Parahaemolyticus Strains Research Articles

Antibacterial Activity of Extracts from Dried and Fresh Herbal Plant (Phyllanthus amarus) Against Pathogens Causing Acute Hepatopancreatic Necrosis Disease (Ahpnd) in White Leg Shrimp (Litopenaeus vannamei) at Thua Thien Hue Province, Vietnam

The study aimed to determine extract yield (%), antibacterial activity, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts from dried and fresh herbal plants (Phyllanthus amarus) against Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in white leg shrimp (L. vannamei). The result showed that the extract yields of dry and fresh herbs reached 11.50% and 2.75%, respectively and the antibacterial activity of the two extracts both are good at concentrations from 250 to 1,000 mg/mL at the same bacterial density of 106 CFU/mL. Specifically, the diameter of the inhibition zone at 250; 500; 750 and 1,000 mg/mL concentration of dried herbal extracts reached 16.75±0.96; 18.50±1.29; 20.75±0.96 and 21.25±0.50 mm, while that of fresh herbal extracts reached 14.50±1.29; 16.25±0.50; 16.75±0.50 and 17.00±0.00 mm, respectively, with a statistically significant difference p<0.05. The result also showed that MIC values of dried and fresh extracts were defined at 125 mg/mL and 250 mg/mL, respectively and that MBC values of the extracts were 500 and 1,000 mg/mL respectively. The GC-MS analysis revealed that there were 19 natural compounds in the dried extract, in which Ethyl Linoleolate (C20H36O2) compound occupied the highest ratio (22.43%), while 2.3-Dihydro-3.5-dihydroxy-6-methy-4H-pyran-4-one (C6H8O4) was the lowest (0.24%).

Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis

The dynamic nature of Vibrio parahaemolyticus epidemiology has presented a unique challenge for disease intervention strategies. Despite the continued rise of disease incidence and outbreaks of vibriosis, as well as the global emergence of pandemic clones and serovariants with enhanced virulence, there is a paucity of molecular methods for the serotyping of V. parahaemolyticus strains to improve disease surveillance and outbreak investigations. We describe the development of a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the simultaneous identification of 11 clinically most common V. parahaemolyticus serotypes spanning a 10-year period. Through extensive sequence analyses using 418 genomes, specific primers and probes were designed for a total of 22 antigen gene targets for the O- and K- serogroups. Additionally, the toxR gene was incorporated into the assay for the confirmation of V. parahaemolyticus. All gene targets were detected by the assay and gave expected Tm values, without any cross reactions between the 11 clinically common serotypes or with 38 other serotypes. The limit of identification for all gene targets ranged from 0.1 to 1 ng/μL. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 1°C and <1% respectively, indicating a highly reproducible assay. A multicenter double-blind clinical study was conducted using the traditional V. parahaemolyticus identification workflow and the MLMA assay workflow in parallel. From consecutive diarrheal stool specimens (n = 6118) collected over a year at 10 sentinel hospitals, a total of 153 V. parahaemolyticus isolates (2.5%) were identified by both workflows. A total agreement (kappa = 1.0) between the serotypes identified by the MLMA assay and conventional serological method was demonstrated. This is the first molecular assay to simultaneously identify multiple clinically important V. parahaemolyticus serotypes, which satisfies the acute need for a practical, rapid and robust identification of V. parahaemolyticus serotypes to facilitate the timely detection of vibriosis outbreaks and surveillance.

Long-distance transmission of pathogenic Vibrio species by migratory waterbirds: a potential threat to the public health

A potential mechanism for the global distribution of waterborne pathogens is through carriage by the migratory waterbirds. However, this mode of transmission has yet been confirmed epidemiologically. Here, we conducted whole genome sequencing of Vibrio spp. collected from waterbirds, sediments, and mollusks in the estuary of the Liaohe River in China to investigate this transmission mode. We found that a V. parahaemolyticus strain isolated from a waterbird was clonally related to the other V. parahaemolyticus strains obtained from the sediments and mollusks, and three V. mimicus strains isolated from bird feces were genomically related to those found in the mollusks and upstream groundwater, suggesting that the bird-carried Vibrio strains were acquired through the direct predation of the local mollusks. Surprisingly, two bird-carried V. parahaemolyticus strains belonging to the same clone were identified in Panjin and Shanghai, which are over 1,150 km apart, and another two were found at two locations 50 km apart, further supporting that waterbirds are capable of carrying and disseminating these pathogens over long distances. Our results provide the first evidence of direct transmission from mollusks to waterbirds and confirm that waterbirds act as disseminating vehicles of waterborne pathogens. Effective surveillance of migratory waterbirds along their routes will be valuable for predicting future epidemics of infectious diseases.

