Thermostable Direct Hemolysin Gene Research Articles

Fitness and transcriptomic analysis of pathogenic Vibrio parahaemolyticus in seawater at different shellfish harvesting temperatures

ABSTRACT To better characterize the population dynamics of Vibrio parahaemolyticus ( Vp ) containing different virulence genes, two Vp strains were inoculated into seawater separately and incubated at temperatures (10°C and 30°C) mimicking winter and summer pre-harvest shellfish rearing seasons. The cellular responses of two Vp strains, one containing the thermostable direct hemolysin ( tdh +) gene and the other one containing tdh -related hemolysin ( trh +) gene, were studied at the transcriptomic level. Results showed that, at 30°C, tdh + and trh + strains reached 6.77 ± 0.20 and 6.14 ± 0.07 log CFU/mL, respectively, after 5 days. During this time, a higher growth rate was observed in the tdh + strain than in the trh + strain. When being kept at 10°C, both Vp strains persisted at ca. 3.0 log CFU/mL in seawater with no difference observed between them. Survival and growth models were then established based on the Baranyi equation. The goodness-of-fit scores ranged from 0.674 to 0.950. RNA sequencing results showed that downregulated central energy metabolism and weakened degradation of branched-chain amino acid were observed only in the trh + strain not in the tdh + strain at 30°C. This might be one reason for the lower growth rates of the trh + strain at 30°C. Histidine metabolism and biofilm formation pathways were significantly downregulated in both strains at 10°C. No significant difference was observed for virulence-associated gene expression between 10°C and 30°C, regardless of the strains. IMPORTANCE Given the involvement of Vibrio parahaemolyticus ( Vp ) in a wide range of seafood outbreaks, a systematical characterization of Vp fitness and transcriptomic changes at temperatures of critical importance for seafood production and storage is needed. In this study, one of each virulent Vp strain ( tdh + and trh +) was tested. While no difference in survival behavior of the two virulent strains was observed at 10°C, the tdh + strain had a faster growth rate than the trh + strain at 30°C. Transcriptomic analysis showed that a significantly higher number of genes were upregulated at 30°C than at 10°C. The majority of differentially expressed genes of Vp at 30°C were annotated to functional categories supporting cellular growth. At 10°C, the downregulation of the biofilm formation and histidine metabolism indicates that the current practice of storing seafood at low temperatures not only protects seafood quality but also ensures seafood safety.

Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus.

Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39-100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 101, 102, and 103 CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.

First isolation of a trh positive V. alginolyticus from Atlantic bluefin tuna (Thunnus thynnus) farmed in Turkey

Popularity of sushi-sashimi (raw-almost raw fish service) is increasing around the globe, and Atlantic bluefin tuna is among the most frequently used fish in these dishes. In this study, trh (Thermostable direct hemolysin-related hemolysin gene) positive Vibrio alginolyticus was isolated from the internal organs of two tuna sent to the laboratory from a farm in Aegean region of Turkey in 2014. Isolation was done by conventional microbiological methods and Vitek 2 Compact fully automatic identification device was used for identification. Isolate confirmation and investigation of trh-tdh (Thermostable direct hemolysin) gene existence were carried out by a Polymerase Chain Reaction (PCR) method. Two V.alginolyticus isolates from two tuna were found to be trh positive and tdh negative. trh is one of the important genes associated with pathogenicity in the Vibrio genus, and raw consumption of tuna infected with trh positive V.alginolyticus can cause serious food poisoning. The aim of this study is to report first isolation of trh positive Vibrio alginolyticus from two bluefin tuna cultured in the Aegean sea, to draw attention to possible dangers in raw-almost raw fish consumption and supply information for further epidemiological studies. However, more samples are necesary for mentioning the prevalence of high virulence food pathogens in tuna. However, due to their economic value, multiple tuna samples are rarely sent to laboratories for diagnostic purposes.

