Parahaemolyticus Strains Research Articles

Genomic epidemiology of domestic and travel-associated Vibrio parahaemolyticus infections in the UK, 2008–2018

Globally, V. parahaemolyticus is the most prevalent food-poisoning bacterium associated with seafood consumption. To date, the epidemiology of Vibrio parahaemolyticus infections in the UK has remained unexplored. Here we analysed archived V. parahaemolyticus strains isolated from domestic infections and travellers into the UK from 2008 to 2018 and who had submitted clinical samples to Public Health England (PHE)'s Gastrointestinal Bacteria Reference Unit. A total of 48 strains were retrieved from the PHE strain collection, confirmatory tested by PCR and analysed by genome-wide phylogeny using a global collection of genomes with representative strains from the major epidemic clones. Most reported infections entering the UK originated from travellers returning from Southeast Asia, however cases were also reported to have potentially originated from the USA, Cuba, India, Turkey, Caribbean, France, Slovenia and also within the UK, highlighting the wide geographical spread of infections. A large genetic diversity of V. parahaemolyticus strains was observed, with sequence type 3 (ST3; pandemic group) strains the most common sequence type (23 of the 48 analysed strains, 47.9%). The majority of strains were tdh positive (25/48, 71%), with 5 isolates positive for both haemolysin genes (tdh and trh), with 2 isolates trh positive only (5.7%). Six isolates did not possess either haemolysin gene. We demonstrate that whole genome sequencing can be effective to type certain human pathogens entering a traditionally considered “non-endemic” county and this was captured via passive epidemiological surveillance systems. Such approaches may potentially provide a useful snapshot of the diversity of a given pathogen group circulating worldwide.

Genomic and histopathological characteristics of Vibrio parahaemolyticus isolated from an acute hepatopancreatic necrosis disease outbreak in Pacific white shrimp (Penaeus vannamei) cultured in Korea

This report describes the detailed microbial and histological characteristics of Vibrio parahaemolyticus strains associated with acute hepatopancreatic necrosis disease (VpAHPND) that infected Pacific white shrimp (Penaeus vannamei) cultured in Korea. Shrimps showing typical symptoms of AHPND, including a whitish hepatopancreas and empty gut, were reported at two private shrimp farms in Korea in 2019. In the hepatopancreas of the field samples, sloughing of undifferentiated cells in the hepatopancreatic tubule epithelium was observed upon histopathological examination, and 2 Vibrio spp.-related toxin genes (pirA and pirB) associated with AHPND were detected in the samples. Among samples obtained from the hepatopancreas, pond water, and feces (pond sediment), the copy number of the virulent AHPND plasmid was the highest in the hepatopancreas samples, followed by that in the pond water and feces (pond sediment) samples. Two VpAHPND strains were isolated from pond water and diseased shrimp. The genome of the virulent AHPND plasmid pVp_Kor-D1–2 (GenBank accession no. CP046414.1), isolated from strain 19–021-D1, was fully sequenced. We confirmed that the Korean VpAHPND strain harbored the Asian-type AHPND plasmid, which was almost identical to the AHPND-associated V. owensii plasmid pVOWZ2 previously identified in China. Furthermore, the pathogenicity of the Korean VpAHPND strains was evaluated in laboratory bioassays, and we verified that those isolates were highly pathogenic to Pacific white shrimp, causing high cumulative mortality within 3 days.

Genomic characterization of filamentous phage vB_VpaI_VP-3218, an inducible prophage of Vibrio parahaemolyticus.

The seawater temperature rise can promote the growth of potentially pathogenic Vibrio species. In the North Sea, V. parahaemolyticus strains have been isolated and characterized. These strains contain prophages that may contribute to the emergence of pathogenic strains in the marine environment. Here, we present the genome structure and possible biological functions of the inducible phage vB_VpaI_VP-3218, a novel filamentous phage carried by the V. parahaemolyticus strain VN-3218. Prophages of the strain VN-3218 were induced with mitomycin C and the DNA from the phage induction was sequenced. Two incomplete prophages were identified, only one complete phage genome with length of 11,082bp was characterized. The phage vB_VpaI_VP-3218 belongs to the Inoviridae family and shows close homology to the Saetivirus genus. This phage can integrate into the chromosomal host genome and carries host-related regions absent in similar phage genomes, suggesting that this phage might integrate in other Vibrio host genomes from the environment. Furthermore, this phage might have a role in pathogenicity due to potential zonula occludens toxin genes. Based on its genomic similarity, the genome of vB_VpaI_VP-3218 phage probably integrates into the lysogen's chromosome and replicates as episome. This study complements prophage induction and bioinformatic studies applied to non-model species of potentially pathogenic Vibrio species. The characterization of this phage provides new insights with respect to the presence of filamentous phages in environmental V. parahaemolyticus strains, which might have a role in the emergence of new pathogenic strains in the North Sea.