Vibrio parahaemolyticus cqsA controls production of quorum sensing signal molecule 3-hydroxyundecan-4-one and regulates colony morphology.

In order to adapt to different environments, Vibrio parahaemolyticus employed a complicated quorum sensing system to orchestrate gene expression and diverse colony morphology patterns. In this study, the function of the putative quorum sensing signal synthase gene cqsA (VPA0711 in V. Parahaemolyticus strain RIMD2210633 genome) was investigated. The cloning and expression of V. parahaemolyticus cqsA in Escherichia coli system induced the production of a new quorum sensing signal that was found in its culture supernatant. The signal was purified by high performance liquid chromatography methods and determined to be 3-hydroxyundecan- 4-one by indirect and direct mass spectra assays. The deletion of cqsA in RIMD2210633 changed V. parahaemolyticus colony morphology from the classical 'fried-egg' shape (thick and opaque in the center, while thin and translucent in the edge) of the wild-type colony to a 'pancake' shape (no significant difference between the centre and the edge) of the cqsA deleted colony. This morphological change could be restored by complementary experiment with cqsA gene or the signal extract. In addition, the expression of opaR, a well-known quorum sensing regulatory gene, could be up-regulated by cqsA deletion. Our results suggested that V. parahaemolyticus used cqsA to produce 3-hydroxyundecan-4-one signal and thereby regulated colony morphology and other quorum sensing-associated behaviors.

Oxidative Disinfectants Activate Different Responses in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a prevalent seafoodborne enteropathogen that has become a global concern since the spread of its pandemic strain in 1996. This study investigates the responses of this pathogen to the oxidative disinfectants hydrogen peroxide, chlorine dioxide, and peracetic acid. Expression of the regulator genes oxyR and rpoS, determined by reverse transcription PCR, in V. parahaemolyticus wild-type, oxyR mutant, and rpoS mutant strains exhibited similar patterns in response to the tested oxidative disinfectants. The transcription of the rpoS gene was markedly enhanced in the oxyR mutant strain in the exponential phase. The expression of catalase KatE1 was tracked by using a LacZ fusion reporter in these strains. The experimental results revealed that KatE1 was a significant scavenger of hydrogen peroxide and peracetic acid in V. parahaemolyticus, and RpoS may partially compensate for the regulatory role of OxyR in the oxyR mutant strain. In contrast to its responses to hydrogen peroxide and paracetic acid, KatE1 was not the primary scavenger of chlorine dioxide in these V. parahaemolyticus strains. This study shows that these disinfectants activated a basic oxidative response in this pathogen with different features.

Antibacterial and antibiofilm activity of coenzyme Q0 against Vibrio parahaemolyticus

Coenzyme Q0 (CoQ0) demonstrates antineoplastic and anti-inflammatory properties; however, its antimicrobial and antibiofilm activities against Vibrio parahaemolyticus have not been extensively examined. Thus, the aim of the current study was to investigate the antimicrobial and antibiofilm activities, along with the underlying mechanism, of CoQ0 against V. parahaemolyticus. The minimum inhibitory concentrations (MICs) of CoQ0 against eight V. parahaemolyticus strains ranged from 4 to 16 μg/mL, while V. parahaemolyticus treated with CoQ0 showed a longer lag phase compared with the untreated control. CoQ0 was effective in destroying cell membrane integrity and inducing morphological changes in the cell. In addition, an antibiotic sensitivity assay showed that CoQ0 caused cell damage associated with DNA, RNA, and proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the action of CoQ0 on destroying proteins or inhibiting proteins synthesis of V. parahaemolyticus. CoQ0 effectively inactivated V. parahaemolyticus in mature biofilm on stainless steel surfaces and inhibited V. parahaemolyticus in fresh shrimp. These findings suggested that CoQ0 could be used as a natural antimicrobial and antibiofilm agent to control V. parahaemolyticus in the seafood supply chain, thereby preventing seafood-associated infections.