Prevalence, antimicrobial resistance and genetic characterization of Vibrio parahaemolyticus isolated from retail aquatic products in Nanjing, China

Vibrio parahaemolyticus, is one of the most frequently reported pathogenic microorganisms that causes foodborne illnesses worldwide. The aims of the current study were to determine the prevalence, virulence genes, antimicrobial resistance, biofilm formation ability (BFA) and genetic characterization of V. parahaemolyticus recovered from retail aquatic products in Nanjing, China. There were 131 samples (71.6%) that tested positive for V. parahaemolyticus. The thermostable direct hemolysin-related hemolysin (trh) gene was found in two isolates (1.5%). Antimicrobial susceptibility tests showed that 46.6% of isolates were multidrug resistant. High resistance was observed to ampicillin (100%), cephalosporin (99.2%), trimethoprim/sulfamethoxazole (38.2%) and tetracycline (16.0%). Ten resistance patterns were found. The crystal violet staining assay showed that 35.1% had strong BFA, and 52.7% had intermediate BFA; notably, five (3.8%) extremely strong BFA strains were obtained from wet markets. According to whole genome sequencing analysis of 59 randomly selected isolates, 46 sequence types (STs) were identified, including 22 novel STs, and ST1042 was the dominant sequence type. It is clear that the V. parahaemolyticus population exhibits a high level of genetic variation. Our findings provide comprehensive insight into the prevalence and phylogenetic relationship of V. parahaemolyticus in aquatic products, suggesting potential hazards to consumers in Nanjing.

Determination of tdh and trh Positive Vibrio alginolyticus Isolates from Black Mussel (Choromytilus meridionalis) in Aegean Sea coast of Turkey

Abstract: Vibrio alginolyticus is one of the important pathogens, especially found in bivalve mollusks and food poisoning in humans. The severity of food poisoning is directly proportional to the virulence genes of V. alginolyticus. Tdh-Related Hemolysin (trh) and Thermostable Direct Hemolysin (tdh) genes have an important place among the virulence genes found in V. alginolyticus. In this research, 17 V. alginolyticus were isolated from 17 orders (80.95%) of 21 sets of black mussels (Choromytilus Meridionalis) samples purchased from local divers in İzmir and Balıkesir regions. While trh gene was detected in 7 (42.17%) of 17 isolates, tdh gene was found in 6 (35.29%) and both trh and tdh genes were found in 2 (11.76%) isolates; no trh or tdh gene was found in 2 isolates (11.76%). The results of the study are also important in terms of public health. Black mussel is a product that is mainly consumed in coastal areas in Turkey and is mostly sold uncontrolled by mussel sellers. Vibrios with virulence genes can cause food poisoning, especially in summer. In addition, V. alginolyticus may be a tdh-trh reservoir for other vibrio species. To clarify this, more detailed research should be done with other vibrio species and other bivalve species.

Vibrio parahaemolyticus O10:K4: An Emergent Serotype with Pandemic Virulence Traits as Predominant Clone Detected by Whole-Genome Sequence Analysis - Beijing Municipality, China, 2021.

IntroductionVibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen which causes gastroenteritis in humans, especially the O3:K6 pandemic clone which is still a prominent serotype in Beijing, China. In this study, we observed a novel serotype O10:K4 isolated from clinical diarrhea cases, which became the most prevalent clone in 2021. Methods73 clinical isolates were collected through sentinel hospitals’ surveillance in 2021. Serum agglutination testing and antimicrobial susceptibility testing were conducted. Whole genome sequencing was applied to characterize 73 V. parahaemolyticus strains and complete phylogenetic analysis. ResultsSeven serotypes were identified among 73 strains. O10:K4 was the most common serotype (83.6%), followed by O2:KUT, O4:KUT, and O1:KUT. Multilocus sequence typing divided the 73 isolates into 10 sequence types (STs) with ST3 as the most prevalent, which covered all O10:K4 strains. Most isolates were sensitive to common antimicrobial agents apart from colistin. All the O10:K4 isolates were positive for the thermostable direct hemolysin gene, toxRS/new, andorf8, and negative for the TDH-related hemolysin gene. The whole genome sequencing-single nucleotide polymorphism phylogenetic analysis revealed O10:K4 strains formed a main genetic lineage, which was genetically distinct from other serotypes. We also demonstrated the presence of two type III secretion system genes (T3SS1 and T3SS2) and β lactamase resistance gene blaCARB-22 in all O10:K4 strains. ConclusionsThe study confirmed the emergence of V. parahaemolyticus O10:K4 possessing virulence factors similar to the O3:K6 pandemic clone, which may have enabled them to become prevalent in Beijing, China.

