Parahaemolyticus Isolates Research Articles

Hygienic-sanitary quality of ready-to-eat salmon sashimi (Salmo Salar)

Fifty sashimi samples from 5 restaurants were characterized for enumeration of Aerobic Psychrotrophic Heterotrophic Bacteria (APHB) and Aerobic Mesophilic Heterotrophic Bacteria (AMHB), Enterobacteriaceae, coliforms at 45ºC (thermotolerant coliforms), coagulase-positive Staphylococci, presence of Vibrio parahaemolyticus and Salmonella sp., and determination of hydrogen potential (pH) and total volatile basic nitrogen (TVB-N). The bacterial isolates were evaluated for their resistance profile to the antimicrobial agents Penicillin, Ampicillin, Cefoxitin, Cefotaxime, Amikacin, Gentamicin, Tetracycline, and Trimethropim-sulfamethoxazole. The results showed that APHB, AMHB and coagulase-positive Staphylococci counts and the determination of pH and TVB-N were in accordance with national and international standards adopted as safe limits for consumption. In contrast, the Enterobacteriaceae and thermotolerant coliforms counts and presence of Salmonella sp. and V. parahaemolyticus were in disagreement with those standards, raising concern about the hygienic-sanitary quality of sashimi. The Staphylococcus aureus and V. parahaemolyticus isolates showed resistance to Penicillin, Ampicillin, Cefoxitin, Cefotaxime, Tetracycline, Gentamicin, and Amikacin, while the Salmonella sp. isolate showed no resistance to all the antimicrobial agents studied. The results showed that 48% of the samples were fit for consumption while 52% had unsatisfactory hygienic-sanitary quality for the parameters evaluated.

Molecular and pathogenic characterization of Vibrio parahaemolyticus isolated from seafood

Gastroenteritis infections in humans are mainly associated with consumption of Vibrio parahaemolyticus contaminated shellfish, which causes health and economic loss. Virulence factor production, antibiotic resistance profile, and biofilm-forming capacity of Vibrio parahaemolyticus isolates on food and food contact surfaces at 30 °C were investigated to evaluate the antibiotic sensitivity and pathogenic level. Strains of V. parahaemolyticus were isolated from shellfish (e.g., Crassostrea gigas, Venerupis philippinarum, Mytilus coruscus, Anadara kagoshimensis) in Korea. When examined for 17 virulence factor-encoding genes, 53.3, 73.1, 87.1, 87.9, and 90.9% of the isolates were positive for genes encoding TDH, T6SS, T3SS1, T3SS2, and Type I pilus, respectively. All isolates showed resistance to vancomycin, tetracyclines, penicillin, nalidixic acid, and doxycycline, among 26 antibiotics tested, with most isolates resistant to kanamycin (93.5%), ampicillin (96.8%), clindamycin (96.8%), tobramycin (88.7%), amikacin (83.97%), and minocycline (80.7%). Biofilm formation, cell–cell attachment, and motility were high in most isolates. These findings may assist in monitoring the epidemics of the pathogen. Continuous monitoring could help to decrease V. parahaemolyticus infections and improve seafood safety.

Incidence, Antibiotic Susceptibility and Characterization of Vibrio parahaemolyticus Isolated from Seafood in Selangor, Malaysia

Vibrio parahaemolyticus is one of the major foodborne pathogens owing to its cause of infectious diseases such as gastroenteritis. These diseases are often associated with the consumption of contaminated seafood. This study aims to investigate the presence of V. parahaemolyticus, their virulence, antibiotic profiles, and plasmid profiles from 77 different kinds of shellfish samples collected from wet markets and supermarkets in Selangor, Malaysia. High densities of Vibrio species ( > 5 log CFU/g) were found in 14/16 groups of shellfish. Among 77 presumptive V. parahaemolyticus isolates, 43 (55.8%) were positive for the toxR gene, confirming the identity of the isolates at the species level. However, none of the V. parahaemolyticus isolates harboured the virulence tdh and trh genes. The antibiotic susceptibility of the V. parahaemolyticus isolates revealed that most of them were resistant to ampicillin (95.3%), ampicillin-sulbactam (81.4%), cefotaxime (37.2%) and imipenem (23.3%). The plasmid profiles of the V. parahaemolyticus isolates showed that 41.9% (18/43) possess at least one plasmid. Our results indicate the V. parahaemolyticus isolates are continuously exposed to various antibiotics in the environments, thus consuming the seafood carries a potential health risk to consumers. The antibiotic resistance conferred by the species necessitates an immediate plan to approach the usage of antibiotics differently.

