, another four ARGs [aph (3′')-Ia, sul1, qacE, and dfrA27] were also identified in this plasmid. Moreover, the ST150 isolate HB23 also harbored this plasmid in 2017.

Genomic analysis of insertion sites of ISVal1 in the chromosomes of V. parahaemolyticus.The discovery of resistant MGEs with ISVal1 in V. parahaemolyticus prompted us to make a thorough investigation of the insertion sites of ISVal1 in the chromosome of V. parahaemolyticus from three farms.

Genomic analysis of 60 sequenced genomes divided them into five clusters (Fig. 3) and identified 61 insertion events in the ST1, ST122, ST150, ST424, ST1166, ST1743, and ST917a isolates, which were scattered around the two chromosomes (Table S3).

FIG 3
FIG 3

Maximum-likelihood (ML) phylogeny and the presence/absence variation (PAV) matrix in the 60 V. parahaemolyticus genomes collected in three shrimp farms; the scale bar denotes substitutions per variable site; the PAV matrix per isolate is ordered alongside the phylogenetic tree. The presence of the gene family is indicated by a block per column (the colors range from red to blue).

TABLE S3

Insertion sites of ISVal1 in V. parahaemolyticus isolates from three farms. Download Table S3, XLSX file, 0.02 MB.
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In the Tianjin data set, 2013 strain VP17 has five ISVal1 insertions in the chromosomes (Table S3), two of which resulted in the truncation of genes encoding aminotransferase class V-fold pyridoxal 5′-phosphate (PLP)-dependent enzyme and hypothetical protein (Fig. S2). Another ST1743 strain in 2013 was negative for both pirABvp and ISVal1. In 2014, ISVal1 was found in the chromosome of a ST424 isolate VP131, which inserted between two hypothetical proteins (Table S3). In 2015, apart from two ST1 strains with resistant MGEs flanked by ISVal1, six isolates were also ISVal1-positive in their chromosomes, of which ST424 strain 20151007009 had three genes disrupted by ISVal1 (Fig. S2). This suggests that ISVal1 has actively inserted into more genomes during the spread of AHPND. In July 2016, ISVal1 was found in the chromosome of ST424, ST1, and three ST917a isolates.

FIG S2

The disruption of functional genes by ISVal1 in ST150 and ST424 strains. Download FIG S2, PDF file, 0.2 MB.
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In the Tangshan data set, only one ST424 isolate harbored ISVal1 in the chromosomes. Subsequently, ISVal1 was identified in the chromosome six genomes from 2015 and 2016. In the Huanghua data set, ISVal1 was found in six genomes distributed in various locations of chromosomes. Remarkably, four and six insertion events of ISVal1 were identified in chromosomes I and II of strain HB23, respectively. Further analysis showed that ISVal1 insertion resulted in the disruption of the gene encoding lipid A biosynthesis lauroyl acyltransferase (Fig. S2) and ATP-dependent DNA helicase PcrA (Fig. S2) in chromosomes I and II, respectively. In addition, in strain HB23, a transposase belonging to the IS4 family was inserted between fliD and flaA within the gene cluster encoding flagellar biosynthesis.

Pan-genomic analysis revealed the HGT of GIs assisted by IS.As MLST revealed that three shrimp farms shared several common STs (e.g., ST424, ST1743, ST864, and ST415), subsequently, we conducted a pan-genomic analysis to identify potential HGT of MGEs. Interestingly, the formation of MGEs possibly associated with virulence and subsequent HGTs was observed in one ST1 and three ST917a and ST1800 isolates. In one sequenced ST1 strain, TJ-20, ISVal1 was inserted into the flanking region next to the trh-nik-ure genomic island (Fig. 4A).

FIG 4
FIG 4

Horizontal transfer of a genomic island. (A) Horizontal transfer of a trh-nik-ure genomic island from the chromosome to the plasmid mediated by ISVal1. (B) Schematic diagram showing the comparative genome analysis of ST917a isolates and the integration of two genomic islands near recA in VP156 and TJ125. (C) Comparative genome analysis between the V. parahaemolyticus strain VP17 and HB23. Linear comparison of an inserted genomic island between V. parahaemolyticus genome HB23 and Vibrio fluvialis genome FDAARGOS_104 showing the acquisition of a nitrate reductase cluster.

Another new MGE with ISVal1 was found in ST917a strains. A genomic analysis of 2015 strain VP156 revealed that it harbors a 19.5-kb genomic island (GI-1) that disrupted the original recA gene with a 14-bp insert repeat. Eleven open reading frames (ORFs) were identified in this genomic island containing a second non-V. parahaemolyticus recA gene, which differed from the indigenous V. parahaemolyticus recA gene (87%/82% coverage/identity). In 2016, compared with strain VP156, strain TJ125 had another 24.3-kb genomic island (GI-2) next to GI-1, which has 16 ORFs, including one copy of ISVal1 and an integrase gene (Fig. 4B). Interestingly, GI-2 was also found in ST150 strains VP17 and TJ107 but without ISVal1.