Genetic Basis of High-Pressure Tolerance of a Vibrio parahaemolyticus Mutant and Its Pathogenicity.

Foodborne pathogens with high-pressure processing (HPP) tolerance and their pathogenicity have gained considerable attention in the field of food safety. However, tolerance to pressure treatment varies among microorganisms and growth phases, and the mechanism by which Vibrio parahaemolyticus can become tolerant of HPP is currently not known. In this study, 183 strains of V. parahaemolyticus were isolated from seafood products, and one strain, C4, carried a thermostable direct hemolysin (tdh) gene. A strain, N11, which was acquired from the C4 strain through adaptive laboratory evolution under HPP stress, could tolerate up to 200 MPa for 10 min. Compared with the C4 strain, the catalase and Na+/K+-ATPase activities in N11 strain were increased by about 2–3 times, and the cells maintained an intact cell membrane structure under HPP treatment. As shown by murine infection trials, the C4 and N11 strains impacted the physiological activities of mice and damaged liver and spleen cells. Comparative genomic analysis showed that 19 nucleotides were mutated in the N11 strain, which led to sustained high expression of mlaC and mlaD genes in this strain. Knockout of these genes confirmed that they were involved in the high-pressure stress response, and also related to pathogenicity of V. parahaemolyticus. Thereby, our findings revealed a HPP tolerance mechanism of V. parahaemolyticus, and the high-pressure-tolerant strain still retained pathogenicity in mice with skin and fur pleating and lethargy, indicating the pressure-tolerant foodborne pathogens present health risks.

Characterization and Analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Pandemic and Non-Pandemic Vibrio parahaemolyticus Isolates from Seafood Sources.

Vibrio parahaemolyticus is one of the significant seafood-borne pathogens causing gastroenteritis in humans. Clustered regularly interspaced short palindromic repeats (CRISPR) are commonly detected in the genomes of V. parahaemolyticus and the polymorphism of CRISPR patterns has been applied as a genetic marker for tracking its evolution. In this work, a total of 15 pandemic and 36 non-pandemic V. parahaemolyticus isolates obtained from seafood between 2000 and 2012 were characterized based on hemolytic activity, antimicrobial susceptibility, and CRISPR elements. The results showed that 15/17 of the V. parahaemolyticus seafood isolates carrying the thermostable direct hemolysin gene (tdh+) were Kanagawa phenomenon (KP) positive. The Multiple Antibiotic Resistance (MAR) index ranged between 0.1 and 0.4, and 45% of the isolates have an MAR index ≥ 0.2. A total of 19 isolates were positive for CRISPR detection, including all tdh+ trh− isolates, two of tdh− trh+, and each of tdh+ trh+ and tdh− trh−. Four spacer types (Sp1 to Sp4) were identified, and CRISPR-positive isolates had at least one type of spacer homolog to the region of Vibrio alginolyticus megaplasmid. It is of interest that a specific CRISPR profile and spacer sequence type was observed with correlations to the hemolysin genotype (tdh/trh). Thus, these provide essential data on the exposure of foreign genetic elements and indicate shared ancestry within different genotypes of V. parahaemolyticus isolates.

Antibiotic resistance and determination of resistant genes among cockle (Anadara granosa) isolates of Vibrio alginolyticus.

A total of 24 strains of Vibrio alginolyticus were isolated from cockles (Anadara granosa) and identified for VibA and gyrB genes. All V. alginolyticus isolates were then tested against nine different antibiotics. In this study, the highest percentage of antibiotic resistance was obtained against penicillin (37.50%), followed by ampicillin, vancomycin (12.50%) and erythromycin (8.33%). All of V. alginolyticus isolates were susceptible against streptomycin, kanamycin, tetracycline, chloramphenicol and sulfamethoxazole. Polymerase chain reaction (PCR) assay has confirmed the presence of four antibiotic resistance genes of penicillin (pbp2a), ampicillin (blaOXA), erythromycin (ermB) and vancomycin (vanB). Out of 24 V. alginolyticus isolates, 2 isolates possessed the tdh-related hemolysin (trh) (strains VA15 and VA16) and none for the thermostable direct hemolysin (tdh) gene. Both strains of the tdh-related hemolysin (trh) were susceptible to all antibiotics tested. The multiple antibiotic resistance (MAR) index ranging between 0.2 and 0.3 with 5 antibiograms (A1-A5) was observed. Combination of enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and antibiotic resistance indicated 18 genome types which showed genetic heterogeneity of those V. alginolyticus isolates. The results demonstrated the presence of V. alginolyticus strain found in cockles can be a potential risk to consumers and can contribute to the deterioration of human health in the study area. Thus, it is essential for local authority to provide the preventive measures in ensuring the cockles are safefor consumption.