Whole genome analysis unveils genetic diversity and potential virulence determinants in Vibrio parahaemolyticus associated with disease outbreak among cultured Litopenaeus vannamei (Pacific white shrimp) in India

ABSTRACT Vibrio parahaemolyticus has caused widespread mortality in Indian shrimp aquaculture in recent years. However, there are insufficient genome data for the isolates from Indian shrimp vibriosis to analyze genetic diversity and track the acquisition of genetic features that could be involved in virulence and fitness. In this study, we have performed genome analysis of V. parahaemolyticus isolated from moribund shrimps collected from shrimp farms along coastal Karnataka, India, for better understanding of their diversity and virulence. Five newly sequenced genomes of V. parahaemolyticus along with 40 genomes retrieved from NCBI were subjected to comparative genome analysis. The sequenced genomes had an overall genome size of 5.2 Mb. MLST analysis and core genome phylogenomic analysis revealed considerable genetic diversity among the isolates obtained from the moribund shrimps. Interestingly, none of the V. parahaemolyticus isolates possessed the classical features (PirAB) of the strains associated with Acute Hepatopancreatic Necrosis Disease (AHPND). This study also revealed the presence of multiple virulence attributes, including ZOT, ACE and RTX toxins, secretion systems, and mobile genetic elements. The findings of this study provide insights into the possible transition of an environmental V. parahaemolyticus to emerge as pathogens of aquaculture species by increasing its virulence and host adaptation. Future studies focusing on continuous genomic surveillance of V. parahaemolyticus are required to study the evolution and transmission of new variants in shrimp aquaculture, as well as to design and implement biosecurity programs to prevent disease outbreaks.

Distribution and antimicrobial resistance of Vibrio parahaemolyticus isolated from fish and shrimp aquaculture farms along the Korean coast

The present study investigated the distribution and antimicrobial susceptibility patterns of Vibrio parahaemolyticus in water samples and aquatic animals (fish and shrimp) from major aquaculture farms along the Korean coast in 2018. V. parahaemolyticus is the most common pathogen causing seafood-borne illness. The strain was detected in 34.7% of all samples tested, and was detected at higher levels during summer to autumn when the water temperature is higher. Although more than 90.0% of V. parahaemolyticus isolates were sensitive to 13 of the 15 antimicrobials tested, which is useful for treating V. parahaemolyticus infectious disease, the isolates exhibited higher resistance to two antibiotics (colistin and ampicillin), which should be excluded as treatment options for these infections. Koreans typically enjoy consuming raw seafood. To reduce the potential human health risk of raw seafood consumption, the prevalence and antimicrobial resistance of V. parahaemolyticus in aquaculture environments should be continuously valuated.

Phylogenetic relationships and antibiotic resistance of Vibrio parahaemolyticus isolates related to acute hepatopancreatic necrosis disease in Korea

