Vibrio Parahaemolyticus Isolates Research Articles

Biofilm inhibitor Con A lectin feed additive protects shrimp against pathogenic Vibrio harveyi and V. parahaemolyticus

Extracellular matrix (ECM) is a major component of all bacterial biofilms that protect the bacterial cells living within. It consists mainly of carbohydrates, proteins, lipids, and nucleic acids. We characterized the ECM of an acute hepatopancreatic necrosis disease (AHPND) causing Vibrio parahaemolyticus isolate 3HP and two luminescent shrimp disease-causing V. harveyi isolates (1114GL or VH0 and its Siphoviridae phage-lysogenized isolate, VH1) using 11 FITC-labeled plant lectins with different terminal sugar specificities. Canavalia ensiformis lectin (Con A), wheat germ lectin (Triticum vulgaris, WGA), Griffonia simplicifolia lectin II (GSL-II), and Maclura pomifera Lectin (MPL) were found to react with the biofilms of all three isolates. Based on these results and availability, Con A and WGA and their respective specific sugars (D-mannose or glucose and N-acetyl-D-glucosamine) were tested for effects on V. harveyi and V. parahaemolyticus biofilm formations. Biofilms of these bacteria were evaluated by thickness after growth with and without the two lectins and with and without their respective sugar targets at different concentrations. Only Con A at 0.1 mg/ml and 0.5 mg/ml (with and without D-mannose or d-glucose) were found to inhibit biofilms of all three Vibrio isolates in a dose-dependent manner. Con A was then tested for efficacy in protecting shrimp against V. harveyi (VH1) and V. parahaemolyticus 3-HP by its addition to shrimp feed at 0.05 mg/g and 0.5 mg/g and fed for seven days before they were immersion challenged with either VH1 or 3HP on day 8. After the challenge, the shrimp continued to be fed with their respective feed, and their health and survival were monitored for another 11 and 12 days for the VH1 and 3HP experiments, respectively. From both experiments, the groups fed with Con A-supplemented feed gave significantly higher survival rates [51.9% and 53.6% (VH1) and 52.8% and 62.9% (3HP)] than those fed with un-supplemented feed [38.3% (VH1) and 21.2% (3HP)] (p < 0.05). These results suggested that D-mannose/d-glucose plays an essential role in V. harveyi and V. parahaemolyticus biofilm formations, and Con A lectin might be helpful in monitoring and controlling biofilms to prevent Vibrio diseases.

Prevalence, virulence characteristics, and antimicrobial resistance of Vibrio parahaemolyticus isolates from raw seafood in a province in Northern Thailand.

Vibrio parahaemolyticus (V. parahaemolyticus) is commonly found in seawater and seafood products, but evidence is limited of its presence in seafood marketed in locations very distant from coastal sources. This study determined the prevalence and characterization of V. parahaemolyticus in seafood from markets in landlocked Phayao province, Northern Thailand. Among 120 samples, 26 (21.7%) were positive for V. parahaemolyticus, being highest in shrimp (43.3%), followed by shellfish (36.7%), and squid (6.7%), but was not found in fish. V. parahaemolyticus comprised 33 isolates that were non-pathogenic and non-pandemic. Almost all isolates from shrimp and shellfish samples were positive for T3SS1. Only five isolates (15.2%) showed two antimicrobial resistance patterns, namely, kanamycin-streptomycin (1) carrying sul2 and ampicillin-kanamycin-streptomycin (4) that carried tetA (2), tetA-sul2 (1), as well as one negative. Antimicrobial susceptible V. parahaemolyticus isolates possessing tetA (67.9%) and sul2 (3.5%) were also found. Six isolates positive for integron class 1 and/or class 2 were detected in 4 antimicrobial susceptible and 2 resistant isolates. While pathogenic V. parahaemolyticus was not detected, contamination of antimicrobial resistance V. parahaemolyticus in seafood in locations distant from coastal areas requires ongoing monitoring to improve food safety in the seafood supply chain.