Prevalence, Virulence Characterization, AMR Pattern and Genetic Relatedness of Vibrio parahaemolyticus Isolates From Retail Seafood of Kerala, India.

Vibrio parahaemolyticus, a halophilic bacterium often found in the marine or estuarine environment is a well-known enteropathogen responsible for foodborne outbreaks associated with seafood. The pathogenic strains of V. parahaemolyticus are marked by the presence of thermostable direct hemoylsin (tdh) and/or TDH related hemolysin (trh) genes. This study aimed to investigate the prevalence and characteristics of potentially pathogenic V. parahaemolyticus in selected retail markets of Cochin, Kerala, along the south-western coast of the Indian subcontinent. One hundred samples collected from 10 retail markets were analyzed for the presence of pathogenic isolates of V. parahaemolyticus. Out of the 721 presumptive isolates, 648 were confirmed to be V. parahaemolyticus by toxR gene amplification, among which 29 were Kanagawa phenomenon (KP) positive. Among these potentially pathogenic isolates, 17 possessed the tdh gene whereas none of them had the trh gene. The faint amplification bands produced during the amplification of tdh gene from two isolates was confirmed by sequencing. Multiplex O serotyping identified O1 serotype as the most prevalent serotype among the 29 potentially pathogenic isolates. Further, studies on the pandemic nature of these isolates revealed that 14 of the 29 were positive for the PGS-PCR, whereas all the isolates were negative for GS-PCR and HU-α PCR. The antibiogram of the isolates revealed that three isolates had significant Multiple Antibiotic Resistance (MAR) index of 0.2 or above. Pathogenic isolates resistant to second, third and fourth generation Cephalosporins were found to be present in the seafood studied. The molecular fingerprinting studies using ERIC-PCR, and PFGE revealed that three of these isolates shared close genetic similarities with the clinical strains. The environmental and seafood isolates that produced faint amplification bands during the amplification of tdh gene suggests that the tdh gene often goes undetected in environmental isolates. The conventional methods used to identify the pathogenic V. parahaemolyticus would be good for clinical isolates, but a more elaborate method is recommended for the detection of tdh gene in environmental isolates. This is the first comprehensive study on pathogenic V. parahaemolyticus in Kerala, India and demonstrates for the first time, the isolation of potentially pathogenic V. parahaemolyticus, carrying tdh gene from seafood collected from retail markets in Kerala.

Isolation and Genomic Characterization of phiVibrioH1 a Myoviridae Phage for Controlling Pathogenic Vibrio parahaemolyticus from Seafood and Human

Vibrio parahaemolyticus is a zoonotic pathogen causing vibriosis in marine fish and associated with food poisoning outbreaks in humans. This study aimed to isolate and characterize bacteriophage against multidrug resistant (MDR) Vibrio parahaemolyticus from seafood and humans, and also to assess the lytic efficacy of phage on growth. Vibrio Parahaemolyticus was isolated from flesh of 80 white shrimp, 70 blue crabs and 50 mullets, and from 50 hand swabs of humans (fish handlers) on Thiosulphate Citrate bile salts sucrose agar media. The suspected colonies were biochemically identified. Pathogenic V. parahaemolyticus carrying thermostable direct hemolysin (tdh+) gene was molecularly detected . The pathogenic tdh+V. parahaemolyticus (n=25) isolates were explored for antimicrobial vulnerability resistant to 12 antimicrobials employing the disc diffusion method. Fifteen isolates of tdh+V. parahaemolyticus were 100% resistant to five antimicrobials. Bacteriophage was isolated from sewage water using spot test and double over layer agar assay. The phiVibrioH1 was belonged to family Myoviridae according to transmission electron microscopy. This study revealed a polyvalent phage infecting wide host range of MDR V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus, Pseudomonas aeruginosa, E. coli O26 and Proteus vulgaris. The genome of phage was sensitive to digestion with BamH1 and ECoR1 restriction enzymes indicating double stranded DNA. From 24 to 42 hours post-treatment of pathogenic V. parahaemolyticus with the PhiVibrioH1 phage showed complete lysis of bacterial cells. This study confirmed that the phiVibrioH1 is a lytic phage and has a high potential to control pathogenic V. parahaemolyticus strains recovered from seafood and humans.