In this study, we performed genotype analysis based on multi-locus sequence typing (MLST) and antibiotic resistance of 27 pirAB-harboring Vibrio parahaemolyticus (VpAHPND) isolates recovered from white leg shrimp (Penaeus vannamei) from acute hepatopancreatic necrosis disease (AHPND) affected farms (in 2016, n = 22) and retail markets (in 2016, n = 5) of Korea. Most VpAHPND isolates were resistant to colistin, ampicillin, and streptomycin but susceptible to other antibiotics. The comparison of their antibiotic resistance to that of non-VpAHPND isolates revealed a significantly higher resistance of VpAHPND isolates to streptomycin. No differences, however, were observed in the minimal inhibitory concentration median (MIC50), 90th percentile (MIC90), and average multiple antibiotic resistance (MAR) index between the two groups. In MLST analysis, sequence types could not be defined for 19 isolates owing to the failure of recA gene sequencing. Seven new STs were found in five and two isolates from retail markets and AHPND affected farms, respectively. Based on the MLST-based phylogenetic tree, all isolates were divided into eight clades; 22 isolates from AHPND affected farms were grouped into three clades, and five isolates from retail markets formed singleton clades. These data suggest that most AHPND outbreaks in Korea may be related to VpAHPND isolates originating from a common clone through the acquisition of pirAB. Further, the detection of VpAHPND from retail shrimp samples suggests that the pathogen may be widely distributed in shrimp aquaculture and/or the environment of Korea.

One health pathogen surveillance demonstrated the dissemination of gut pathogens within the two coastal regions associated with intensive farming

BackgroundIntensive aquaculture farming has caused significant degradation of coastal wetlands and has been proposed as a reservoir for pathogenic Vibrio spp.ResultsGut pathogens including Vibrio spp., Salmonella spp., and Klebsiella spp. were isolated from bird feces, shrimp and wetland water in two typical coastal regions of China in 2015 and 2017 and were subsequently subjected to whole-genome sequencing. Meanwhile, local patient isolates were also selected to confirm the epidemiological links. Bacterial community composition analyses of the sediments that were sampled in 2015 and 2017 were conducted by the hypervariable region 4 of the 16S rRNA gene. Together with the local clinical isolates, we observed highly related Vibrio isolates from waterbirds, wetlands and shrimp. Phylogenetic genome comparisons also demonstrated that sequence types ST3 and ST2414 Vibrio parahaemolyticus isolates obtained from aquatic animals were clonally related to patient isolates. Likewise, three Salmonella typhimurium isolates were also genomically related to one clinical strain. The results showed that farming activities significantly altered the community composition and resulted in the emergence of several pathogens, including Acinetobacter, Mycobacterium and Legionella.ConclusionsIn conclusion, our results demonstrated that intensive shrimp farming in wetlands has two devastating impacts: pathogen dissemination from aquatic animals into migratory birds and transmission of foodborne pathogens into local communities.

Surveillance of enteric pathogens in imported seafood and environmental surfaces in five seafood markets before the outbreak of COVID-19

To monitor the presence of enteric pathogens in imported seafood, a total of 140 seafood samples imported from eight overseas countries were collected from Beijing, Dalian, Shanghai, Guangzhou, and Wuhan seafood markets from June to November 2019. Additionally, 116 viral, environmental swab samples were also collected from the Wuhan and Guangzhou seafood markets. Five typical enteric bacterial pathogens (Aeromonas spp., Shigella spp., Salmonella spp., Vibrio spp., and Listeria monocytogenes) and four viruses (Rotavirus, Norovirus, Astrovirus, and Sapovirus) were detected positive. Results showed that eight Vibrio parahaemolyticus isolates appeared in seafood imported to Dalian, Wuhan, Shanghai, Guangzhou, and Beijing. In contrast, Vibrio fluvialis and Aeromonas were isolated in another two samples. Norovirus was detected in one oyster sample imported from France and environmental surface in Guangzhou. The remaining pathogens were negative in all the samples being tested. With 120 V. parahaemolyticus isolates from the above countries, the genomic analysis revealed that sequence type ST1152 isolates imported from Canada were clustered with two V. parahaemolyticus isolates from Canada. This study presented the first microbiological analysis of the Wuhan seafood market before the outbreak of COVID-19, which demonstrated that supervision should be strengthened to prevent enteric pathogens via imported seafood.