A new emerging serotype of Vibrio parahaemolyticus in China is rapidly becoming the main epidemic strain

ObjectivesDuring surveillance, we found a new type of Vibrio parahaemolyticus named ‘O4:KUT-recAin’ and studied the phenotypic, pathogenic and epidemiological characteristics of O4:KUT-recAin. MethodsV. parahaemolyticus were isolated from acute diarrhoeal patients in coastal hospitals of China. Serum agglutination test, specific PCR assay, growth curves under different conditions and rabbit diarrhoeal models were using to characterize O4:KUT-recAin. ResultsThe O4:KUT-recAin strain has a new type of K antigen and a 25 043-bp-large fragment encoding 20 proteins inserted in the housekeeping gene recA. Retrospective analysis found that only one O4:KUT-recAin strain was detected in 563 V. parahaemolyticus strains in 2014; then the proportion increased rapidly and reached 17.8% (105/590) in 2016 and 31.1% (224/721) in 2017, making O4:KUT-recAin the second dominant serotype following O3:K6. O4:KUT-recAin strains (100%, 14/14) exhibited increased acid resistance and could reproduce in medium at pH 4.9, while 92.9% (13/14) of the O3:K6 strains could not grow at this pH value. O4:KUT-recAin could cause diarrhoea and small intestinal tissue lesions in infant rabbits, but its diarrhoeal (93.1%, 27/29) and mortality (78.6%, 22/28) rates were slightly lower than those of O3:K6 (100% 16/16, 100% 16/16). Based on diarrhoea patients, there were no significant differences in the two groups for most clinical symptoms and laboratory results, except media age, haemoglobin and the number of red blood cells in stool samples. ConclusionsO4:KUT-recAin had enhanced acid resistance, was capable of causing infectious diarrhoea in both rabbits and humans, and has become widespread during a short period of time in China.

Rapid and accurate detection of viable Vibrio parahaemolyticus by sodium deoxycholate-propidium monoazide-qPCR in shrimp

Vibrio parahaemolyticus is an important human pathogen causing a variety of life-threatening diseases and is widely distributed in marine and estuarine environments. The objective of this study was to develop a sensitive, specific, and accurate method by using sodium deoxycholate (SD)-propidium monoazide (PMA)-qPCR (SD-PMA-qPCR) for selective detection of viable V. parahaemolyticus cells in shrimp. A qPCR assay was developed by targeting a unique fragment in the toxR gene in V. parahaemolyticus. The qPCR assay demonstrated superior specificity (100%) on V. parahaemolyticus strains (n = 70) and non-V. parahaemolyticus strains (n = 37) examined in the inclusivity and exclusivity tests; and the limit of detection (LOD) of the assay reached 5 × 101 CFU/ml. To remedy the drawback of PCR, SD-PMA treatment was incorporated with the qPCR assay. The optimized PMA treatment conditions were determined as follows, 40 μM PMA and 3-min light exposure at 40 w. The maximum removal efficiency of non-viable cell DNA was achieved by an optimal amplicon (262 bp) of qPCR for PMA treatment with SD at an optimal concentration (0.02% wt/vol). Furthermore, we have applied the SD-PMA-qPCR assay for detection of viable V. parahaemolyticus cells in shrimp. Consequently, the SD-PMA-qPCR assay could accurately detect as low as 5 × 101 CFU/g of V. parahaemolyticus in the presence of a large number of non-viable cells (5 × 107 CFU/g) in spiked shrimp with a 4-h enrichment. In summary, the qPCR assay based on the target gene, toxR, is sensitive and specific; treatment of non-viable cells with SD and PMA improved the removal efficiency of DNA of non-viable cells; and the SD-PMA-qPCR assay developed in this study is a specific and accurate detection method for viable V. parahaemolyticus, providing an effective and rapid means for detection of viable V. parahaemolyticus in food.