Occurrence and Abundance of Pathogenic Vibrio Species in Raw Oysters at Retail Seafood Markets in Northwestern Mexico

Seafood has frequently been associated with foodborne illness because pathogens are easily introduced during seafood cultivation, handling, and processing. Vibrio parahaemolyticus and Vibrio cholerae are human pathogens that cause gastroenteritis and cholera, respectively, and Vibrio vulnificus can cause fatal wound infections and septicemia. However, information about the occurrence of these pathogens in oysters from the Pacific coast of Mexico is limited to V. parahaemolyticus. In the present study, we evaluated the presence and abundance of these three Vibrio species in 68 raw oysters (Crassostrea corteziensis) obtained from retail seafood markets in Sinaloa, Mexico. The most probable number (MPN)-PCR assay was used for amplification of the tlh (thermolabile hemolysin), ompW (outer membrane protein), and vvhA (hemolytic cytolysin) genes that are specific to V. parahaemolyticus, V. cholerae, and V. vulnificus, respectively. All oyster samples were positive for at least one Vibrio species. V. parahaemolyticus, V. cholerae, and V. vulnificus prevalences were 77.9, 8.8, and 32.3% overall, respectively, and most species were present in all sample periods with increased prevalence in period 3. The tdh (thermostable direct hemolysin) gene was detected in 30.1%, trh (TDH-related hemolysin) was detected in 3.7%, and tdh/trh was detected in 7.5% of the total tlh-positive samples (53 of 68), whereas the pandemic serotype O3:K6 (orf8 positive) was detected in only 1 sample (1.8%). The total prevalence of tdh and/or trh was 41.5%. In none of the samples positive for V. cholerae were the cholera toxin (ctxA) and cholix (chxA) toxigenic genes or the rfb gene encoding the O1 and O139 antigens amplified, suggesting the presence of non-O1 non-O139 V. cholerae strains. Our results clearly indicated a high prevalence of pathogenic Vibrio species in raw oysters from retail seafood markets in Mexico. Consumption of these raw oysters carries the potential risk of foodborne illness, which can be limited by cooking.

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<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.

Preliminary investigation on the microbiological quality of edible marine gastropods of the Adriatic Sea, Italy

According to the European Legislation, marine gastropods placed unprocessed on the market must comply with the same requirements established for live bivalve molluscs but, being considered not filterfeeding and unable to concentrate fecal contaminants, they may be harvested outside the classified areas. Despite this statement, little scientific information is available on the microbiological quality of these animals. The aim of the present study was to investigate 28 batches of edible snails of the Adriatic Sea, namely Nassarius mutabilis and Bolinus brandaris, with respect to i) smell and viability, by a method here reported; ii) the bacterial component of the whole body referred to E. coli, Vibrio spp., V. parahaemolyticus, V. vulnificus, V. cholerae and V. alginolyticus. A total of 21 batches of N. mutabilis and 7 batches of B. brandaris were analyzed. Batches of both species retrieved from the primary production were all largely composed of viable animals, had saltwater/neutral smell, and showed mean value of Vibrio spp. of 5,34 and 5,79 log10 UFC g-1 in N. mutabilis and B. brandaris respectively. 47% of the batches of N. mutabilis retrieved from the market, were largely composed of dead animals, had acrid/nasty smell, and showed mean value of Vibrio spp. of 6,53 log10 UFC g-1. E. coli, V. vulnificus and V. cholerae were never detected, but all samples were positive for V. alginolyticus. One sample of B. brandaris was positive for V. parahaemolyticus genotyped by PCR at the specie level (ToxR+) and positive for the thermostable direct hemolysin gene (tdh+).