Identification of Pathogenic Variations in Seafood Vibrio parahaemolyticus Isolates by Comparing Genome Sequences

An investigation of the pathogenic characteristics of isolates of Vibrio parahaemolyticus was conducted by identifying the pathogenic tdh gene and then performing adherence and cytotoxicity assays. Genome sequences of the seafood isolates were analyzed using the Illumina HiSeq 2500 platform. The isolated strains were then mapped by comparing the genomes to the reference genome, and variations in the nucleotide sequences and amino acids were identified with the CLC Genomics Workbench program. The tdh gene was identified in four isolates of V. parahaemolyticus, of which three-SMFM201809-CPC7-3, SMFM201809-CF8-2, and SMFM201809-CF8-3-showed high cytotoxicity and differences in cell adhesion. These isolates were selected to identify virulence factors and genomic variations. All three isolates had the same virulence factors, such as adherence, secretion system, and toxin. In addition, amino acid variants were identified in the regions of type IV pilus, T3SS1 and T3SS1 secreted effectors, and thermolabile hemolysin. These results indicate that variations in amino acids found in regions related to adherence and cytotoxicity result in differences in adhesion efficiency and cytotoxicity; therefore, the isolates with these variations may cause more serious foodborne illness compared with other strains.

Genotypic characterization and antimicrobial resistance of Vibrio cholerae and Vibrio parahaemolyticus isolated from milk, dairy products, and humans with respect to inhibitory activity of a probiotic Lactobacillus rhamenosus

Several species of Vibrio were identified in 4% of total examined samples with the highest isolation in human stool samples. Vibrio cholerae (V. cholerae) was highly isolated in raw milk (66.7%), whereas Vibrio parahaemolyticus (V. parahaemolyticus) was highly isolated in yoghurt (75%) out of the positive samples. The tested cholera toxin (ctxAB), haemolysin toxin (hlyA) and the toxin-coregulated pilus (tcpA) associated-virulence genes were identified in all V. cholera isolates. However, the thermostable direct haemolysin (tdh) and the thermostable direct haemolysin-related haemolysin (trh) associated-virulence genes were identified in 33.3% and 16.7% of the examined V. parahaemolyticus isolates, respectively. The multiple antibiotic resistance (MAR) index ranged from 0.15 to 0.54. Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction showed a higher respective discriminatory power to V. cholerae and V. parahaemolyticus (D = 0.93 and D = 1, respectively) isolates. Lactobacillus rhamnosus (L. rhamnosus) probiotic activity on V. cholerae and V. parahaemolyticus can eliminate or significantly reduce the number of bacteria. In conclusion, most V. cholerae and V. parahaemolyticus isolates exhibited resistance to multiple antimicrobial classes. However, tetracycline, trimethoprim–sulfamethoxazole, chloramphenicol, and meropenem were recommended to treat vibriosis. L. rhamnosus could be used as an effective and applicable method for controlling antimicrobial-resistant Vibrio isolates.

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.

Survey of clinical and environmental Vibrio parahaemolyticus isolates for putative virulence factors and cytotoxicity of extracellular products to a CHO-K1 cell line

The halophilic bacterium Vibrio parahaemolyticus is widely distributed as a natural inhabitant of marine and estuarine environments throughout the world. Many strains are avirulent in humans, but virulent strains are the causative agent of gastroenteritis acquired through consumption of contaminated raw or undercooked seafood. Therefore, it is likely that most V. parahaemolyticus isolates from the marine environment are harmless to humans. Virulent isolates are difficult to differentiate from the majority of avirulent environmental isolates. A total of 55 V. parahaemolyticus isolates (22 clinical, 31 environmental and 2 reference strains) were examined for the carriage of plasmids, whole cell protein profiles; their haemolytic, lipase, phospholipase, protease and urease activities; and the cytotoxicity of their extracellular products (ECPs). Analysis of variance for V. parahaemolyticus isolates showed that Kanagawa haemolysis, phospholipase, protease and urease activities were significantly related to cytotoxicity as assayed by measuring relative extracellular lactate dehydrogenase as an indicator of damage of CHO-K1 cells. The difference between the mean values of cytotoxicity of ECPs of clinical and environmental isolates was significant at a p value of < 0.05.