We further compared the genome structures between two ST150 strains. Results showed that strains VP17 and HB23 possess two and one unique prophage remnants, respectively (Fig. 4C). Additionally, strain VP17 processes an integrating conjugative element (ICE), which is widely found in Vibrio cholerae, Proteus mirabilis, Shewanella spp., and other species. Interestingly, strain HB23 harbored a 17.8-kb genomic island with 99% DNA similarity to a Vibrio fluvialis strain which encodes respiratory nitrate reductase, nitrate/nitrite transporter NarK, nitrate/nitrite sensor protein NarX, and nitrate/nitrite response regulator protein NarL (Fig. 4C). A denitrification assay showed that strain HB23 is able to grow in denitrification agar plates, which is not observed for another two ST150 isolates (Table S5). This observation suggested that the acquisition of this 17.8-kb genomic island might confer strain HB23 denitrification capacity under aerobic conditions.

TABLE S4

Homology of integrating conjugative element identified in strain VP17. Download Table S4, XLSX file, 0.01 MB.
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TABLE S5

Denitrification assay of ST150 strains. Download Table S5, XLSX file, 0.01 MB.
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In 2017, a 15-kb trh-nik-ure genomic island with ISVal1 was also identified in the plasmid of an ST424 strain, TJ187 (Fig. S3), suggesting that the genomic island has horizontally transferred to ST424.

FIG S3

Schematic diagrams of three plasmids identified in strain TJ187. Genes are denoted by arrows and colored based on gene function classification. The innermost two circles indicate the GC skew [(G – C)/(G + C)] and the GC content. The gene content of the plasmids is illustrated with DNAplotter. Download FIG S3, TIF file, 2.5 MB.
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To determine whether the trh-nik-ure genomic island was also presented in other STs, we searched public genomes and found that one Vibrio diabolicus strain and 27 V. parahaemolyticus strains were positive for this genomic island, which was located on both chromosome 1 and chromosome 2 (Table S6). These V. parahaemolyticus strains could be divided into 10 STs, including ST34, ST631, ST417, and ST36, the majority of which were found in clinical samples. These observations suggest that trh-nik-ure genomic islands were frequently transferred via plasmids.

TABLE S6

Distribution of the trh-nik-ure genomic island in the public genomes of Vibrio spp. Download Table S6, XLSX file, 0.01 MB.
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Plasmid profile of sequenced isolates.The formation of trh-positive plasmids promoted us to take a survey of the plasmid profiles of all sequenced isolates to understand whether plasmids were horizontally transferred. For the isolates with multiple plasmids, Nanopore sequencing technology was employed to obtain the complete plasmid sequence. Comparative genome analysis of 60 sequenced strains from three shrimp farms also found that nine plasmids were variably presented, namely, pVP17-1 (70.1 kb), pVP17-2 (78.0 kb), pVP17-3 (68.7 kb), pTJ187-2 (71.7 kb), pTJ187-3 (86.5 kb), pVPTJ15-2 (83.3 kb), p 114-2 (36.9 kb), pLH80-1 (83.3 kb), and pVP156-1 (106.2 kb) (Table S7).

TABLE S7

General information of plasmids identified in this study. Download Table S7, XLSX file, 0.01 MB.
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Horizontal transfer of plasmids was also commonly observed among the sequenced genomes (Fig. 5). The pirABvp-positive plasmid pVP17-1 was found in one ST150, three ST1743, five ST415, and four ST424 isolates (Table S1). The remaining five pirABvp-negative plasmids were variably presented. pVP17-2, with 99% DNA similarity to p1937-2, was first found in ST150 and then identified in ST122, ST1749, and ST809. pVP17-3 (no significant hit with public genome data) was initially found in an ST150 strain but later was identified in ST1 strains in 2015. All of these results pointed to isolate VP17 as a potential genetic donor for the remaining isolates. The introduction of an ST150 strain might have led to the subsequent transfer of ISVal1 and plasmids into the ST917a, ST1743, and ST424 in 2014 (Fig. 3).

FIG 5
FIG 5

Schematic diagrams of evolutionary events observed in the Tianjin, China, shrimp farm. Horizontal gene transfer observed in the Tianjin shrimp farm from 2013 to 2017. The sequence types obtained from different years are indicated in the circles, which are placed in a sequential order. Horizontal transfer of plasmids, insertion sequences (IS), and genomic islands (GI) are indicated on the black lines connecting the circles. The dashed lines indicate speculative transfer routes.

ST917a isolate VP156 has a 106.2-kb plasmid with 95%/99% (coverage/similarity) to plasmid pVPE61b, which was found in all ST917a strains and some ST1743 strains. pTJ187-2 was exclusively found in ST424 strains which contain an f237 prophage harboring zot and ace. This prophage was also found in the chromosome of all ST424 strains, indicating prophage transfer between the plasmid and chromosome. pTJ187-3 was only identified in ST424 strains TJ102, TJ108, TJ124, TJ187, and LH24, of which strain TJ187 received the 15-kb PAIs.