Shewanella algae and Vibrio spp. strains isolated in Italian aquaculture farms are reservoirs of antibiotic resistant genes that might constitute a risk for human health

The aquatic environment can represent a reservoir of antimicrobial resistance genes. In the present study, phenotypical, biochemical and molecular techniques were used to screen a collection of marine strains isolated in Italian aquaculture farms to investigate their beta-lactam resistance profiles. The genome of 12 carbapenemase and/or beta-lactamase producing strains was sequenced and a phylogenetic analysis of the beta-lactamases found in their chromosomes was performed. Gene annotation and prediction revealed the presence of blaAmpC and blaOXA-55-like in all the Shewanella algae isolates whereas in Vibrio anguillarum and Vibrio parahaemolyticus strains, blaAmpC and blaCARB-19 were found, respectively. Multiple alignments of OXA-55-like and AmpC protein sequences showed different point mutations. Finally, comparisons between enzyme phylogeny and strain clusterization based on sampling sites and dates indicate the diffusion of specific Multi Drug Resistant (MDR) Shewanella algae clones along the Italian Adriatic coast.

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.

Recombinase polymerase amplification-lateral flow (RPA-LF) assay combined with immunomagnetic separation for rapid visual detection of Vibrio parahaemolyticus in raw oysters.

This study was the first attempt to optimize a recombinase polymerase amplification (RPA) and lateral flow (LF) assay combined with immunomagnetic separation (IMS) for the detection of Vibrio parahaemolyticus in raw oysters. The newly developed IMS-RPA-LF assay effectively combines sample preparation, amplification, and detection into a single platform. Under optimal conditions, the average capture efficiency (CE) for 104colony forming units (CFU)/mL of four V. parahaemolyticus strains with 0.4mg of immunomagnetic beads within 45min was 80.3%. After optimization, the RPA-LF assay was able to detect V. parahaemolyticus within 15min, comprising DNA amplification with RPA for 10min at 37°C and visualization of the amplicons through LF strips for 5min. The RPA-LF assay exhibited good specificity by showing a test line for eight V. parahaemolyticus strains with different serotypes but no cross-reaction with 12 non-V. parahaemolyticus bacteria. RPA-LF assay was found to be sensitive and detected as low as 10pg genomic DNA of V. parahaemolyticus. For spiked oyster samples, the detection sensitivity of V. parahaemolyticus was improved to 2CFU/g by IMS-RPA-LF after enrichment for 4h; in contrast, the IMS-PCR method required 8h. Hence, even when V. parahaemolyticus was present in very low numbers in samples, the IMS-RPA-LF assay could be completed within half a workday. Because of the high sensitivity, specificity, and speed of the IMS-RPA-LF assay, this newly developed method opens a novel pathway for rapid diagnostic screening of V. parahaemolyticus in seafood, which is an increasingly important health issue worldwide. Graphical abstract.

Comparison on the Growth Variability of Vibrio parahaemolyticus Coupled With Strain Sources and Genotypes Analyses in Simulated Gastric Digestion Fluids.

Vibrio parahaemolyticus is a food-borne pathogen that causes pathogenic symptoms such as diarrhea and abdominal pain. Currently no studies have shown that either pathogenic and non-pathogenic V. parahaemolyticus possess growth heterogeneity in a human environment, such as in gastric and intestinal fluids. The tlh gene is present in both pathogenic and non-pathogenic V. parahaemolyticus strains, while the tdh and trh genes are only present in pathogenic strains. This study firstly applied simulated human gastric fluids to explore growth variability of 50 strains of V. parahaemolyticus at 37°C. The bacterial growth curves were fitted by primary modified Gompertz model, and the maximum growth rate (μmax), lag time (LT), and their CV values were calculated to compare the stress response of pathogenic and non-pathogenic V. parahaemolyticus to simulated human gastric fluids. Results showed that the simulated human gastric fluids treatment significantly increased the μmax of pathogenic strains and shortened the lag time, while decreased the μmax of non-pathogenic strains and prolonged the lag time. Meanwhile, the CV values of genotypes (tlh+/tdh+/trh–) evidently increased, showing that the pathogenic genotype (tlh+/tdh+/trh–) strains had strong activity to simulated gastric fluids. All of the results indicated that the V. parahaemolyticus strains exhibited a great stress-resistant variability and growth heterogeneity to the simulated gastric fluids, which provides a novel insight to unlock the efficient control of pathogenic V. parahaemolyticus.