Isolation of Lytic Bacteriophages infected Indonesian-strain Vibrio parahaemolyticus and its Protective Effects on Brine Shrimp (Artemia sp.)

Highlight Research vB_Vp_TSK01 and vB_Vp_JKT02 a specific parahaemolyticus bacteriophage were isolated from the shrimp aquaculture water. Compared to control vB_Vp_TSK01 reduced 11.46±1.35% of bacterial growth, and by 9.86±5.92% for vB_Vp_JKT02 treatment. Their cocktail had the highest parahemolyticus growth inhibition by 30.92±3.89%. The phage treatment increased the survival of the Artemia by 28.57% compared to infection control. Abstract Acute Hepatopancreatic Necrosis Disease (AHPND) caused by V. parahaemolyticus infection was one of the major diseases in shrimp culture in recent years. The Vibrio could also affect the survival of Artemia as the shrimp’s main live feed in the hatchery and they become the possible carrier for the AHPND. Phage therapy in shrimp aquaculture could reduce the application of the antibiotic as an antibacterial agent for the AHPND. The present study aimed to isolate the specific lytic phage for the Indonesian strain of V. parahaemolyticus (Vp) and evaluate the phage therapy for the brine shrimp Artemia infected with the Vp. The Vp-specific phage was isolated from the shrimp farm’s water at Tasikmalaya, and North Jakarta City, Indonesia. After isolation and plaque assay, brine shrimp were used as a model to evaluate the phages’ anti-Vibrio activity The Vp-lytic phage was successfully isolated from shrimp culture water at North Jakarta and Tasikmalaya (Vb_Vp_TSK01 and Vb_Vp_JKT01, respectively) and the results showed that both isolated phages and their cocktails were capable to inhibit the growth of Vp with the highest inhibition shown at the cocktail treatment (p&lt;0.05). The survival of Artemia was higher in the phage treatments (p&lt;0.005) compared to the infected control. Infected control had 68.33% of brine shrimp survival, and the Vb_Vp_TSK01, Vb_Vp_JKT01, and their cocktail had similar average brine shrimp survival of 91.11%. In conclusion, phage therapy proved effective in preventing vibriosis in brine shrimp under the conditions tested.

Occurrence of virulence determinants in vibrio cholerae, vibrio mimicus, vibrio alginolyticus, and vibrio parahaemolyticus isolates from important water resources of Eastern Cape, South Africa

BackgroundVirulence determinants are crucial to the risk assessment of pathogens in an environment. This study investigated the presence of eleven key virulence-associated genes in Vibrio cholerae (n = 111) and Vibrio mimicus (n = 22) and eight virulence determinants in Vibrio alginolyticus (n = 65) and Vibrio parahaemolyticus (n = 17) isolated from six important water resources in Eastern Cape, South Africa, using PCR techniques. The multiple virulence gene indexes (MVGI) for sampling sites and isolates as well as hotspots for potential vibriosis outbreaks among sampling sites were determined statistically based on the comparison of MVGI.ResultThe PCR assay showed that all the V. cholerae isolates belong to non-O1/non-O139 serogroups. Of the isolates, Vibrio Cholera (84%), V. mimicus (73%), V. alginolyticus (91%) and V. parahaemolyticus (100%) isolates harboured at least one of the virulence-associated genes investigated. The virulence gene combinations detected in isolates varied at sampling site and across sites. Typical virulence-associated determinants of V. cholerae were detected in V. mimicus while that of V. parahaemolyticus were detected in V. alginolyticus. The isolates with the highest MVGI were recovered from three estuaries (Sunday river, Swartkopps river, buffalo river) and a freshwater resource (Lashinton river). The cumulative MVGI for V. cholerae, V. mimicus, V. alginolyticus and V. parahaemolyticus isolates were 0.34, 0.20, 0.45, and 0.40 respectively. The targeted Vibrio spp. in increasing order of the public health risk posed in our study areas based on the MVGI is V. alginolyticus > V. parahaemolyticus > V. cholerae > V. mimicus. Five (sites SR, PA5, PA6, EL4 and EL6) out of the seventeen sampling sites were detected as the hotspots for potential cholera-like infection and vibriosis outbreaks.ConclusionsOur findings suggest that humans having contact with water resources in our study areas are exposed to potential public health risks owing to the detection of virulent determinants in human pathogenic Vibrio spp. recovered from the water resources. The study affirms the relevancy of environmental Vibrio species to the epidemiology of vibriosis, cholera and cholera-like infections. Hence we suggest a monitoring program for human pathogenic Vibrio spp. in the environment most especially surface water that humans have contact with regularly.