Genomic characterization of Vibrio parahaemolyticus from Pacific white shrimp and rearing water in Malaysia reveals novel sequence types and structural variation in genomic regions containing the Photorhabdus insect-related (Pir) toxin-like genes.

The Malaysian and global shrimp aquaculture production has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND) typically caused by Vibrio parahaemolyticus harboring the pVA plasmid containing the pirAVp and pirBVp genes, which code for Photorhabdus insect-related (Pir) toxin. The limited genomic resource for V. parahaemolyticus strains from Malaysian aquaculture farms precludes an in-depth understanding of their diversity and evolutionary relationships. In this study, we isolated shrimp-associated and environmental (rearing water) V. parahaemolyticus from three aquaculture farms located in Northern and Central Malaysia followed by whole-genome sequencing of 40 randomly selected isolates on the Illumina MiSeq. Phylogenomic analysis and multilocus sequence typing (MLST) reveal distinct lineages of V. parahaemolyticus that harbor the pirABVp genes. The recovery of pVA plasmid backbone devoid of pirAVp or pirABVp in some V. parahaemolyticus isolates suggests that the toxin genes are prone to deletion. The new insight gained from phylogenomic analysis of Asian V. parahaemolyticus, in addition to the observed genomic instability of pVa plasmid, will have implications for improvements in aquaculture practices to diagnose, treat or limit the impacts of this disease.

First detection of the plasmid-mediated colistin resistance gene mcr-1 in virulent Vibrio parahaemolyticus

The plasmid-mediated colistin resistance gene mcr-1 has been identified in various Enterobacteriaceae species, which poses a great challenge to the public health. The present study aimed to investigate the prevalence of mcr-1 in Vibrio parahaemolyticus isolated from food samples in China, and to conduct a comprehensive analysis on the molecular characterization of V. parahaemolyticus isolate carrying mcr-1-harboring plasmid. A total of 646 V. parahaemolyticus strains isolated from 2531 food samples collected in retail markets in 34 different cities in China were screened for colistin resistance. Of the 646 V. parahaemolyticus isolates tested, 25 (2.5%) exhibited colistin resistance. The mcr-1 gene was detected in one colistin-resistant V. parahaemolyticus isolate, VP181, obtained from a shrimp sample collected in Hong Kong. The mcr-1 gene was located on a transferable IncX4 plasmid with size of ~40 kb. A Class A β-lactamase gene, blaCARB-17 and the plasmid-mediated quinolone resistance (PMQR) gene qnrVC5 were detected in the mcr-1-positive V. parahaemolyticus isolate VP181. Virulence gene assays indicated that tdh was detected in VP181 by PCR. This is the first report of the occurrence of plasmid-encoded mcr-1 in virulent V. parahaemolyticus strain. Our findings indicate horizontal transfer of this gene to non-Enterobacteriaceae gram-negative bacteria, which warrants further investigation because of the public health threat it poses.

Characteristics of and germicidal efficacy at different temperatures against Vibrio parahaemolyticus