DISCUSSION

IS facilitated the formation of resistance and pathogenicity islands (PAIs).V. parahaemolyticus is one of the most significant bacterial pathogens, mostly because of its exceptional ability to adapt to marine environments and infectivity to humans and a wide range of marine animals, such as shrimp (22), fish, and mollusks (27). Previous studies suggested that the serotype O3:K6 clone acquired several genomic islands and subsequently caused the pandemic worldwide since 1996. Our previous study found that ISVal1 can transfer between plasmids and chromosomes both in vivo and in vitro (22). In this study, we first observed the formation of resistant MGEs mediated by ISVal1, as a result of selection pressure from the use of antibiotics. As AHPND in shrimp spread, various antibiotics were used for treatment, which might activate the transposition of ISVal1. The stepwise acquisition of resistance MGEs in the plasmid of ST1 and ST122 strains mirrors the usage of antibiotics. Application of florfenicol in 2014 resulted in the independent acquisition of floR-carrying plasmids in 2015. Thereafter, subsequent application of enrofloxacin, streptomycin, and sulfamethoxazole resulted in the emergence of multidrug resistance (MDR) plasmids in 2016.

Interestingly, our data indicate that the simultaneous use of different classes of drugs has limited benefits due to the occurrence of the horizontal acquisition of MGEs, leading to acquisition of MDR in one event. The rapid and diverse acquisition events of resistance MGEs point out the potential of the environment as a reservoir for resistance genes. Thus, our results indicate that the simultaneous usage of over two antibiotics during the outbreak has, in fact, hastened the emergence of multidrug resistance (28).

Although no in vitro experimental evidence is presented in this study, the activation of MGEs by antibiotics at subinhibitory concentrations has been reported in several previous studies. For instance, Beaber et al. demonstrated that SXT elements from Vibrio cholerae are induced by antibiotics triggering the SOS response (29). Likewise, Scornec et al. found that subinhibitory concentrations of tetracycline activated the transfer of MGEs in Enterococcus faecalis through an antiattenuation mechanism (30).

Next, we looked for the other signatures of microevolution among the V. parahaemolyticus genomes mediated by insertion sequence ISVal1. A thorough search of the insertion sites of ISVal1 in the 60 genomes identified 61 types of insertion sites, of which the trh-nik-ure genomic island flanked by ISVal1 was found in both the chromosome of ST1 in 2016 and the plasmid of ST424 in 2017. blastn searches showed that the trh genes were flanked by different ISs in numerous V. parahaemolyticus genomes (Table S6), indicating the horizontal transferability of this genomic island. The genomic island containing the tdh or trh genes flanked by ISs in V. parahaemolyticus was found previously (26, 31). The HGT of this type of genomic island among different Vibrio spp. was subsequently found by Nishibuchi et al. (32). In their study, the tdh gene was transferred between different replicons by transposition, and plasmids could have been the vehicles for the tdh gene transfer between different organisms, suggesting the tdh gene was transferred first from the tdh-bearing non-Vibrio organisms to a Vibrio species. Likewise, Xu et al. found that one human-pathogenic variant (ST631) evolved independently through acquisition of distinct PAIs containing trh, suggesting that nonpathogenic V. parahaemolyticus harbored the regulatory mechanisms involved in managing newly acquired virulence elements (33). These studies suggested that the coordinated expression of tdh/trh is an ancient mechanism and that horizontally acquired genomic islands can incorporate into existing regulatory networks with sufficient expression (33).

Acquisition of PAIs or fitness islands via HGT provides an essential mechanism for the bacterial adaptation to different niches. Another interesting horizontal transfer event is the disruption of recA by a genomic island in an ST917a strain followed by the second insertion from a second genomic island, which might come from ST150 strains. The potential roles of two genomic islands will be examined in a future study. However, it has been shown that the inserted recA could protect against DNA damage induced by UV irradiation in V. cholerae (34). In another study, Chen et al. also identified an O4:KUT-recAin type strain with an insertion of a genomic island in recA alleles, which has become a new emerging serotype since 2014 and exhibited strong acid resistance at pH 4.9 (35). Additionally, strain HB23, obtained from the Huanghua farm, acquired a fitness island, which confers to it the denitrifying capacity under aerobic conditions. These observations suggested that genomic surveillance at the farm would facilitate the early warning about the new virulent genotype.

Real-time genomic surveillance of pathogens provided important insights into the prediction of emergent populations.In this study, we selected three shrimp farms to observe the in vivo evolution of V. parahaemolyticus. Despite the three farms exhibiting distinct V. parahaemolyticus populations and the surveillance periods varying from 6 to 8 years, the three data sets exhibited similar evolutionary trajectories—frequent transposition activities resulting in the HGT of virulence or fitness islands.