Effect of sublethal dose of chloramphenicol on biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates are generally very sensitive to chloramphenicol. However, it is usually necessary to transfer a plasmid carrying a chloramphenicol resistance gene into V. parahaemolyticus to investigate the function of a specific gene, and the effects of chloramphenicol on bacterial physiology have not been investigated. In this work, the effects of sublethal dose of chloramphenicol on V. parahaemolyticus were investigated by combined utilization of various phenotypic assays and RNA sequencing (RNA-seq). The results showed that the growth rate, biofilm formation capcity, c-di-GMP synthesis, motility, cytoxicity and adherence activity of V. parahaemolyticus were remarkably downregulated by the sublethal dose of chloramphenicol. The RNA-seq data revealed that the expression levels of 650 genes were significantly differentially expressed in the response to chloramphenicol stress, including antibiotic resistance genes, major virulence genes, biofilm-associated genes and putative regulatory genes. Majority of genes involved in the synthesis of polar flagellum, exopolysaccharide (EPS), mannose-sensitive haemagglutinin type IV pilus (MSHA), type III secretion systems (T3SS1 and T3SS2) and type VI secretion system 2 (T6SS2) were downregulated by the sublethal dose of chloramphenicol. Five putative c-di-GMP metabolism genes were significantly differentially expressed, which may be the reason for the decrease in intracellular c-di-GMP levels in the response of chloramphenicol stress. In addition, 23 genes encoding putative regulators were also significantly differentially expressed, suggesting that these regulators may be involved in the resistance of V. parahaemolyticus to chloramphenicol stress. This work helps us to understand how chloramphenicol effect on the physiology of V. parahaemolyticus.

Prevalence of multi-antibiotics resistant (MAR) Vibrio parahaemolyticus in shrimp farms in Sarawak, Malaysia

The shrimp farming industry is constantly under threat due to outbreaks of infectious diseases and environmental problems. Vibrio parahaemolyticus is an important foodborne pathogen causing significant economic losses within the shrimp aquaculture industry worldwide. This research aimed to determine the prevalence of V. parahaemolyticus from different shrimp farms from the stocking to harvesting period and assess the antibiotic susceptibility of V. parahaemolyticus using the antimicrobial susceptibility test (AST). In this study, a total of 288 samples comprising twenty-four from each sample consisting of shrimp, water, effluent, and sediment samples were collected aseptically from three (n = 3) shrimp farms located at Telaga Air Farm 1 (Pond 6), Telaga Air Farm 2 (Pond 9) and Santubong Farm (Pond 7), Kuching, Sarawak. A molecular approach by polymerase chain reaction was used to confirm the presence of regulator gene, toxR, V. parahaemolyticus. A total of 14 antibiotics, including spectinomycin (SH100), imipenem (IPM10), amoxycillin/ clavulanic acid (AMC30), enrofloxacin (ENR5), bacitracin (B10), meropenem (MEM10), cephalothin (KF30), penicillin G (P10), tetracycline (TE30), kanamycin (K30), streptomycin(S25), rifampicin (RD2), erythromycin (E15), and nalidixic acid (NA30) were used. The results obtained showed that 51/288 (17.71%) of the collected samples with regulator gene, toxR V. parahaemolyticus. As a whole, this includes 31.25% (30/288) from sediment samples, 4.17% (4/288) from shrimp samples, 15.63% (15/288) from water samples, and 2.08% (2/288) from effluent water samples. A total of 54.9% (28/51) of V. parahaemolyticus acquired multiple antibiotic resistance (MAR). The resistance of antibiotics was profiled, and the multiple antibiotic resistance (MAR) indexes and classified into ten patterns. The MAR index of V. parahaemolyticus isolates ranged from 0.11 to 0.36. Vibrio parahaemolyticus isolates showed 31.38% with a MAR index &gt; 0.2, indicating that these isolates might be originated from high-risk sources. The data obtained from this study is helpful to monitor the presence of V. parahaemolyticus in the aquaculture farm management system to mitigate the hazard potentially arising from the environmental factor that causes shrimp diseases and shrimp infection.