Objective To study the biological characteristics of, antibiotic resistance in and germicidal efficacy at different temperatures against Vibrio parahaemolyticus (V.parahaemolyticus) strains isolated from different sources in order to provide reference for clinical treatment and prevention. Methods Stool samples from patients with diarrhea and small seafood product specimens from markets were collected and analyzed with biochemical identification method, serotyping analysis, drug susceptibility test and germicidal efficacy test at different temperatures. Results There were 107 V. parahaemolyticus strains isolated from 1 166 stool samples of patients with foodborne diarrhea with a positive rate of 9.18% and 42 from 72 seafood product samples with a positive rate of 58.30%. V. parahaemolyticus strains isolated from the foodborne diarrhea cases were divided into eight serogroups and among them, O3 and O4 were the predominant serogroups, accounting for 49.53% and 34.58%, respectively. Most of the O antigens in small seafood products belonged to serogroups of O4, O1 and O3, and four strains of O3∶K6 were isolated. Results of the drug susceptibility test showed that both of the clinical isolates and marine product isolates were highly resistant to ampicillin and the drug resistance rates were 94.39% and 88.10%, respectively. Antibiotic-sensitive strains to monocyclic β-lactams, aminoglycosides, quinolones, carbapenems, tetracyclines and sulfonamides accounted for over 90.00% or even nearly 100.00%. There were 17 (15.89%) clinical strains and three (7.14%) marine product isolates resistant to three or more kinds of antibiotics. At 80℃, the bactericidal rate for marine product isolates was 85.71% in 60 s and reached 100.00% in 90 s. At 90℃ and 100℃, these isolates could be completely killed in 40 s and 30 s. It took 120 s, 90 s and 50 s to kill clinical isolates at 80℃, 90℃ and 100℃, respectively. Conclusions This study systematically analyzed and compared the drug-resistant phenotypes of and the bactericidal efficacy at different temperatures on V. parahaemolyticus strains isolated from clinical samples and marine products. It would provide reference for preventing and controlling the spread of V. parahaemolyticus and hospital infection and for studying treatment strategies. Key words: Vibrio parahaemolyticus; Antibiotic susceptibility; Germicidal efficacy; Temperature

The Fate of Bacteria in Human Digestive Fluids: A New Perspective Into the Pathogenesis of Vibrio parahaemolyticus.

Vibrio parahaemolyticus causes the most seafood-attributed gastroenteritis outbreaks worldwide and studies on its pathogenesis during passage through the human digestive fluids are limited. An in vitro continuous model system mimicking passage through saliva, gastric and intestinal fluid was used to study the survival, morphology and virulence-related gene expression of a total of sixty pathogenic, and non-pathogenic V. parahaemolyticus strains. The changes to these three characteristics for the sixty V. parahaemolyticus strains were minimal on passage through the saliva fluid. No V. parahaemolyticus strains survived passage through gastric fluid with low pH values (2.0 and 3.0) and the cells, examined microscopically, were severely damaged. However, when the pH of gastric fluid increased to 4.0, the bacterial survival rate was 54.70 ± 1.11%, and the survival rate of pathogenic strains was higher when compared to non-pathogenic strains. Even though the bactericidal effect of intestinal fluid was lower than gastric fluid, virulence-related gene expression was enhanced in the intestinal fluid. Seafood matrices can significantly raise the pH level of gastric fluid and thus aid the survival of V. parahaemolyticus through passage from human gastric acid and progression of pathogenesis in the intestinal fluid. We confirmed these phenomena in the in vitro continuous digestion model.

Antimicrobial residues, non-typhoidal Salmonella, Vibrio spp. and associated microbiological hazards in retail shrimps purchased in Ho Chi Minh city (Vietnam)

We investigated antimicrobial residues, non-typhoidal Salmonella (NTS), Vibrio spp. and their associated antimicrobial resistance (AMR), in shrimps locally purchased in Ho Chi Minh City (Vietnam). In addition, we investigated the relationship between AMR in NTS, Vibrio spp. and antimicrobial residue in the same sample. A total of 40 samples of shrimp heads/shells from different retail sources was cultured using ISO 6579–1:2017 (NTS) and ISO/TS 21872–1:2007 (Vibrio spp.). Phenotypic antimicrobial susceptibility was investigated using Vitek (NTS, 34 antimicrobials) and disk diffusion (Vibrio spp., 12 antimicrobials). A total of 9 (22.5%) samples contained antimicrobial residue, including tetracyclines, fluoroquinolones, sulfonamides and macrolides (in 7.5%, 7.5%, 2.5% and 2.5% of samples, respectively). Shrimp samples from supermarkets had a higher prevalence of antimicrobial residue than those purchased in street markets (50% vs. 13.3%) (p = 0.049). A total of 30 (75%) samples were contaminated with NTS. All samples contained Vibrio spp., with V. parahaemolyticus being most common (87.5% samples). A total of 58.9% NTS isolates were multidrug resistant. With regards to the highest priority, critically important antimicrobials, the highest resistance corresponded to quinolones (14.4–47.8%), followed by 3rd and 4th generation cephalosporins (3.3–7.8%). Vibrio spp. isolates were characterised by their high resistance against ampicillin (82.7%) and 3rd generation cephalosporins (8.3–16.5%). Extended Spectrum Beta-Lactamase (ESBL) activity was detected in 28.1% V. parahaemolyticus isolates. Half of ESBL-positive V. parahaemolyticus strains harboured blaCTX-M1. We found an association between the presence of residues and the number of resistances for NTS (p = 0.075) and Vibrio spp. isolates (p = 0.093) from the same sample. These findings suggest that the presence of residues may contribute to the selection of AMR in foodborne pathogens in shrimps. Authorities should strengthen policies aiming at restricting inappropriate antimicrobial usage in shrimp farming, and step up monitoring of antimicrobial residues and food-borne pathogens at retail in Vietnam.