Thus, we summarized that there were four stages of the microevolution of V. parahaemolyticus (Fig. 6A). In stage I (from 2011 to 2014), occasional outbreaks occurred on the shrimp farm accompanied with the transfer of ISVal1 from plasmid to chromosome. Then AHPND outbreaks spread into more ponds, resulting in wide use of antibiotics. In stage II (2015), antibiotic usage facilitated the formation of resistant MGEs mediated by insertion sequences. Resistant strains started to emerge with resistant MGEs thanks to ISVal1. In stage III (2016), ISVal1 was inserted into the chromosomes of more STs, resulting in the formation of virulent MGEs. One ISVal1 was inserted into the flanking regions of trh. Meanwhile, another genomic island might horizontally transfer from ST150 to ST917a, conferring on them a stronger resistance to UV treatment. In stage IV (2017), horizontal transfers of GIs associated with pathogenicity and fitness were found in ST424, ST917a, and ST150 strains.

FIG 6
FIG 6

(A and B) Four milestone evolutionary stages of bacterial pathogens (A) and suggested prevention strategies (B). Evolutionary trajectories were divided into the following four stages: stage 1, transfer of ISVal1 from plasmid to chromosome; stage 2, formation of resistant MGEs; stage 3, formation of virulent MGEs; stage 4, horizontal transfer of MGEs and plasmid. The decline of the outbreak-related pond is due to the discarding of the pond.

The HGT of pathogenicity islands can be suggested as a signal of early warning for the emergence of virulent populations. Therefore, various prevention measures can be adopted at the different stages to avoid the epidemic deterioration. For instance, at stage III, as antibiotics trigger the transposition activity of resistance islands, antibiotics should be used with caution. At stage IV, critical measures should be adopted to prevent the spillover of the pathogen. Subsequently, we conducted 6-month genomic surveillance on another shrimp farm, which prevented the spread of the outbreaks and controlled the formation of virulence MGEs.

Continuous pathogen surveillance is necessary to understand the health risks of a certain pathogen and to help predict future evolutionary consequences, which serves as the basis for an intermediate intervention. Our recent genomic surveillance at a chicken farm identified an anthrax toxin-positive Bacillus cereus strain in a probiotic product used as a feed supplement, which was transferred into the groundwater and to a nearby fish farm (36). Continuous surveillance of the groundwater subsequently monitored the contamination of groundwater and led to the early prevention of a potential epidemic (36).

HGT has had a major role in the acquisition of genes associated with virulence in Vibrio vulnificus (37). Our study showed that the activity of HGT mediated by IS could be used as an indicator to predict potential human pathogens. The emergence of a recent Shiga toxin-producing E. coli O104:H4 clone in Germany was another example caused by horizontal transfer of a toxin-producing phage into an enteroaggregative E. coli strain (12).

Our results also suggest that clinical surveillance is not sufficient to warn about an oncoming epidemic at an early stage. Three levels of surveillance program to monitor, respectively, the animal infection at the farm or in wildlife, prepandemic transmission at the interface between animals and humans, and sporadic infection at sentinel hospitals should be set up (Fig. 6B). Information should also be shared between agriculture and public health sectors to address the emerging epidemic in a timely manner. Further studies should examine the possibility of real-time metagenomic surveillance for all pathogens. Efficient monitoring of the interfaces between the environment, wildlife and farm animals, and humans may contribute to reducing the dissemination of pathogens and ultimately to detect early signals of an epidemic.

Conclusion.Real-time genomic surveillance of V. parahaemolyticus in a shrimp-farming region found that ISVal1 mediated the HGT of prophages, plasmids, and genomic islands among V. parahaemolyticus in response to adverse external environmental and selection pressure. Analysis of evolutionary events in this study has led to a better understanding of the underlying mechanisms of pathogen emergence. It is a matter of urgency that we need to strength the surveillance of zoonotic infectious agents at the interfaces between wildlife, farm animals, the environment, and humans.

MATERIALS AND METHODS

Sample collection, bacterium isolation, and identification in shrimp farms.From 2011 to 2018, 60 shrimp were collected in July or April annually from three shrimp-farming regions near Tianjin (from 2011 to 2018), Tangshan (from 2011 to 2017), and Huanghua City (from 2011 to 2016), China (Fig. S1). Hepatopancreas from shrimp were aseptically disaggregated in 100 ml alkaline peptone water and streaked on thiosulfate citrate bile salts sucrose (TCBS) plates at 28°C for 24 h. The bacterial identification of suspected colonies obtained from TCBS was conducted as described previously (22).

MLST was conducted as described by Gonzalez-Escalona et al. (23). PCR detection of the virulence genes tdh, trh, pirABvp, and ISVal1 was conducted as previously described (24, 25). The presence of T3SS1 and T3SS2 was verified by PCR of the genes vcrD1 and vscC2, respectively (26).

Whole-genome sequencing, de novo assembly, and annotation.Total DNA was extracted from overnight culture of the isolates with the Wizard Genomic DNA kit (Promega, Madison, WI, USA) for whole-genome sequencing. High-throughput genome sequencing was carried out on Illumina platforms or a Nanopore platform at Novogene (Tianjin, China). The draft genome was assembled de novo with SPAdes version 3.0 (38). RAST was used to annotate the sequences of each genome determined with next-generation sequencing (39). PIRATE was used to build a comprehensive pan-genome of the population and identify accessory genes (40). Outputs of the pan-genome were analyzed with R version 3.2.3 (41).

Identification of SNPs and phylogenetic analyses.The core genome of V. parahaemolyticus defined by Gonzalez-Escalona et al. was used as the reference genome to call the single-nucleotide polymorphisms (SNPs) for V. parahaemolyticus (42). SNP calling was performed with a previously developed pipeline to guarantee that only genuine SNPs were included in the analysis (43).

RAxML version 7.8.6 was used with the generalized time-reversible model and a Gamma distribution to model site-specific rate variation (the GTR+⌈ substitution model; GTRGAMMA in RAxML) (44). Support for the ML phylogeny was assessed by 100 bootstrap pseudoanalyses of the alignment data, and the final tree was visualized in FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

Denitrification assay.A denitrification medium (DM) was used to confirm the denitrification capacity of test strains under aerobic conditions. The composition of the DM was described previously (45). Strains HB23, TJ107, and VP17 were suspended in 0.9% saline solution and streaked onto the DM agar plates (with g · liter−1 NO3−-N 0.72). A Luria-Bertani (LB) medium was also inoculated as the control. The aerobic denitrification ability was assessed after incubation at 28°C for 48 h. All experiments were carried out in triplicate.

Data availability.The raw sequencing data were submitted to GenBank (NCBI) under the BioProject no. PRJNA503785.

ACKNOWLEDGMENTS

We thank Huiqin Tian, Shibo Jin, and Lei Zhang for sampling assistance. We thank Jaime Martinez-Urtaza for insightful comments.

This research was supported by the National Natural Science Foundation of China (81903372) and the National Science and Technology Major Project (2018ZX10714-002).

S.F. and B.P. designed the study and coordinated the project. B.P., D.W., Q.W., B.Q., and Y.L. isolated the strains and conducted the MLST. S.F. and Q.Y. conducted the genomic analysis. S.F., R.L., and Q.Y. wrote the manuscript. All authors approved the final version of the manuscript.

We declare no competing interests.

FOOTNOTES

    • Received December 3, 2020.
    • Accepted December 28, 2020.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

REFERENCES

  1. 1.↵
    1. Wolfe ND,
    2. Dunavan CP,
    3. Diamond J
    . 2007. Origins of major human infectious diseases. Nature 447:279–283. doi:10.1038/nature05775.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Wilder-Smith A,
    2. Chiew CJ,
    3. Lee VJ
    . 2020. Can we contain the COVID-19 outbreak with the same measures as for SARS? Lancet Infect Dis 20:e102–e107. doi:10.1016/S1473-3099(20)30129-8.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Morens DM,
    2. Fauci AS
    . 2013. Emerging infectious diseases: threats to human health and global stability. PLoS Pathog 9:e1003467. doi:10.1371/journal.ppat.1003467.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Jones KE,
    2. Patel N,
    3. Levy M,
    4. Storeygard A,
    5. Balk D,
    6. Gittleman JL,
    7. Daszak P
    . 2008. Global trends in emerging infectious diseases. Nature 451:990–993. doi:10.1038/nature06536.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Daszak P,
    2. Cunningham AA,
    3. Hyatt AD
    . 2000. Emerging infectious diseases of wildlife: threats to biodiversity and human health. Science 287:443–449. doi:10.1126/science.287.5452.443.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Woolhouse M,
    2. Gaunt E
    . 2007. Ecological origins of novel human pathogens. Crit Rev Microbiol 33:231–242. doi:10.1080/10408410701647560.
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    1. Karesh WB,
    2. Dobson A,
    3. Lloyd-Smith JO,
    4. Lubroth J,
    5. Dixon MA,
    6. Bennett M,
    7. Aldrich S,
    8. Harrington T,
    9. Formenty P,
    10. Loh EH,
    11. Machalaba CC,
    12. Thomas MJ,
    13. Heymann DL
    . 2012. Ecology of zoonoses: natural and unnatural histories. Lancet 380:1936–1945. doi:10.1016/S0140-6736(12)61678-X.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    1. Morse SS,
    2. Mazet JA,
    3. Woolhouse M,
    4. Parrish CR,
    5. Carroll D,
    6. Karesh WB,
    7. Zambrana-Torrelio C,
    8. Lipkin WI,
    9. Daszak P
    . 2012. Prediction and prevention of the next pandemic zoonosis. Lancet 380:1956–1965. doi:10.1016/S0140-6736(12)61684-5.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Walker TM,
    2. Kohl TA,
    3. Omar SV,
    4. Omar SV,
    5. Hedge J,
    6. Del Ojo Elias C,
    7. Bradley P,
    8. Iqbal Z,
    9. Feuerriegel S,
    10. Niehaus KE,
    11. Wilson DJ,
    12. Clifton DA,
    13. Kapatai G,
    14. Ip CLC,
    15. Bowden R,
    16. Drobniewski FA,
    17. Allix-Béguec C,
    18. Gaudin C,
    19. Parkhill J,
    20. Diel R,
    21. Supply P,
    22. Crook DW,
    23. Smith EG,
    24. Walker AS,
    25. Ismail N,
    26. Niemann S,
    27. Peto TEA
    , Modernizing Medical Microbiology (MMM) Informatics Group. 2015. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study. Lancet Infect Dis 15:1193–1202. doi:10.1016/S1473-3099(15)00062-6.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Gardy JL,
    2. Loman NJ
    . 2018. Towards a genomics-informed, real-time, global pathogen surveillance system. Nat Rev Genet 19:9–20. doi:10.1038/nrg.2017.88.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Scheutz F,
    2. Moller Nielsen E,
    3. Frimodt-Moller J,
    4. Dahbi G,
    5. Mamani R,
    6. Pita JM,
    7. Alonso MP,
    8. Llovo J,
    9. Bernárdez MI,
    10. Blanco JE,
    11. Blanco M,
    12. Blanco J
    . 2011. Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. Euro Surveill 16:19889. https://www.eurosurveillance.org/content/10.2807/ese.16.24.19889-en.
    OpenUrlPubMed
  12. 12.↵
    1. Boisen N,
    2. Melton-Celsa AR,
    3. Scheutz F,
    4. O’Brien AD,
    5. Nataro JP
    . 2015. Shiga toxin 2a and Enteroaggregative Escherichia coli: a deadly combination. Gut Microbes 6:272–278. doi:10.1080/19490976.2015.1054591.
    OpenUrlCrossRef
  13. 13.↵
    1. Baker-Austin C,
    2. Oliver JD,
    3. Alam M,
    4. Ali A,
    5. Waldor MK,
    6. Qadri F,
    7. Martinez-Urtaza J
    . 2018. Vibrio spp. infections. Nat Rev Dis Primers 4:8. doi:10.1038/s41572-018-0005-8.
    OpenUrlCrossRef
  14. 14.↵
    1. Crim SM,
    2. Griffin PM,
    3. Tauxe R,
    4. Cieslak PR,
    5. Dunn J,
    6. Hurd S,
    7. Jervis R,
    8. Lathrop S,
    9. Muse A,
    10. Ryan P,
    11. Smith K,
    12. Tobin-D’Angelo M,
    13. Vugia DJ,
    14. Holt KG,
    15. Wolpert BJ,
    16. Tauxe R,
    17. Geissler AL
    . 2015. Preliminary incidence and trends of infection with pathogens transmitted commonly through food - foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2006–2014. MMWR Morb Mortal Wkly Rep 64:328–332.
    OpenUrlPubMed
  15. 15.↵
    1. Espejo RT,
    2. Garcia K,
    3. Plaza N
    . 2017. Insight into the origin and evolution of the vibrio parahaemolyticus pandemic strain. Front Microbiol 8:1397. doi:10.3389/fmicb.2017.01397.
    OpenUrlCrossRef
  16. 16.↵
    1. Han D,
    2. Yu F,
    3. Chen X,
    4. Zhang R,
    5. Li J
    . 2019. Challenges in Vibrio parahaemolyticus infections caused by the pandemic clone. Future Microbiol 14:437–450. doi:10.2217/fmb-2018-0308.
    OpenUrlCrossRef
  17. 17.↵
    1. Harris SR,
    2. Feil EJ,
    3. Holden MT,
    4. Quail MA,
    5. Nickerson EK,
    6. Chantratita N,
    7. Gardete S,
    8. Tavares A,
    9. Day N,
    10. Lindsay JA,
    11. Edgeworth JD,
    12. de Lencastre H,
    13. Parkhill J,
    14. Peacock SJ,
    15. Bentley SD
    . 2010. Evolution of MRSA during hospital transmission and intercontinental spread. Science 327:469–474. doi:10.1126/science.1182395.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Grenfell BT,
    2. Pybus OG,
    3. Gog JR,
    4. Wood JL,
    5. Daly JM,
    6. Mumford JA,
    7. Holmes EC
    . 2004. Unifying the epidemiological and evolutionary dynamics of pathogens. Science 303:327–332. doi:10.1126/science.1090727.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Du X,
    2. Bayliss SC,
    3. Feil EJ,
    4. Liu Y,
    5. Wang C,
    6. Zhang G,
    7. Zhou D,
    8. Wei D,
    9. Tang N,
    10. Leclercq SO,
    11. Feng J
    . 2019. Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm. Environ Microbiol 21:1113–1123. doi:10.1111/1462-2920.14531.
    OpenUrlCrossRef
  20. 20.↵
    1. Lee CT,
    2. Chen IT,
    3. Yang YT,
    4. Ko TP,
    5. Huang YT,
    6. Huang JY,
    7. Huang MF,
    8. Lin SJ,
    9. Chen CY,
    10. Lin SS,
    11. Lightner DV,
    12. Wang HC,
    13. Wang AH,
    14. Wang HC,
    15. Hor LI,
    16. Lo CF
    . 2015. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. Proc Natl Acad Sci U S A 112:10798–10803. doi:10.1073/pnas.1503129112.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Xiao J,
    2. Liu L,
    3. Ke Y,
    4. Li X,
    5. Liu Y,
    6. Pan Y,
    7. Yan S,
    8. Wang Y
    . 2017. Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species. Sci Rep 7:42177. doi:10.1038/srep42177.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Fu S,
    2. Wei D,
    3. Yang Q,
    4. Xie G,
    5. Pang B,
    6. Wang Y,
    7. Lan R,
    8. Wang Q,
    9. Dong X,
    10. Zhang X,
    11. Huang J,
    12. Feng J,
    13. Liu Y
    . 2020. Horizontal plasmid transfer promotes the dissemination of Asian acute hepatopancreatic necrosis disease and provides a novel mechanism for genetic exchange and environmental adaptation. mSystems 5:e00799-19. doi:10.1128/mSystems.00799-19.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Gonzalez-Escalona N,
    2. Martinez-Urtaza J,
    3. Romero J,
    4. Espejo RT,
    5. Jaykus LA,
    6. DePaola A
    . 2008. Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing. J Bacteriol 190:2831–2840. doi:10.1128/JB.01808-07.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Tada J,
    2. Ohashi T,
    3. Nishimura N,
    4. Shirasaki Y,
    5. Ozaki H,
    6. Fukushima S,
    7. Takano J,
    8. Nishibuchi M,
    9. Takeda Y
    . 1992. Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol Cell Probes 6:477–487. doi:10.1016/0890-8508(92)90044-x.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Tran L,
    2. Nunan L,
    3. Redman RM,
    4. Mohney LL,
    5. Pantoja CR,
    6. Fitzsimmons K,
    7. Lightner DV
    . 2013. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis Aquat Organ 105:45–55. doi:10.3354/dao02621.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Okada N,
    2. Iida T,
    3. Park KS,
    4. Goto N,
    5. Yasunaga T,
    6. Hiyoshi H,
    7. Matsuda S,
    8. Kodama T,
    9. Honda T
    . 2009. Identification and characterization of a novel type III secretion system in trh-positive Vibrio parahaemolyticus strain TH3996 reveal genetic lineage and diversity of pathogenic machinery beyond the species level. Infect Immun 77:904–913. doi:10.1128/IAI.01184-08.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Austin B
    . 2010. Vibrios as causal agents of zoonoses. Vet Microbiol 140:310–317. doi:10.1016/j.vetmic.2009.03.015.
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Hughes D,
    2. Andersson DI
    . 2017. Evolutionary trajectories to antibiotic resistance. Annu Rev Microbiol 71:579–596. doi:10.1146/annurev-micro-090816-093813.
    OpenUrlCrossRef
  29. 29.↵
    1. Beaber JW,
    2. Hochhut B,
    3. Waldor MK
    . 2004. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72–74. doi:10.1038/nature02241.
    OpenUrlCrossRefPubMedWeb of Science
  30. 30.↵
    1. Scornec H,
    2. Bellanger X,
    3. Guilloteau H,
    4. Groshenry G,
    5. Merlin C
    . 2017. Inducibility of Tn916 conjugative transfer in Enterococcus faecalis by subinhibitory concentrations of ribosome-targeting antibiotics. J Antimicrob Chemother 72:2722–2728. doi:10.1093/jac/dkx202.
    OpenUrlCrossRef
  31. 31.↵
    1. Terai A,
    2. Baba K,
    3. Shirai H,
    4. Yoshida O,
    5. Takeda Y,
    6. Nishibuchi M
    . 1991. Evidence for insertion sequence-mediated spread of the thermostable direct hemolysin gene among Vibrio species. J Bacteriol 173:5036–5046. doi:10.1128/jb.173.16.5036-5046.1991.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Nishibuchi M,
    2. Janda JM,
    3. Ezaki T
    . 1996. The thermostable direct hemolysin gene (tdh) of Vibrio hollisae is dissimilar in prevalence to and phylogenetically distant from the tdh genes of other vibrios: implications in the horizontal transfer of the tdh gene. Microbiol Immunol 40:59–65. doi:10.1111/j.1348-0421.1996.tb03304.x.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Xu F,
    2. Gonzalez-Escalona N,
    3. Drees KP,
    4. Sebra RP,
    5. Cooper VS,
    6. Jones SH,
    7. Whistler CA
    . 2017. Parallel evolution of two clades of an Atlantic-endemic pathogenic lineage of Vibrio parahaemolyticus by independent acquisition of related pathogenicity islands. Appl Environ Microbiol 83:e01168-17. doi:10.1128/AEM.01168-17.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Rapa RA,
    2. Islam A,
    3. Monahan LG,
    4. Mutreja A,
    5. Thomson N,
    6. Charles IG,
    7. Stokes HW,
    8. Labbate M
    . 2015. A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage. Environ Microbiol 17:1090–1102. doi:10.1111/1462-2920.12512.
    OpenUrlCrossRef
  35. 35.↵
    1. Chen X,
    2. Li Y,
    3. Yao W,
    4. Wu T,
    5. Zhu Q,
    6. Zhang Y,
    7. Ye H,
    8. Wang R,
    9. Zheng S,
    10. Yu F,
    11. Chen W,
    12. Zhu Z,
    13. Mao L,
    14. Hu Q,
    15. Tang Z,
    16. Chen H,
    17. Liu Y,
    18. Chen Y
    . 2020. A new emerging serotype of Vibrio parahaemolyticus in China is rapidly becoming the main epidemic strain. Clin Microbiol Infect 26:644.e1–644.e7. doi:10.1016/j.cmi.2019.09.024.
    OpenUrlCrossRef
  36. 36.↵
    1. Fu S,
    2. Yang Q,
    3. He F,
    4. Lan R,
    5. Hao J,
    6. Ni P,
    7. Liu Y,
    8. Li R
    . 2019. National safety survey of animal-use commercial probiotics and their spillover effects from farm to human: an emerging threat to public health. Clin Infect Dis 70:2386–2395. doi:10.1093/cid/ciz642.
    OpenUrlCrossRef
  37. 37.↵
    1. Efimov V,
    2. Danin-Poleg Y,
    3. Raz N,
    4. Elgavish S,
    5. Linetsky A,
    6. Kashi Y
    . 2013. Insight into the evolution of Vibrio vulnificus biotype 3’s genome. Front Microbiol 4:393. doi:10.3389/fmicb.2013.00393.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Bankevich A,
    2. Nurk S,
    3. Antipov D,
    4. Gurevich AA,
    5. Dvorkin M,
    6. Kulikov AS,
    7. Lesin VM,
    8. Nikolenko SI,
    9. Pham S,
    10. Prjibelski AD,
    11. Pyshkin AV,
    12. Sirotkin AV,
    13. Vyahhi N,
    14. Tesler G,
    15. Alekseyev MA,
    16. Pevzner PA
    . 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi:10.1089/cmb.2012.0021.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Aziz RK,
    2. Bartels D,
    3. Best AA,
    4. DeJongh M,
    5. Disz T,
    6. Edwards RA,
    7. Formsma K,
    8. Gerdes S,
    9. Glass EM,
    10. Kubal M,
    11. Meyer F,
    12. Olsen GJ,
    13. Olson R,
    14. Osterman AL,
    15. Overbeek RA,
    16. McNeil LK,
    17. Paarmann D,
    18. Paczian T,
    19. Parrello B,
    20. Pusch GD,
    21. Reich C,
    22. Stevens R,
    23. Vassieva O,
    24. Vonstein V,
    25. Wilke A,
    26. Zagnitko O
    . 2008. The RAST server: Rapid Annotation using Subsystems Technology. BMC Genomics 9:75. doi:10.1186/1471-2164-9-75.
    OpenUrlCrossRefPubMed
  40. 40.↵
    1. Bayliss SC,
    2. Thorpe HA,
    3. Coyle NM,
    4. Sheppard SK,
    5. Feil EJ
    . 2019. PIRATE: a fast and scalable pangenomics toolbox for clustering diverged orthologues in bacteria. Gigascience 8:giz119. doi:10.1093/gigascience/giz119.
    OpenUrlCrossRef
  41. 41.↵
    R Core Team. 2017. R: a language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. https://www.R-project.org/.
  42. 42.↵
    1. Gonzalez-Escalona N,
    2. Jolley KA,
    3. Reed E,
    4. Martinez-Urtaza J
    . 2017. Defining a core genome multilocus sequence typing scheme for the global epidemiology of Vibrio parahaemolyticus. J Clin Microbiol 55:1682–1697. doi:10.1128/JCM.00227-17.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Chan CHS,
    2. Octavia S,
    3. Sintchenko V,
    4. Lan R
    . 2016. SnpFilt: a pipeline for reference-free assembly-based identification of SNPs in bacterial genomes. Comput Biol Chem 65:178–184. doi:10.1016/j.compbiolchem.2016.09.004.
    OpenUrlCrossRef
  44. 44.↵
    1. Stamatakis A
    . 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690. doi:10.1093/bioinformatics/btl446.
    OpenUrlCrossRefPubMedWeb of Science
  45. 45.↵
    1. Wan C,
    2. Yang X,
    3. Lee DJ,
    4. Du M,
    5. Wan F,
    6. Chen C
    . 2011. Aerobic denitrification by novel isolated strain using NO2− “–” N as nitrogen source. Bioresour Technol 102:7244–7248. doi:10.1016/j.biortech.2011.04.101.
    OpenUrlCrossRefPubMed
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KEYWORDS

acute hepatopancreatic necrosis disease (AHPND)
Vibrio parahaemolyticus
mobile genetic elements (MGEs)
evolutionary trajectories
ISVal1

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