Prevalence and genetic basis of tetracycline resistance in Vibrioparahaemolyticus isolates recovered from food products in Shenzhen, China during 2013 to 2021

Understanding tetracycline resistance in Vibrio parahaemolyticus from food products is crucial for effective control measures against this foodborne pathogen. This study aimed to investigate the prevalence, evolution routes, and mechanism of transmission of tetracycline resistance in Vibrio parahaemolyticus isolates collected from food products in Shenzhen, China. A total of 2342 non-duplicate Vibrio parahaemolyticus were isolated from 3509 food samples during the period 2013–2021. Among these 2342 Vibrio parahaemolyticus strains, 530 (21.37 %) were resistant to tetracycline. These tetracycline-resistant Vibrio parahaemolyticus strains were mainly isolated from shrimp samples, with the highest resistance rate (46.9 %) observed in 2019. Phylogenetic and genomic analyses of 387 isolates carrying the tet genes revealed that five different types of tet genes (tet(34), tet(A), tet(B), tet(M), and tet(E)) were present. The tet(A) gene was the most common (65 % of isolates), while tet(E) and tet(M) genes were only detected in specific years. Although tet(A) is the most commonly detected gene, it only encodes resistance in a low percentage of strains (47/129). On the other hand, the resistance rate is highest in isolates carrying tet(B) (41/55). Interestingly, V. parahaemolyticus carrying the tet genes were not necessarily tetracycline-resistant, and vice versa. A total of six different types of plasmids and two transposable units were found to carry the tet genes. V. parahaemolyticus strains that harbored these plasmids were often resistant to multiple antibiotics, indicating that horizontal transfer of antibiotic resistance genes is common among V. parahaemolyticus strains. Our findings suggest a high prevalence of tetracycline resistance in Vibrio parahaemolyticus strains recovered from food products in Shenzhen, China. These results provide valuable insight into the evolution and transmission of tetracycline resistance in foodborne Vibrio parahaemolyticus isolates and highlight the need for effective control measures to prevent the spread of antibiotic resistance.

Non-clinical isolates of Vibrio parahaemolyticus harbouring traits of potential pathogenicity and fitness: A molecular analysis

Here we investigated the distribution of virulence and fitness attributes V. parahaemolyticus isolated from marine environment (n = 105). We discovered ∼1% of isolates positive for tdh, 8.57% for trh, and 4.76% had tdh and trh genes. More than 50% of the isolates had pathogenicity islands specific to pandemic clones and secretion systems which are detected partially or entirely. VPaI-1 found in 59.04%; VPaI-4 in 60%; VPaI-5 in 34.28%; VPaI-2 in 99.04%; VPaI-3 in 91.42% and VPaI-6 in 99.04% isolates. Also, 34.28% of the isolates harboured T3SS2 encoding VPaI 7; T3SS1 in 98.09%; T6SS2 in 99.04% isolates and T6SS1 in 60.95% isolates. The cytotoxicity analysis showed a significant effect by causing when infected with trh+ environmental isolates. The expression of the trh, VopC, and VopA genes during infection showed a significant upregulation. This suggests the presence of virulence traits among V. parahaemolyticus that could threaten public health.

Diarrhea-Causing Bacteria and Their Antibiotic Resistance Patterns Among Diarrhea Patients From Ghana.

Diarrheal disease remains a major global health problem particularly in children under 5 years and the emergence of antibiotic-resistant strains of causative pathogens could slow control efforts, particularly in settings where treatment options are limited. This surveillance study conducted in Ghana aimed to determine the prevalence and antimicrobial susceptibility profile of diarrhea-causing bacteria. This was a cross-sectional study carried out in five health facilities in the Ga West Municipality of Ghana between 2017 and 2021. Diarrheic stool samples from patients were collected and cultured on standard differential/selective media and isolates identified by standard biochemical tests, MALDI-TOF assay, and serological analysis. The antibiogram was determined using Kirby-Bauer disk diffusion and Microscan autoScan4 MIC panels which were used for extended-spectrum beta-lactamase (ESBL) detection. Bacteria were isolated from 97.5% (772/792) of stool samples, and 167 of the isolates were diarrheagenic and met our inclusion criteria for antimicrobial resistance (AMR) analysis. These included Escherichia coli (49.1%, 82/167), Salmonella species (23.9%, 40/167), Vibrio species (16.8%, 28/167), and Shigella species (10.2%, 17/167). Among 24 Vibrio species, we observed resistances to cefotaxime (21/24, 87.5%), ceftriaxone (20/24, 83.3%), and ciprofloxacin (6/24, 25%), including four multi-drug resistant isolates. All 13 Vibrio parahaemolyticus isolates were resistant to cefazolin. All 17 Shigella isolates were resistant to tetracycline with resistance to shigellosis drugs such as norfloxacin and ciprofloxacin. Salmonella isolates were highly susceptible to norfloxacin (40/40, 100%) and tetracycline (12/34, 35%). Two ESBL-producing E. coli were also identified with marked susceptibility to gentamicin (66/72, 91.7%) and amikacin (57/72, 79.2%) prescribed in the treatment of E. coli infections. This study showed the different bacteria implicated in diarrhea cases in Ghana and the need for differential diagnoses for better treatment outcomes. Escherichia coli, Shigella, Salmonella, and Vibrio have all been implicated in diarrhea cases in Ghana. The highest prevalence was E. coli and Salmonella with Shigella the least prevalent. Resistance to commonly used drugs found in these isolates may render bacteria infection treatment in the near future nearly impossible. Routine antimicrobial susceptibility testing, effective monitoring, and nationwide surveillance of AMR pathogens should be implemented to curb the increase of antimicrobial resistance in Ghana.

Culture medium from a marine endophytic fungus protects shrimp against acute hepatopancreatic necrosis disease (AHPND)

Because endophytic fungi are widely known for producing secondary metabolites and bioactive compounds, they are of interest to pharmaceutical and biotechnology industries. Many species are found in mangrove forests that are habitats for marine animals, especially in their early life stages. In this research, we screened 42 endophytic fungi originating from Thai mangrove forests for inhibitors of biofilm and planktonic cell growth of 7 Vibrio isolates. Some of these isolates were collected from shrimp ponds exhibiting early mortality syndrome (EMS) and diagnosed as outbreaks of acute hepatopancreatic necrosis disease (AHPND) that is caused by specific isolates of Vibrio parahaemolyticus (VPAHPND). Cell-free supernatants (CFS) of two fungal isolates (MCR433 and MCR 440) that inhibited biofilm growth for at least 5 out of the 7 Vibrio isolates were chosen for further study in shrimp feeding trials. One (MCR433) had strong biofilm inhibition and the other (MCR 440) weak biofilm inhibition for VPAHPND. Feed supplementation with CFS of MCR 433 protected shrimp against AHPND (72% mean survival) when compared to control shrimp given un-supplemented feed (20% mean survival) or given feed supplemented with CFS of MCR 440 (0% survival). Histological analysis of the hepatopancreas confirmed that shrimp given feed supplemented with MCR 433 showed little or no signs of AHPND pathology while shrimp in the other two groups showed histopathology typical AHPND. Using a multigene molecular typing approach, both MCR 433 and MCR 440 were identified as distinct members of the order Muyocopronales, class Dothideomycetes and phylum Ascomycota with differing biological activities. Further research is needed on the lead compounds generated by these strains. Those from MCR 433 have potential for use as feed additives against VPAHPND as an alternative to antibiotics.