Prevalence and Antibiogram of Vibrio parahaemolyticus and Aeromonas hydrophila in the Flesh of Nile Tilapia, with Special Reference to Their Virulence Genes Detected Using Multiplex PCR Technique.

Vibrio parahaemolyticus and Aeromonas hydrophila are major public health problems and the main cause of bacterial disease in Nile tilapia (Oreochromis niloticus). This study was conducted to determine the prevalence, antibiotic resistance and some virulence genes of both V. parahaemolyticus and A. hydrophila isolates from Nile tilapia. From Manzala Farm at Dakahlia governorate, 250 freshwater fish samples were collected. The confirmed bacterial isolates from the examined Nile tilapia samples in the study were 24.8% (62/250) for V. parahaemolyticus and 19.2% (48/250) for A. hydrophila. multiplex PCR, revealing that the tlh gene was found in 46.7% (29/62) of V. parahaemolyticus isolates, while the tdh and trh virulence genes were found in 17.2% (5/29). Meanwhile, 39.5% (19/48) of A. hydrophila isolates had the 16s rRNA gene and 10.5% (2/19) had the aerA and ahh1 virulence genes. The Multiple Antibiotic Resistance indices of V. parahaemolyticus and A. hydrophila were 0.587 and 0.586, respectively. In conclusion, alternative non-antibiotic control strategies for bacterial infections in farmed fish should be promoted to avoid multidrug-resistant bacteria. Therefore, it is suggested that farmers should be skilled in basic fish health control and that molecular detection methods are more rapid and cost-effective than bacteriological methods.

Antimicrobial and Genetic Profiles of Vibrio vulnificus and Vibrio parahaemolyticus Isolated From the Maryland Coastal Bays, United States.

Vibrio vulnificus and V. parahaemolyticus, found naturally in marine and estuarine environments, are the leading cause of seafood associated gastrointestinal illness and death. Consumption of improperly cooked crabs and handling of live crabs are potential routes of exposure to pathogenic bacteria such as V. vulnificus and V. parahaemolyticus. Little information is available on serotype genetic and antimicrobial profiles of V. vulnificus and V. parahaemolyticus recovered from Maryland estuaries. The aim of the present study was to determine the serotype of V. parahaemolyticus, evaluate antimicrobial susceptibility and genetic profiles of V. vulnificus and V. parahaemolyticus isolated from water and blue crab (Callinectes sapidus) samples collected from the Maryland Coastal Bays. One hundred and fifty (150) PCR confirmed V. parahaemolyticus including 52 tdh+ (pathogenic) and 129 V. vulnificus strains were tested for susceptibility to twenty (20) different antibiotics chosen by clinical usage for Vibrio species. The O serogroups were determined using an agglutination test with V. parahaemolyticus antisera. Pulsed-field gel electrophoresis (PFGE) was used for molecular subtyping to investigate the genetic diversity among tested strains. The most prevalent serotypes were O5 (33.3%), O3 (18.7%) and O1 (14.7%). More than 41% of all tested Vibrio isolates were resistant to three or more antibiotics. Cephalothin showed the highest resistance (42% and 61%), followed by cefoxitin (42% and 31%) and ceftazidime (36% and 29%) for V. vulnificus and V. parahaemolyticus, respectively. Most strains (99–100%) were susceptible to ampicillin/sulbactam, levofloxacin, piperacillin, piperacillin/tazobactam, and tetracycline. Fifty percent (50%) of the cephalothin resistant strains were crab isolates. Vibrio vulnificus and V. parahaemolyticus isolates demonstrated a high genetic diversity and 31% of V. vulnificus and 16% of V. parahaemolyticus strains were PFGE untypeable. No correlations were found between the V. parahaemolyticus serotype, pathogenicity, genetic and antimicrobial resistance profiles of both species of Vibrio. The observed high multiple drug resistance of V. vulnificus and V. parahaemolyticus from blue crab and its environment is of public health concern. Therefore, there is a need for frequent antibiotic sensitivity surveillance for Vibrio spp.