Genomic Features ofVibrio parahaemolyticusfrom Lebanon and Comparison to Globally Diverse Strains by Whole-Genome Sequencing

Gastroenteritis is a disease that can be caused by virulent strains of Vibrio parahaemolyticus in humans upon the consumption of contaminated seafood. In summer 2017, a sudden increase in the number of patients suffering from gastroenteritis due to a V. parahaemolyticus infection was observed at the Middle East Institute of Health University Hospital in Lebanon. The aim of this study was to analyze the isolates recovered from stool specimens, and to compare them using different phenotypic assays, genomic profiling techniques, and whole-genome sequencing, to achieve a better understanding of the current V. parahaemolyticus strains available in Lebanon. Virulence potential was analyzed based on the detection of the hemolysins: thermostable direct hemolysin (tdh), thermostable direct hemolysin-related hemolysin (trh), and thermolabile hemolysin (tlh). Resistance was determined by testing antibiotic susceptibility and performing PCR assays for β-lactamases and quinolone resistance determinants. Genetic relatedness was verified by multilocus sequence typing, pulsed-field gel electrophoresis, and whole genome-based single nucleotide polymorphism analysis. All of the isolates had the tdh+, trh-, group-specific PCR+ genotype, which is a characteristic of the O3:K6 pandemic clone. The isolates were resistant to ampicillin (100%), ceftazidime (86%), ticarcillin (14%), and amikacin (14%), belonged to the sequence type ST3, and had very similar phylogenetic fingerprints. The isolates undertaken in this study exhibited almost identical resistance, virulence, and phylogenetic patterns, confirming an outbreak linked to the spread of the pandemic O3:K6 serotype in the country.

Clonal relationship among Vibrio parahaemolyticus isolated from Mediterranean mussels (Mytilus galloprovincialis) and grooved carpet shells (Ruditapes decussatus) harvested in Sardinia (Italy)

The aim of the present study was to investigate the genetic variability of Vibrio parahaemolyticus strains isolated from naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) and Grooved carpet shells (Ruditapes decussatus) from three harvesting areas of Sardinia (Italy) using a combination of different typing methods: traditional phenotypic systems and molecular techniques. Ninety-nine putative V. parahaemolyticus strains isolated from shellfish collected before and after purification were included in the study. Seventy-two isolates were confirmed as V. parahaemolyticus and were submitted to REP, ERIC and BOX PCRs. The combined dendrogram showed the similarity of the data set of the three typing methods and demonstrates how the different techniques grouped the strains in two clusters in accordance with each singular dendrogram. Several strains rendered a unique pattern regardless of the typing method, which indicates the high discriminatory power of the methods. Moreover, the use of multiple typing methods allowed a more accurate characterization of the genetic profiles of isolates and the identification of clones hardly revealed through the common techniques. The intraspecific typing of environmental V. parahaemolyticus can be of great interest in order to recognize clonal relationships between environmental contamination, foodborne disease, and geographical/temporal distribution of this pathogen. The comparative analysis focusing on the obtained genetic profiles supports the possibility for typing methods to discriminate strains with similar phenotypic profile, identifying the level of genetic correlation among the strains and the presence of genetic clones.

Isolation, detection of virulence genes, antibiotic resistance genes, plasmid profile, and molecular typing among Vibrio parahaemolyticus isolated in Malaysian seawater from recreational beaches and fish.