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.

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.

Novacq™ improves survival of Penaeus vannamei when challenged with pathogenic Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND) caused by pathogenic isolates of Vibrio parahaemolyticus (VPAHPND) carrying a plasmid encoding two pir-like toxins, is one of the most serious diseases affecting shrimp aquaculture. This study investigated if the microbial feed ingredient “Novacq™” improves resilience of Penaeus vannamei to VPAHPND infection and mortality. Two independent challenge trials were conducted where shrimp were fed a diet containing Novacq™ or a control diet for either 10 or 15-days (for trial 1 and trial 2 respectively) prior to exposure to VPAHPND. Mortalities were monitored every 3 h over the challenge periods (trial 1–99 h and trial 2–120 h) and foregut samples were collected from individual shrimp at different stages of the disease challenge to compare pirA and pirB toxin expression and V. parahaemolyticus abundance via qPCR. Shrimp fed Novacq™ had higher survival compared to control fed shrimp in both trial 1 and trial 2 with the relative percent survival (RPS) compared to the control 34% and 51% respectively. However, differences in Kaplan Meier survival estimates between the two diet treatments were only significant in trial 2. There was no significant difference in expression of pirA or pirB toxin genes or V. parahaemolyticus abundance between the two diet treatments in either trial, however, both toxin expression and V. parahaemolyticus abundance significantly changed throughout the disease time course. Results of this study along with previous studies of Novacq™ effects on a viral pathogen and other performance traits (i.e. growth), suggests Novacq™ may promote a general resilience phenotype that offers greater protection against both bacterial and viral agents of which the mode of action is still unknown.

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.

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.

Biogenic zinc oxide, copper oxide and selenium nanoparticles: preparation, characterization and their anti-bacterial activity against Vibrio parahaemolyticus

The present study investigates biogenic preparation of zinc oxide (ZnO), copper oxide (CuO) and selenium (Se) nanoparticles using the marine brown alga Sargassum swartzii. The prepared nanomaterials were characterized using X-ray diffraction pattern (XRD), scanning electron microscopy (SEM) equipped with energy dispersive X-ray analysis (EDAX), transmission electron microscopy (TEM) and UV–Vis diffuse reflectance spectroscopy (DRS-UV) analysis. The particle size of biogenic ZnO, CuO and Se nanoparticles was ca. 32, 32 and 21 nm, respectively. The isolation of bacterial pathogenic strain Vibrio parahaemolyticus (V. parahaemolyticus) from the diseased shrimp and virulent genes (toxR and tlh) confirmed by PCR technique. Further, the molecular characterized using 16S ribosomal RNA gene sequences and identified new strain V. parahaemolyticus strain (GRCORNICRA001). Anti-bacterial activity of biogenic nanomaterials (ZnO, CuO and Se) was investigated against isolated V. parahaemolyticus using well diffusion method and growth inhibitory assay. The minimum inhibitory concentration (MIC) was 25, 25 and 10 µg mL−1 of ZnO, CuO and Se nanoparticles, respectively. The results show that there is a strong bacterial inhibition in a dose-dependent manner. Further, SEM analysis revealed that the interaction of nanomaterials with V. parahaemolyticus, resulted in a surface tension change that leads to membrane depolarization, formation of abnormal textures such as membrane rupture, membrane blebs, membrane clumping, and also caused cell death. Results of this effort highlighted the way for the future that these nanomaterials incorporated with shrimp feed for the management of aquatic diseases.