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp.

The online version contains supplementary material available at 10.1007/s12088-021-00934-6.

Characterization of Pathogenic Vibrio parahaemolyticus Isolated From Fish Aquaculture of the Southwest Coastal Area of Bangladesh.

Vibrio parahaemolyticus is a major foodborne pathogen responsible for significant economic losses in aquaculture and a threat to human health. Here, we explored the incidence, virulence potential, and diversity of V. parahaemolyticus isolates from aquaculture farms in Bangladesh. We examined a total of 216 water, sediment, Oreochromis niloticus (tilapia), Labeo rohita (rui), and Penaeus monodon (shrimp) samples from the aquaculture system where 60.2% (130/216) samples were positive for V. parahaemolyticus. Furthermore, we identified 323 V. parahaemolyticus strains from contaminated samples, 17 of which were found positive for trh, a virulence gene. Four isolates out of the 17 obtained were able to accumulate fluid in the rabbit ileal loop assay. The correlation between the contamination of V. parahaemolyticus and environmental factors was determined by Pearson correlation. The temperature and salinity were significantly correlated (positive) with the incidence of V. parahaemolyticus. Most of the pathogenic isolates (94.1%) were found resistant to ampicillin and amoxicillin. O8: KUT was the predominant serotype of the potentially pathogenic isolates. ERIC-PCR reveals genetic variation and relatedness among the pathogenic isolates. Therefore, this region-specific study establishes the incidence of potential infection with V. parahaemolyticus from the consumption of tilapia, rui, and shrimp raised in farms in Satkhira, Bangladesh, and the basis for developing strategies to reduce the risk for diseases and economic burden.

A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis.

The serotyping of Vibrio parahaemolyticus, which is crucial to the surveillance and detection of outbreaks of vibriosis infection, has been widely used in many countries. In this study, we developed a molecular assay, named multiplex ligation reaction based on probe melting curve analysis (MLMA), for simultaneous identification of V. parahaemolyticus 57 K-serogroups. Based on the previous genomes of 418 strains including 39 K-serogroups and the 18 K-serogroups sequences from public databases, we obtained 57 K-serogroups specific gene sequences for designing primers and probes. The developed MLMA assay for identifying the V. parahaemolyticus 57 K-serogroups showed high reproducibility, with the intra- and inter-assay standard deviations and coefficients of variation of no more than 1°C and 1%, respectively. The limit of detection for all gene targets ranged from 0.1 to 1.0 ng/µl. We validated the MLMA assay with a double-blind test identifying 595 V. parahaemolyticus isolates using conventional serotyping methods for comparison. The results showed the kappa value between the MLMA assay and the traditional serological method was 0.936 and that there was a 96.97% consistency rate with conventional serotyping methods for all detected isolates. Additionally, five rare K-serogroups were identified using the MLMA assay, as well as 18 strains that could not be identified using the traditional serotyping method. Thus, the MLMA assay provides a rapid, robust, and promising tool for the molecular serotyping of V. parahaemolyticus K-serogroups and has the potential application to the detection of outbreaks and surveillance of V. parahaemolyticus infection.

Prevalence, antibiogram and virulence characterization of Vibrio isolates from fish and shellfish in Egypt: a possible zoonotic hazard to humans.