Comparative Survival and the Cold-Induced Gene Expression of Pathogenic and Nonpathogenic Vibrio Parahaemolyticus from Tropical Eastern Oysters during Cold Storage.

Expression of the regulatory stress rpoS gene controls the transcription of cspA genes, which are involved in survival and adaptation to low temperatures. The purpose of this study was to assess the growth kinetics of naturally occurring V. parahaemolyticus in shellstock oysters and in vitro and the cold-shock-induced expression of the rpoS and cspA gene response in vitro during postharvest refrigeration. Naturally contaminated eastern oysters (Crassostrea virginica) and pathogenic (Vp-tdh) and nonpathogenic (Vp-tlh) isolates were stored at 7 ± 1 °C for 168 h and 216 h, respectively. The regulatory stress (rpos) and cold-shock (cspA) gene expressions were determined by reverse transcription PCR. At 24 h, the (Vp-tdh) strain grew faster (p < 0.05) than the (Vp-tlh) strain in oysters (λ = 0.33, 0.39, respectively) and in vitro (λ = 0.89, 37.65, respectively), indicating a better adaptation to cold shock for the (Vp-tdh) strain in live oysters and in vitro. At 24 h, the (Vp-tdh) strain rpoS and cspA gene expressions were upregulated by 1.9 and 2.3-fold, respectively, but the (Vp-tlh) strain rpoS and cspA gene expressions were repressed and upregulated by −0.024 and 1.9-fold, respectively. The V. parahaemolyticus strains that were isolated from tropical oysters have adaptive expression changes to survive and grow at 7 °C, according to their virulence.

Environmental conditions steer phenotypic switching in acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus, affecting PirAVP /PirBVP toxins production.

Bacteria in nature are widely exposed to differential fluid shears which are often a trigger for phenotypic switches. The latter mediates transcriptional and translation remodelling of cellular metabolism impacting among others virulence, antimicrobial resistance and stress resistance. In this study, we evaluated the role of fluid shear on phenotypic switch in an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain under both in vitro and in vivo conditions. The results showed that V. parahaemolyticus M0904 grown at lower shaking speed (110 rpm constant agitation, M0904/110), causing low fluid shear, develop cellular aggregates or floccules. These cells increased levan production (as verified by concanavalin binding) and developed differentially stained colonies on Congo red agar plates and resistance to antibiotics. In addition, the phenotypic switch causes a major shift in the protein secretome. At 120 rpm (M0904/120), PirAVP /PirBVP toxins are mainly produced, while at 110 rpm PirAVP /PirBVP toxins production is stopped and an alkaline phosphatase (ALP) PhoX becomes the dominant protein in the protein secretome. These observations are matched with a very strong reduction in virulence of M0904/110 towards two crustacean larvae, namely, Artemia and Macrobrachium. Taken together, our study provides substantial evidence for the existence of two phenotypic forms in AHPND V. parahaemolyticus strain displaying differential phenotypes. Moreover, as aerators and pumping devices are frequently used in shrimp aquaculture facilities, they can inflict fluid shear to the standing microbial agents. Hence, our study could provide a basis to understand the behaviour of AHPND-causing V. parahaemolyticus in aquaculture settings and open the possibility to monitor and control AHPND by steering phenotypes.

Do acute hepatopancreatic necrosis disease-causing PirABVP toxins aggravate vibriosis?