Antibacterial spectrum of synthetic herbal-based polyphenols against Vibrio parahaemolyticus isolated from diseased Pacific whiteleg shrimp (Penaeus vannamei) in Thailand

Acute hepatopancreatic necrosis disease (AHPND) was first reported as a new shrimp disease in 2009, which caused serious disease in shrimp culture with global economic losses and high rates of mortality. Vibrio parahaemolyticus (VP) was recently identified as the leading causative agent of AHPND that contains Pir toxin bearing plasmid coding toxins (PirA and PirB). Classic antibiotic treatments have been used for the control of infections, however, the frequent use of antibiotics has led to the emergence of antibiotic-resistant bacterial strains of public health concern. The present study aimed to investigate the potential use of synthetic herbal-based polyphenols compounds as natural, eco-friendly antimicrobial agents for disease control. Polyphenols and their active ingredients were investigated against 96 VP isolates retrieved from Pacific whiteleg shrimp (Penaeus vannamei) in Thailand and compared with various commercial antibiotics using a broth microdilution method, time-kill assay, and scanning electron microscope (SEM). All VP isolates were identified using conventional bacteriology and molecular-based tools. Bacteriological examination revealed that all isolates harbored toxR gene with specific amplicons of 368 bp. Only 56 isolates were positive to AP3, a toxin-encoding gene of AHPND-VP with a fragment size of 336 bp. In terms of susceptibility, all isolates were entirely resistant to ampicillin and amoxicillin, whereas they showed different susceptibility patterns to the other tested antibiotics. Among the polyphenols, there was no apparent activity of syringic acid and rutin, while vanillic acid showed higher bactericidal activity against VP isolates at MICs and MBCs of 1024–2048 μg mL−1. Pyrogallol exhibited the strongest antibacterial activity even at very low concentrations with a MIC and MBC of 32–256 μg mL−1, and the majority of cells showed disruption and an indistinguishable cell wall structure by SEM analysis. The present study offers pyrogallol as a potential synthetic herbal-based antimicrobial agent for the adoption of inventory management practices and control of V. parahemolyticus causing AHPND in shrimp producing sectors.

Increased robustness of postlarvae and juveniles from non-ablated Pacific whiteleg shrimp, Penaeus vannamei, broodstock post-challenged with pathogenic isolates of Vibrio parahaemolyticus (VpAHPND) and white spot disease (WSD)

The maturation and reproduction of Pacific whiteleg shrimp, Penaeus vannamei, through the practice of unilateral eyestalk ablation though common is an animal welfare concern. This study assessed the resilience of offspring from non-ablated P. vannamei when challenged with an isolate of Vibrio parahaemolyticus (Vp) causing acute hepatopancreatic necrosis disease (VpAHPND), and with white spot syndrome virus (WSSV). VpAHPND and WSSV challenges were conducted using PL and juveniles under controlled conditions, with both trials using four groups (i.e. shrimp from either ablated or non-ablated females and then either challenged with the pathogen or not challenged). For the VpAHPND challenge, ten replicate 20 L tanks (five replicates for each population) each containing 100 PL 17 (average weight 14 mg) in 15 ppt, 29.05 ± 0.13 °C water were challenged with 2 mL of 2.0 × 108 CFU mL−1 culture of V. parahaemolyticus. A further ten replicate tanks (five per population) served as the corresponding non-challenged controls. The shrimp mortalities were assessed every 3 h over the following 96 h. For the WSSV challenge, individual 1.4 g (average weight) shrimp (50 individuals per population) were housed in 1 L tanks and fed 0.1 g WSSV infected tissue (av. 2.02 × 109 WSSV). A further 50 shrimp per population served as non-challenged controls. The shrimp were maintained at 15 ppt, 26.3 ± 0.71 °C water and assessed every 3 h post-infection over the subsequent 168 h and mortalities at each time point noted. Postlarvae from non-ablated females had significantly (p = 2.4E-23) better survival (70.4%) than those from ablated females (38.8%) at 96 h post-challenge with VpAHPND. Both challenged populations had significantly (p ≤1.3E-36) lower survival than the control groups. The survival of the juveniles from non-ablated females (62%) at 168 h post-infection with WSSV was not significantly higher than that of the juveniles from ablated female (48%) although the difference was significantly different at 65 to 75 h. Both challenged populations also had significantly (p ≤1.0E-5) lower survival rates than the control groups. The study demonstrates that postlarvae and juveniles from non-ablated females are more resilient to typical pathogens (VpAHPND and WSSV) and may show higher survival rates during a disease outbreak.