Infection of seafood with pathogenic species of the genus Vibrio causes human food-borne illnesses. This study was executed to examine the antimicrobial resistance phenotypes, biofilm-forming capability and virulence-associated genes of Vibrio from fish and shellfishes. Three hundred fresh water and marine fish and shellfish samples were collected from wet markets and supermarkets in Mansoura, Egypt. Bacteriological examination and PCR amplification identified 92 Vibrio spp., including 42 Vibrio parahaemolyticus and 50 Vibrio alginolyticus isolates from the examined fish and shellfish (infection rate: 30·67%). However, V. vulnificus was not found in this study. Vibrio spp. exhibited variable frequencies of antimicrobial resistance with higher percentages to ampicillin and penicillin. Multidrug resistance (MDR) was detected in 69·04 and 38% of V. parahaemolyticus and V. alginolyticus respectively. PCR testing of virulence genes, tdh, trh and tlh revealed the presence of tlh and trh in 100 and 11·9% of V. parahaemolyticus isolates respectively and none of V. alginolyticus carried any of these genes. Biofilm-forming capability was displayed by 76% of V. parahaemolyticus and 73·8% of V. alginolyticus isolates. Both V. parahaemolyticus and V. alginolyticus showed nonsignificant weak positive correlations (r<0·4) between antimicrobial pairs belonging to different classes; however, a significant positive correlation (P<0·05) between trh and resistance to erythromycin (r=0·45) and imipenem (r=0·38) was only identified in V. parahaemolyticus. This study reports the existence of MDR strains of V. parahaemolyticus and V. alginolyticus from the common types of fishes and shellfishes in Egypt. Furthermore, the presence of virulence genes in these isolates and the ability to produce a biofilm in vitro pose potential health hazards to consumers. Frequent monitoring of seafood for the presence of Vibrio spp. and their antimicrobial susceptibility, virulence determinants and biofilm-forming capability is important for assessing the risk posed by these organisms to the public and for improving food safety.

Assessment of Vibrio parahaemolyticus levels in oysters (Crassostrea virginica) and seawater in Delaware Bay in relation to environmental conditions and the prevalence of molecular markers to identify pathogenic Vibrio parahaemolyticus strains.

This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.

Characterization of vibrio parahaemolyticus organisms isolated from shrimps and shrimp fields located in the coastal areas of Bangladesh

Background: In recent years, this group isolated trh positive Vibrio parahaemolyticus serotype 01:K56 from fresh water sediments around Dhaka city, Bangladesh. V. parahaemolyticus is a well known marine bacterium with pandemic effects of causing enteric diseases and the presence of trh positive V. parahaemolyticus serotype 01;K56 in the rivers of Bangladesh imply great public health concern. On the other hand, shrimp export sector is the second largest export earner for Bangladesh and contamination of shrimp with pathogenic V. parahaemolyticus could be a threat to the shrimp export sector. Therefore, this study was carried out to characterize V. parahaemolyticus isolates from shrimps and shrimp fields located in the coastal areas of Bangladesh. Methods and materials: Thirty four V. parahaemolyticus organisms were isolated and confirmed by standard biochemical, serological and molecular techniques. All these isolates belonged to 10 serogroups (O10:KUT, O1:K25, OUT:K61, O11:K33, O2:K28, O1:K33, O1:KUT, O5:KUT, O11:KUT and O8:KUT) confirmed by duplicate testing. Along with the occurrence of thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH), the incidence of two sets of gene clusters that encode the type 3 secretion system apparatus (T3SS1 & T3SS2α) which contribute in pathogenicity of V. parahaemolyticus were also investigated. Results: All V. parahaemolyticus isolates were found to be negative for both tdh and trh genes, which were confirmed by PCR. T3SS1 was found to be present in all V. parahaemolyticus strains as confirmed by the PCR. All these environmental isolates were found to be negative for T3SS2α, which confirmed that T3SS2α is not associated with non pathogenic environmental strains. We also phenotypically characterized the environmental isolates for the enterotoxicity test, which showed no fluid accumulation in rabbit ileal loop assay. All the above mentioned tests were done twice and the variances were less than 5 percent. Conclusion: All these findings clearly indicate that the V.parahaemolyticus organisms, which are associated with the shrimp and shrimp fields of the coastal areas are non pathogenic in nature and thus, would not be a threat for shrimp export sector, as long as the concerned bacterium is V. parahaemolyticus.