ABSTRACT Gram-negative marine bacterium Vibrio parahaemolyticus is an important aquatic pathogen and has been demonstrated to be the causative agent of acute hepatopancreatic necrotic disease (AHPND) in shrimp aquaculture. The AHPND-causing V. parahaemolyticus strains contain a pVA1 plasmid encoding the binary PirAVP and PirBVP toxins, are the primary virulence factor that mediates AHPND and mortality in shrimp. Since PirABVP toxins are secreted extracellularly, one can hypothesize that PirABVP toxins would aggravate vibriosis in the aquatic environment. To address this, in vivo and in vitro experiments were conducted. Germ-free Artemia franciscana were co-challenged with PirABVP toxins and 10 Vibrio spp. The in vivo results showed that PirABVP toxin interact synergistically with MM30 (a quorum sensing AI-2 deficient mutant) and V. alginolyticus AQ13-91, aggravating vibriosis. However, co-challenge by PirABVP toxins and V. campbellii LMG21363, V. parahaemolyticus CAIM170, V. proteolyticus LMG10942, and V. anguillarum NB10 worked antagonistically, increasing the survival of Artemia larvae. The in vitro results showed that the addition of PirABVP toxins significantly modulated the production of the virulence factors of studied Vibrio spp. Yet these in vitro results did not help to explain the in vivo results. Hence it appears that PirABVP toxins can aggravate vibriosis. However, the dynamics of interaction is strain dependent.

Comparison of Extracellular Proteins from Virulent and Avirulent Vibrio parahaemolyticus Strains to Identify Potential Virulence Factors

Vibrio parahaemolyticus is a leading seafood-borne pathogen that causes gastroenteritis, septicemia, and serious wound infections due to the actions of virulence-associated proteins. We compared the extracellular proteins of nonvirulent JHY20 and virulent ATCC 33847 V. parahaemolyticus reference strains. Eighteen extracellular proteins were identified from secretory profiles, and 11 (68.75%) of the 16 proteins in ATCC 33847 are associated with virulence and/or protection against adverse conditions: trigger factor, chaperone SurA, aspartate-semialdehyde dehydrogenase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, glutamate 5-kinase, alanine dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, outer membrane protein OmpV, ribosome-associated inhibitor A, chaperone protein Skp, and universal stress protein. Two nontoxic-related proteins, amino acid ABC transporter substrate-binding protein and an uncharacterized protein, were identified in JHY20. The results provide a theoretical basis for supporting safety risk assessment of aquatic foods, illuminate the pathogenic mechanisms of V. parahaemolyticus, and assist the identification of novel vaccine candidates for foodborne pathogens.

A Novel Mouse Model of Enteric Vibrio parahaemolyticus Infection Reveals that the Type III Secretion System 2 Effector VopC Plays a Key Role in Tissue Invasion and Gastroenteritis.

The Gram-negative marine bacterium Vibrio parahaemolyticus is a common cause of infectious gastroenteritis due to the ingestion of contaminated seafood. Most virulent V. parahaemolyticus strains encode two type III secretion systems (T3SS1 and T3SS2); however, the roles they and their translocated effectors play in causing intestinal disease remain unclear. While studies have identified T3SS1 effectors as responsible for killing epithelial cells in culture, the T3SS2 effectors caused massive epithelial cell disruption in a rabbit ileal loop model. Additional models are thus needed to clarify the pathogen-host interactions that drive V. parahaemolyticus-associated gastroenteritis. Germfree mice were infected with a pathogenic clinical isolate of V. parahaemolyticus, RIMD2210633 (RIMD). The pathogen was found to adhere to as well as invade the cecal mucosa, accompanied by severe inflammation and dramatic mucosal damage, including widespread sloughing of infected epithelial cells. Mice infected with a V. parahaemolyticus strain lacking the T3SS1 (POR2) also developed severe pathology, similar to that seen with RIMD. In contrast, the ΔT3SS2 strain (POR3) appeared unable to invade the intestinal mucosa or cause any mucosal pathology. Confirming a role for TS332 effectors, a strain expressing the T3SS2 but lacking VopC (POR2ΔvopC), a T3SS2 effector implicated in epithelial cell invasion in culture, was strongly attenuated in invading the intestinal mucosa and in causing gastroenteritis, although infection with this mutant resulted in more pathology than the ΔT3SS2 strain. We thus present an experimental system that enables further characterization of T3SS effectors as well as the corresponding host inflammatory response involved in the gastroenteritis caused by invasive V. parahaemolyticusIMPORTANCEVibrio parahaemolyticus causes severe gastroenteritis following consumption of contaminated seafood. Global warming has allowed this pathogen to spread worldwide, contributing to recent outbreaks. Clinical isolates are known to harbor an array of virulence factors, including T3SS1 and T3SS2; however, the precise role these systems play in intestinal disease remains unclear. There is an urgent need to improve our understanding of how V. parahaemolyticus infects hosts and causes disease. We present a novel mouse model for this facultative intracellular pathogen and observe that the T3SS2 is essential to pathogenicity. Moreover, we show that the T3SS2 effector VopC, previously shown to be a Rac and Cdc42 deamidase that facilitates bacterial uptake by nonphagocytic cells, also plays a key role in the ability of V. parahaemolyticus to invade the intestinal mucosa and cause gastroenteritis. This experimental model thus provides a valuable tool for future elucidation of virulence mechanisms used by this facultative intracellular pathogen during in vivo infection.