Spatiotemporal distribution of Vibrio parahaemolyticus in relation to environmental parameters in a coastal lagoon on the Pacific coast of northwestern Mexico

Vibrio parahaemolyticus has been responsible for the increasing number of diarrhea cases in Sinaloa, Mexico, since 2003. We investigated the presence, distribution, and content of V. parahaemolyticus toxigenic genes detected in water, zooplankton, and sediment samples in relation to environmental variables in Caimanero Lagoon (Mazatlán, Sinaloa). Samples were analyzed by PCR to detect the presence of V. parahaemolyticus and its toxigenic factors. Of all the samples analyzed, 57.5% tested positive for thermolabile hemolysin (tlh), a gene indicative of the species. The thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) genes, which are both pathogenicity markers of this species, were detected in 9% and 6% of the samples, respectively. The orf8 fragment, which has been recently detected in pandemic strains (O3:K6), was detected in 11% of the samples. Vibrio parahaemolyticus was detected more frequently in zooplankton, with the highest incidence observed in February. Salinity was positively correlated with V. parahaemolyticus; however, no correlation was found between V. parahaemolyticus and temperature. The presence of toxigenic V. parahaemolyticus during the different seasons indicates the need to maintain continuous sanitary inspection of fish products from Caimanero Lagoon.

Regulatory effects of Shewanella putrefaciens isolated from shrimp Penaeus orientalis on the virulence factors of Vibrio parahaemolyticus and evaluation of the role of quorum sensing in virulence factors regulation.

As an aquatic pathogen widely present in aquatic food, Vibrio parahaemolyticus causes outbreaks of gastroenteritis across the globe. Virulence factors of V. parahaemolyticus increases with the amount of spoilage in aquatic organisms including shrimp, but mechanisms regulating its virulence factors are not well understood. In this study, five spoilage bacteria isolated from shrimp were investigated for their regulatory effects on the virulence factors including haemolysin and biofilm of V. parahaemolyticus. Among these isolates, Shewanella putrefaciens induced haemolytic activity in V. parahaemolyticus in a time-dose-temperature-dependent manner and we found the main component responsible for this effect to be the supernatant or cell-free extract of S. putrefaciens. Total haemolytic activity, expression of the thermostable direct haemolysin gene tdh and biofilm production of V. parahaemolyticus were significantly up-regulated by S. putrefaciens, but also by deletion of quorum-sensing luxM or luxS gene of V. parahaemolyticus. However, this regulation by S. putrefaciens was significantly impaired by deletion of the luxM gene, but not by deletion of the luxS gene. Further study showed that S. putrefaciens exhibited a strong degradation ability on the signalling molecule acylated homoserine lactone (AHL) synthesised by the LuxM enzyme. This study revealed a novel virulence regulatory mechanism that S. putrefaciens can significantly increase the virulence factors of V. parahaemolyticus via interfering with the luxM- type quorum-sensing signalling pathway through its AHL-degradation ability.

Regulation of Thermostable Direct Hemolysin and Biofilm Formation of Vibrio parahaemolyticus by Quorum-Sensing Genes luxM and luxS.

Vibrio parahaemolyticus is a seafood opportunistic pathogen. There are evidences suggesting that virulence skills, including hemolytic activity and biofilm formation, are regulated by the luxM/luxS-dependent quorum-sensing system in V. parahaemolyticus, and their regulatory mechanism is not well understood. To better understand the virulence regulatory mechanism of V. parahaemolyticus, the luxM deletion (△luxM) and luxS deletion (△luxS) mutants were constructed and their impacts on growth, hemolysin activity, and biofilm were investigated. Results show that both luxM and luxS are involved in the adaptation to environmental conditions in early adaptive-log phase growth of V. parahaemolyticus. Thermostable direct hemolysin gene (tdh) was negatively regulated by luxM and positively regulated by luxS. The biofilm formation was negatively regulated by both luxS and luxM. This study provides an insight into some aspects of V. parahaemolyticus virulence regulation by luxM/luxS-dependent quorum-sensing system.