Background and Aim:Despite the importance of the global emergence of Vibrio parahaemolyticus infections worldwide, there has been scanty information on its occurrence in Malaysian seawaters and fish. This study aimed to determine the occurrence of V. parahaemolyticus isolates using polymerase chain reaction targeted at toxin operon gene, thermostable direct hemolysin (tdh), and tdh-related hemolysin genes and to determine antibiotic resistance pattern, genes, and plasmid profile of V. parahaemolyticus from Malaysian seawaters and fish.Materials and Methods:Samples were collected from four recreational beaches in Malaysia (Port Klang; Bachok; Port Dickson; and Mersing). Thiosulfate-citrate-bile salts-sucrose (TCBS) agar and chromogenic Vibrio agar were used for isolation and identification. Colonies with yellow color on TCBS and green color on chromogenic vibrio (CV) agar were considered to be V. parahaemolyticus and they were subjected to biochemical tests. All V. parahaemolyticus isolates were further subjected to identification using seven specific gene markers.Results:Seventy-three Vibrio isolates were recovered. Only one gene thermostable direct hemolysin (tdh) from seawater isolates of Vibrio has high virulence gene percentage (95.23%). Two genes alkaline serine protease (asp) and (tdh) had high percentage of virulence (83.87% and 80.64%, respectively) from fish. Comparatively, fish isolates have a higher virulence percentage compared to seawater isolates. Only gene streptomycin resistance B (strB) from seawater had 100% of the resistance genes. All isolates were multi-antibiotic resistant. Seventeen antibiotic resistance patterns were observed. The isolates had plasmids of varying sizes ranging from 2.7 kb to 42.4 kb. Dendrogram based on antibiotic resistance patterns of V. parahaemolyticus isolates discriminated the isolates into three clusters.Conclusion:This study demonstrated the occurrence of pathogenic, multi-antibiotic-resistant V. parahaemolyticus strains in Malaysian coastal waters and fish, and this could constitute potential public health risks.

Notch1a can widely mediate innate immune responses in zebrafish larvae infected with Vibrio parahaemolyticus

The Notch signaling pathway is known to regulate innate immunity by influencing macrophage function and interacting with the Toll-like receptor (TLR) signaling pathway. However, the comprehensive role of the Notch signaling pathway in the innate immune response remains unknown. To assess the function of Notch1a in immunity, we examined the innate immune responses to Vibrio parahaemolyticus strain Vp13 of wild-type (WT) and notch1a-/- zebrafish larvae generated using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system. The median lethal dose (LD50) of V. parahaemolyticus was significantly lower in notch1a-/- larvae than in WT larvae 3 days post fertilization (dpf). Transcriptome data analysis revealed 359 significantly differentially expressed genes (DEGs), including 246 significantly down-regulated genes and 113 significantly up-regulated genes, in WT infected groups compared with WT control groups. In contrast, 986 significantly DEGs were found in notch1a-/- infected groups compared with notch1a-/- control groups, of which 82 genes were significantly down-regulated and 904 genes were significantly up-regulated. These DEGs belonged to the tumor necrosis factor (TNF), complement, nuclear factor kappa B (NF-κB), cathepsin, interleukin (IL), chemokine, serpin peptidase inhibitor, matrix metallopeptidase, innate immune cells, pattern recognition receptor (PRR), and other cytokine families. Our results indicate that Notch1a plays roles in inhibiting many immunity-related genes and could comprehensively mediate the innate immune response by regulating TLRs, nucleotide-binding-oligomerization-domain-like receptors (NLRs), lectins, complement, ILs, chemokines, TNF, cathepsin, and serpin. Further studies are required to understand the specific mechanism of Notch1a in innate immunity.