Attenuation of Vibrio parahaemolyticus Virulence Factors by a Mixture of Natural Antimicrobials.

Reducing acute mortality in aquatic crustaceans using natural alternatives to antibiotics has become a necessity, firstly for its positive impact on the aquaculture industry and, secondly, because the extensive use of antibiotics may lead to increased levels of drug resistance in pathogenic microorganisms. This study aimed to investigate the effect of a mixture of natural antimicrobials on the in vitro and in vivo virulence abilities of Type VI secretion system (T6SS)-positive Vibrio parahaemolyticus (A3 and D4), strains known as having potentially harmful health consequences for aquatic crustaceans and consumers. Herein, we report that a natural antimicrobial mixture (A3009) was capable of significantly reducing the virulence of V. parahaemolyticus strains A3 and D4 in an in vitro infection model, using the fish cell line CHSE-214, an effect which correlates with the bacterial downregulation of hcp1 and hcp2 gene expression and with the ability of the antimicrobial to efficiently retain low cytotoxic levels (p < 0.001). We show for the first time that a natural antimicrobial is able to significantly reduce the mortality of shrimps in a challenge experiment and is able to significantly attenuate H2O2 release during infection (p < 0.001), indicating that it could harbor positive intestinal redox balance effects.

Characterization of class 1 integrons harboring blaVEB-1 in Vibrio parahaemolyticus isolated from ready-to-eat foods in China

The aim of this study is to investigate the prevalence of integrons and integron-associated antibiotic resistance in V. parahaemolyticus strains collected from RTE foods in China, and to carry out a comprehensive analysis on the molecular characterization of V. parahaemolyticus strains carrying blaVEB-1-positive class 1 integron. Of the 51 V. parahaemolyticus strains isolated from RTE food samples, none of the isolates was found to carry integrase genes intI2 and IntI3. However, all 51 strains were positive to integrase gene intI1, and only 2 of 51 (3.92%) intI1-positive isolates yielded polymerase chain reaction (PCR) products of gene cassette amplification. Sequence data and BLAST analysis indicated the gene cassette arrays of class 1 integron in VP007 is dfrA14-blaVEB-1-aadB, while the gene cassette arrays of class 1 integron in V187 is blaVEB-1-aadB-arr2-cmlA-blaOXA-10-aadA1. Antimicrobial susceptibility testing showed that the two V. parahaemolyticus isolates harboring class 1 integrons exhibited multi-drug resistance to various antibiotics. S1-PFGE and Southern blot analysis confirmed the class 1 integron harboring blaVEB-1 gene in V187 was located on the plasmid of ~175 kb and transferrable to the recipient strain by conjugation. This is the first detection of class 1 integrons harboring the ESBL gene blaVEB-1 in V. parahaemolyticus. To the best of our knowledge, this is also the first report of VEB-producing V. parahaemolyticus from RTE foods. Our findings revealed that class 1 integron on conjugative plasmid contributes significantly to the dissemination of VEB-producing V. parahaemolyticus, which warrants further investigation because of the public health threat it poses.

Associations of Environmental Conditions and Vibrio parahaemolyticus Genetic Markers in Washington State Pacific Oysters.