Virulence and Antibiotic Resistance of Vibrio parahaemolyticus Isolates from Seafood from Three Developing Countries and of Worldwide Environmental, Seafood, and Clinical Isolates from 2000 to 2017

Vibrio parahaemolyticus is a leading cause of seafood-associated illness. This study investigated the prevalence, virulence, and antibiotic resistance of V. parahaemolyticus in three low- and middle-income countries. Freshly caught fish samples (n = 330) imported to Jordan from Yemen, India, and Egypt were tested. The overall prevalence of V. parahaemolyticus was 15% (95% confidence interval: 11 to 19%). Three isolates (6%) were positive for the thermostable direct hemolysin-related (trh) gene, and all isolates was negative for the thermostable direct hemolysin (tdh) gene. All isolates were resistant to colistin sulfate, neomycin, and kanamycin, and 51 and 43% of isolates were resistant to tetracycline and ampicillin, respectively. Only 4% of the isolates were resistant to cefotaxime and chloramphenicol, and no isolates were resistant to sulfamethoxazole-trimethoprim, streptomycin, gentamicin, ciprofloxacin, and nalidixic acid. All isolates were resistant to two classes of antibiotics, and 86% were multidrug resistant (resistant to at least one drug in three or more classes of antibiotics). A literature review of clinical, seafood, and environmental V. parahaemolyticus isolates worldwide revealed high rates of gentamicin and ampicillin resistance, emerging resistance to third-generation cephalosporins, and limited resistance to sulfamethoxazole-trimethoprim, ciprofloxacin, nalidixic acid, and chloramphenicol. Thus, last-resort antibiotics could be ineffective for treating V. parahaemolyticus infections. Several global reports also documented illness outbreaks in humans caused by trh- and tdh-negative V. parahaemolyticus strains. More research is needed to determine whether the presence of these genes is sufficient to classify the strains as virulent.

Detection of Total and Pathogenic Vibrio parahaemolyticus in Shellfish Growing along the South Yellow Sea and the East China Sea

This study was conducted to monitor the densities of total and pathogenic Vibrio parahaemolyticus in 300 samples of nine shellfish species harvested from the coasts of the South Yellow Sea and the East China Sea (N 23° to 34°, E 116° to 124°), People's Republic of China, between May and October 2015. Total V. parahaemolyticus densities were measured, and V. parahaemolyticus isolates were biochemically identified with probes for the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh). We found that 202 of the 300 samples were positive for V. parahaemolyticus from all the sites: 58 of the 100 samples from the Fujian province, 71 of the 100 samples from the Zhejiang province, and 73 of the 100 samples from the Jiangsu province. In most (170) of the 300 samples, V. parahaemolyticus densities were 0.3 to 10 most probable number (MPN)/g; five lots exceeded 110 MPN/g, and two lots were estimated at 110 MPN/g. Among the 202 V. parahaemolyticus strains, only one was trh positive. Densities of V. parahaemolyticus in these shellfish were temperature dependent, with highest densities in June and July. Among the nine mollusk species, V. parahaemolyticus was most abundant in the agemaki clam (Sinonovacula constricta). The highest and lowest V. parahaemolyticus prevalences were found in oriental cyclina (Cyclina sinensis, 93.8%) and mussels (Mytilus edulis, 28.1%), respectively. Overall, although V. parahaemolyticus is widely distributed in marine environments, the density of V. parahaemolyticus was low and the prevalence of the main virulence factor was very low in shellfish along the coasts of the South Yellow Sea and East China Sea, which is important from a public health perspective. Data presented here will be useful for correlational research and can be utilized for developing risk management plans that establish food safety guidelines for V. parahaemolyticus in Chinese shellfish.

Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes

Vibrio parahaemolyticus, found frequently in oysters, is the most prevalent gastroenteritis-causing pathogen in Korea and in several other Asian countries. This study monitored changes in the environmental parameters and occurrence of V. parahaemolyticus in oyster aquaculture sites. Of the 44 presumed V. parahaemolyticus isolates obtained, when tested against 16 antibiotics, 90.9, 86.4, and 75.0% of the 44 isolates exhibited resistance to vancomycin, ampicillin, and streptomycin, respectively. PCR analysis for the presence of the toxR gene confirmed 31 of the 44 isolates as being positive V. parahaemolyticus strains. The toxR positive isolates were tested for the presence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) virulence genes. Only 9.1% toxR positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The occurrence of multi drug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields.