Genome Sequence of Vibrio parahaemolyticus VP152 Strain Isolated from Penaeus indicus in Malaysia

Vibrio parahaemolyticus is a Gram-negative bacterium that naturally occurs in marine associated aquatic environments (Letchumanan et al., 2014; Malcolm et al., 2015). This bacterium causes highest number of seafood-associated gastroenteritis in many countries including United States and Asian countries (Scallan et al., 2011; Newton et al., 2012). V. parahaemolyticus is often been isolated from aquatic environments such as seawater and marine sediment, as well as from vertebrate and invertebrate seafood products (Shen et al., 2009). The most likely route of infection in humans is reported to be associated with consumption of raw or improperly cooked seafood (Daniels et al., 2000; Jun et al., 2014; Hazen et al., 2015; Raghunath, 2015; Law et al., 2015). 
 
Recently, V. parahaemolyticus has been demonstrated to be a major source of infection in the aquaculture industry (Letchumanan et al., 2014; Soto-Rodriguez et al., 2015; Tey et al., 2015). Aquaculture farmers rely on a wide range of antibiotics to prevent (prophylactic use) and treat (therapeutic use) bacterial infections in fish and invertebrates (Cabello et al., 2013). The extensive use of antibiotics and other chemotherapeutics in aquaculture has led to the emergence of multidrug resistant strains in the biosphere (Letchumanan et al., 2015a, 2016; Rao and Lalitha, 2015). Multidrug resistant V. parahaemolyticus strains have been isolated and detected from shrimp in Thailand (Yano et al., 2014), Malaysia (Al-Othrubi et al., 2011; Sani et al., 2013; Letchumanan et al., 2015b,c) and China (Peng et al., 2010; Xu et al., 2014). Resistance toward clinically used antibiotics will eventually hamper the treatment of bacterial infections in humans and potentially increase the fatality rate (Daniels et al., 2000). Therefore, monitoring Vibrio species in aquaculture surroundings is crucial for both human health and the aquaculture industry. 
 
In our previous study, we have isolated environmental V. parahaemolyticus strains from two types of Malaysian shrimp, Penaeus indicus and Solenocera subnuda. We detected the thermostable direct hemolysin (tdh) and thermostable direct related hemolysin (trh) virulence genes through a PCR based assay and studied the antibiotic resistance profile of all the isolated strains (Letchumanan et al., 2015c). V. parahaemolyticus VP152 was isolated from Penaeus indicus (Banana prawn) and originated from a supermarket sample. This strain did not possess both the tdh and trh virulence genes, which are responsible for causing diseases in humans and marine animals. Despite the fact that V. parahaemolyticus VP152 strain does not have tdh and trh virulence genes properties, the strain cannot be ignored in light of the fact that it exhibits multidrug resistance profiles toward 11/14 antibiotics tested. Based on the antibiotic susceptibility phenotype, the strain exhibited multiple-antibiotic resistance toward ampicillin, oxytetracycline, nalidixic acid, ampicillin/sulbactam, tetracycline, third generation cephalos porins (cefotaxime and ceftazidime), aminoglycosides (amikacin, kanamycin, and gentamicin) and trimethoprim/sulfameth oxazole (Letchumanan et al., 2015c). 
 
This is a worrying situation as the antibiotic resistant profiles shown by V. parahaemolyticus VP152 include the recommended antimicrobial agents used in treatment of Vibrio spp. infections, including third generation cephalosporin, fluoroquinolones, aminoglycosides, tetracycline, gentamicin, trimethoprim/sulfamethoxazole (Daniels and Shafaie, 2000; Shaw et al., 2014). Therefore, the whole genome sequence of V. parahaemolyticus VP152 was studied with respect to the multidrug resistance profiles to gain a better understanding of the antibiotic resistant patterns. The availability of this genome sequence of V. parahaemolyticus VP152 will aid as a basis for further in-depth analysis of the antibiotic resistance profile of environmental V. parahaemolyticus.


INTRODUCTION
Vibrio parahaemolyticus is a Gram-negative bacterium that naturally occurs in marine associated aquatic environments (Letchumanan et al., 2014;Malcolm et al., 2015). This bacterium causes highest number of seafood-associated gastroenteritis in many countries including United States and Asian countries (Scallan et al., 2011;Newton et al., 2012). V. parahaemolyticus is often been isolated from aquatic environments such as seawater and marine sediment, as well as from vertebrate and invertebrate seafood products (Shen et al., 2009). The most likely route of infection in humans is reported to be associated with consumption of raw or improperly cooked seafood Jun et al., 2014;Hazen et al., 2015;Raghunath, 2015;Law et al., 2015).
Recently, V. parahaemolyticus has been demonstrated to be a major source of infection in the aquaculture industry (Letchumanan et al., 2014;Soto-Rodriguez et al., 2015;Tey et al., 2015). Aquaculture farmers rely on a wide range of antibiotics to prevent (prophylactic use) and treat (therapeutic use) bacterial infections in fish and invertebrates (Cabello et al., 2013). The extensive use of antibiotics and other chemotherapeutics in aquaculture has led to the emergence of multidrug resistant strains in the biosphere (Letchumanan et al., 2015a(Letchumanan et al., , 2016Rao and Lalitha, 2015). Multidrug resistant V. parahaemolyticus strains have been isolated and detected from shrimp in Thailand (Yano et al., 2014), Malaysia (Al-Othrubi et al., 2011;Sani et al., 2013;Letchumanan et al., 2015b,c) and China (Peng et al., 2010;Xu et al., 2014). Resistance toward clinically used antibiotics will eventually hamper the treatment of bacterial infections in humans and potentially increase the fatality rate . Therefore, monitoring Vibrio species in aquaculture surroundings is crucial for both human health and the aquaculture industry.
In our previous study, we have isolated environmental V. parahaemolyticus strains from two types of Malaysian shrimp, Penaeus indicus and Solenocera subnuda. We detected the thermostable direct hemolysin (tdh) and thermostable direct related hemolysin (trh) virulence genes through a PCR based assay and studied the antibiotic resistance profile of all the isolated strains (Letchumanan et al., 2015c). V. parahaemolyticus VP152 was isolated from Penaeus indicus (Banana prawn) and originated from a supermarket sample. This strain did not possess both the tdh and trh virulence genes, which are responsible for causing diseases in humans and marine animals. Despite the fact that V. parahaemolyticus VP152 strain does not have tdh and trh virulence genes properties, the strain cannot be ignored in light of the fact that it exhibits multidrug resistance profiles toward 11/14 antibiotics tested. Based on the antibiotic susceptibility phenotype, the strain exhibited multiple-antibiotic resistance toward ampicillin, oxytetracycline, nalidixic acid, ampicillin/sulbactam, tetracycline, third generation cephalosporins (cefotaxime and ceftazidime), aminoglycosides (amikacin, kanamycin, and gentamicin) and trimethoprim/sulfamethoxazole (Letchumanan et al., 2015c).
This is a worrying situation as the antibiotic resistant profiles shown by V. parahaemolyticus VP152 include the recommended antimicrobial agents used in treatment of Vibrio spp. infections, including third generation cephalosporin, fluoroquinolones, aminoglycosides, tetracycline, gentamicin, trimethoprim/sulfamethoxazole (Daniels and Shafaie, 2000;Shaw et al., 2014). Therefore, the whole genome sequence of V. parahaemolyticus VP152 was studied with respect to the multidrug resistance profiles to gain a better understanding of the antibiotic resistant patterns. The availability of this genome sequence of V. parahaemolyticus VP152 will aid as a basis for further in-depth analysis of the antibiotic resistance profile of environmental V. parahaemolyticus.

Genome Sequencing and Assembly
Genomic DNA of VP152 strain was extracted using Masterpure TM DNA purification kit (Epicenter, Illumina Inc, Madison, WI, USA) and subjected to RNase (Qiagen, USA) treatment (Ser et al., 2015). The DNA quality was quantified using NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and a Qubit version 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Illumina sequencing library of genomic DNA was prepared using Nextera TM DNA Sample Preparation kit (Illumina, San Diego, CA, USA) and library quality was validated by a Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Palo Alto, CA, USA) prior to sequencing. The genome of VP152 strain was sequenced on MiSeq platform with MiSeq Reagent Kit 2 (2 × 250 bp; Illumina Inc, San Diego, CA, USA). The trimmed sequences were de novo assembled with CLC Genomic Workbench version 5.1 (CLC Bio, Denmark).