Antibiotic resistance pattern and its correlation to the presence of tdh gene and CRISPR-Cas system in Vibrio parahaemolyticus strains isolated from seafood

Vibrio parahaemolyticus is a pathogen native to the aquatic environment. In this study, 46 environmental V. parahaemolyticus isolates were subjected to a correlational analysis to find the association between their antimicrobial susceptibility pattern, prevalence of CRISPR-Cas system and thermostable direct hemolysin (tdh) gene. Antibiotic resistance profiling against eleven antibiotics revealed the isolates to be multidrug resistant. Isolates exhibited highest resistance to vancomycin (97.8%) followed by ampicillin (91.3%), cefotaxime (69.6%), ceftazidime/clavulanic acid (54.4%), ceftazidime (45.7%) and gentamicin (39.1%). CRISPR loci and tdh gene were detected in 47.83 and 58.7% of strains respectively. No significant correlation was observed between antibiotic resistance to presence of CRISPR, except in the case of gentamicin wherein, a negative correlation was seen (r=-0.272, p&lt;0.10). Similarly, tdh did not correlate to antibiotic resistance. Seventeen strains in this study harboured the CRISPR loci as well as tdh gene, the association of which was found to be statistically significant.

Genetic Characterization of bla CTX-M-55 -Bearing Plasmids Harbored by Food-Borne Cephalosporin-Resistant Vibrio parahaemolyticus Strains in China.

This study aims to investigate and compare the complete nucleotide sequences of the multidrug resistance plasmids pVb0267 and pVb0499, which were recovered from foodborne Vibrio parahaemolyticus isolates, and analyze the genetic environment of blaCTX–M–55 to provide insight into the dissemination mechanisms of this resistance element. Analysis of the sequences of plasmids pVb0267 (166,467 bp) and pVb0499 (192,739 bp) revealed that the backbones of these two plasmids exhibited a high degree of similarity with pR148, a recognized type 1a IncC plasmid recovered from Aeromonas hydrophila (99% identity). The resistance genes, found in both plasmids, included qacH, aadB, arr2, blaOXA–10, aadA1, sul1, tet(A), and blaCTX–M–55, which were mostly arranged in a specific region designated ARI-A. Plasmid pVb0499 was found to possess a larger size of ARI-A than pVb0267, which lacked a mer determination region, a qnr A segment, an aacC3 gene and several mobility-encoding genes. Comparative analysis of resistance island (RI) of these plasmids and others revealed the potential evolution route of these RI sequences. In conclusion, plasmids harboring the blaCTX–M–55 gene has been recovered in Vibrio parahaemolyticus strains of food origin. It is alarming to find that IncC plasmids harboring resistance islands are disseminating in aquatic bacterial strains. The continuous evolution of resistance genes in conjugative plasmid in aquatic bacteria could be due to bacterial adaption to aquaculture environment, where antibiotics were increasingly used.

Vibrio parahaemolyticus and Vibrio harveyi causing Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei (Boone, 1931) isolated from Malaysian shrimp ponds

Acute hepatopancreatic necrosis disease (AHPND) first emerged as a new shrimp disease in 2009 that heavily affected shrimp industry leading to global economic losses. The etiological agent was previously identified as Vibrio parahaemolyticus that carries a plasmid containing toxins (PirA and PirB). However, recent researches revealed that V. parahaemolyticus is not the only bacterial species capable of causing AHPND, thus this study screened on bacterial strains with AHPND toxins from Penaeus vannamei shrimps in Malaysia. Out of the 86 isolated total strains, 12 AHPND positive strains were arbitrarily selected and were evaluated in in vivo assay using Artemia franciscana as a model organism. All the 12 AHPND positive strains with PirA and PirB genes demonstrated significant mortalities (P < 0.05) of A. franciscana compared to the negative control. The 12 AHPND positive strains were identified using molecular methods of 16S rRNA, RctB and RpoD region amplifications belonged to the Harveyi clade and were closely related to V. parahaemolyticus and Vibrio harveyi. Further test showed that the yellow colony V. harveyi strain BpShHep24 was found to be more virulent than the green colony V. parahaemolyticus strain BpShHep31 in shrimp P. vannamei challenge test. Histological examination of shrimp hepatopancreas challenged with yellow colony V. harveyi strain BpShHep24 showed massive sloughing of hepatopancreas tubules of epithelial cells into the lumen, haemocyte infiltrations, proximal-to-distal lesion of hepatopancreas and collapsed tubule epithelia within 24 h.