Genome Annotation
Gene prediction was carried out using Prodigal 2.6, while rRNA and tRNA were analyzed using RNAmmer and tRNAscan SE version 1.21 (Lowe and Eddy, 1997;Lagesen et al., 2007;Hyatt et al., 2010). Gene prediction and annotation were performed using Rapid Annotation Search Tool (RAST; Aziz et al., 2008). Antibiotic resistance genes were analyzed using antibiotic resistance genes-ANNOTation (ARG-ANNOT; Gupta et al., 2014).

Genome Characteristics
The genome of V. parahaemolyticus VP152 consists of 4,982,021 bp with mean genome coverage of 183.46-fold and with an average G+C content of 53.4% (Table 1). A total of 4809 genes was predicted of which 4638 were identified as protein coding genes. There are 91 RNA genes consisting of 11 rRNAs and 80 tRNAs.

Virulence and Antimicrobial Resistance Genes
The analysis obtained from RAST server revealed 573 subsystems (Figure 1). The annotated genome has 97 genes responsible for resistance to antibiotic and toxic compounds including seven genes for mdtABCD multidrug resistance cluster, 19 genes for multidrug resistance efflux pumps, four genes for β-lactamase and two genes aminoglycoside adenylyltransferases. The genome sequence of V. parahaemolyticus VP152 was compared with three environmental V. parahaemolyticus strains, in order to delineate the similarities between the four strains. The genome size of V. parahaemolyticus VP152 was similar to strains of V. parahaemolyticus and contained several antibiotic resistance genes as shown in Table 1. Also, further comparison of hemolysin genes present in V. parahaemolyticus VP152 and the selected strains revealed no significant differences.
The genome analysis on ARG-ANNOT noted the presences of tetracycline resistant gene, Tet and Tet-2 gene within the genome. The presence of these genes is closely related to the phenotypic resistance shown by the strain toward oxytetracycline and tetracycline. Furthermore, β-lactam resistance-related gene, bla gene of VP152 exhibited 99% similarities when compared to other V. parahaemolyticus strain and Vibrio species. The phenotypic resistance shown by V. parahaemolyticus VP152 toward ampicillin, ampicillin/sulbactam, cefotaxime and ceftazidime is closely related to the gene coding β-lactamase in the genome. The gene coding aminoglycosides adenylyltransferase of V. parahaemolyticus VP152 confers resistance phenotype observed toward amikacin, kanamycin, and gentamicin. Based on the annotation tools and detailed analysis of V. parahaemolyticus VP152 genome using PlasmidFinder, the genome of V. parahaemolyticus VP152 did not recover any plasmid sequence. Even though these genes were commonly found in plasmids, some of the Vibrio species including V. coralliilyticus and V. alginolyticus carry these genes in their chromosomes (Costa et al., 2015). Therefore, the resistant genes observed in V. parahaemolyticus VP152 are chromosome mediated.
The multidrug resistance profile seen in the phenotype and genes of V. parahaemolyticus VP152 genome illustrates how extensive antibiotics have been utilized in the aquaculture industry. The resistance phenotype observed in this strain could be triggered by the extensive use of permitted antibiotics in the Asian aquaculture industry namely oxytetracycline, tetracycline, quinolone, sulphonamides, and trimethoprim (Rico et al., 2012;Yano et al., 2014). The resistance toward third generation cephalosporins seen in V. parahaemolyticus VP152 would further hamper the treatment of Vibrio species infection in future. This situation is cause for concern, and warrants more stringent surveillance in the use of antibiotics, as well as the resultant antibiotic resistance in clinically important bacterial species. In summary, the whole genome sequence of V. parahaemolyticus VP152 will be useful in future studies to determine antimicrobial resistance and virulence attributes as well as mechanisms that enhance its environmental or host fitness.

Nucleotide Sequence Accession Numbers
This genome sequence data of VP152 strain sequenced under this study has been deposited in DDBJ/EMBL/GenBank under Accession No. LCUL00000000. The version described in this paper is the first version, LCUL01000000. The genome sequences data are available in FASTA, annotated GenBank flat file, graphical and ASN.1 formats.

AUTHOR CONTRIBUTIONS
The experiments, data analysis and manuscript writing were performed by VL and H-LS, while W-ST, N-SA, B-HG, K-GC, and L-HL provided vital guidance, technical support, and proofreading for the work. The research project was founded by L-HL.