Vibrio parahaemolyticus is a naturally occurring bacterium in estuarine waters and is a major cause of seafood-borne illness. The bacterium has been consistently identified in Pacific Northwest waters and elevated illness rates of vibriosis in Washington State have raised concerns among growers, risk managers, and consumers of Pacific oysters (Crassostrea gigas). In order to better understand pre-harvest variation of V. parahaemolyticus in the region, abundance of total and potentially pathogenic strains of the bacterium in a large number of Washington State Pacific oyster samples were compared with environmental conditions at the time of sampling. The Washington Department of Health regularly sampled oysters between June and September at over 21 locations from 2014 to 2018, resulting in over 946 samples. V. parahaemolyticus strains carrying three genetic markers, tlh, trh, and tdh, were enumerated in oyster tissue using a most probable number-PCR analysis. Tobit regressions and seemingly unrelated estimations were used to formally assess relationships between environmental measures and genetic markers. All genetic markers were found to be positively associated with temperature, independent of the abundance of other genetic markers. Surface water temperature displayed a non-linear relationship, with no association observed between any genetic marker in the warmest waters. There were also stark differences between surface and shore water temperature models. Salinity was not found to be substantially associated with any of the genetic variables. The relative abundance of tdh+ strains given total V. parahaemolyticus abundance (pathogenic ratio tdh:tlh) was negatively associated with water temperature in colder waters and decreased exponentially as total V. parahaemolyticus abundance increased. Strains carrying the trh gene had a pronounced positive association with strains carrying the tdh gene but was also negatively associated with the tdh:tlh pathogenic ratio. These results suggest that there are ecological relationships of competition, growth, and survival for V. parahaemolyticus strains in the oyster tissue matrix. This work also improves the overall understanding of environmental associations with V. parahaemolyticus in Washington State Pacific oysters, laying the groundwork for future risk mitigation efforts in the region.

Selection of Lactic Acid Bacteria (LAB) Antagonizing Vibrio Parahaemolyticus: The Pathogen of Acute Hepatopancreatic Necrosis Disease (AHPND) in Whiteleg Shrimp (Penaeus Vannamei).

Acute hepatopancreatic necrosis disease (AHPND) has recently emerged as a serious disease of cultured shrimp. A total of 19 lactic acid bacteria (LAB) strains isolated from shrimp samples were characterized based on morphological characteristics, biochemical tests, sequencing analysis, and their ability to antagonize Vibrio parahaemolyticus, which causes AHPND in whiteleg shrimp. Results from the agar well diffusion method indicated that 3 out of 19 isolated LAB strains showed the highest antagonizing ability against AHPND V. parahaemolyticus strain with an inhibition zone diameter ranging from 18 to 20 mm. Experiments where shrimps were given feed supplemented with these LAB strains and challenged with AHPND strain showed high survival rates (approximately 80.0%), which were not significantly different as compared to those recorded in the negative control treatment (86.6%), but significantly different to those recorded in the positive control treatment (40.6%) after 16 days of the experiment. However, the histological images of shrimp hepatopancreas indicated that the infection rate significantly reduced from 60.0% to 11.1% in shrimps fed with LAB-supplemented feeds and challenged with AHPND V. parahaemolyticus strain as compared to those in the positive control treatment. A polymerase chain reaction (PCR) and 16S rRNA gene sequencing confirmed the identification of LAB strain. These results can be applied in further experiments to investigate the ability of L. plantarum in preventing AHPND in intensively cultured whiteleg shrimp.

Genomic and evolutionary features of two AHPND positive Vibrio parahaemolyticus strains isolated from shrimp (Penaeus monodon) of south-west Bangladesh

BackgroundDue to its rapid lethal effect in the early development stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) has been causing great economic losses, since its first outbreak in southeast China in 2009. Vibrio parahaemolyticus, carrying the pirA and pirB toxin genes is known to cause AHPND in shrimp. The overall objective of this study was to sequence the whole genome of AHPND positive V. parahaemolyticus strains isolated from shrimp (Peneaus monodon) of the south-west region of Bangladesh in 2016 and 2017 and characterize the genomic features and emergence pattern of this marine pathogen.ResultsTwo targeted AHPND positive V. parahaemolyticus strains were confirmed using PCR with 16S rRNA, ldh, AP3 and AP4 primers. The assembled genomes of strain MSR16 and MSR17 were comprised of a total of 5,393,740 bp and 5,241,592 bp, respectively. From annotation, several virulence genes involved in chemotaxis and motility, EPS type II secretion system, Type III secretion system-1 (T3SS-1) and its secreted effectors, thermolabile hemolysin were found in both strains. Importantly, the ~ 69 kb plasmid was identified in both MSR16 and MSR17 strains containing the two toxin genes pirA and pirB. Antibiotic resistance genes were predicted against β-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains.ConclusionsThe findings of this research may facilitate the tracking of pathogenic and/or antibiotic-resistant V. parahaemolyticus isolates between production sites, and the identification of candidate strains for the production of vaccines as an aid to control of this devastating disease. Also, the emergence pattern of this pathogen can be highlighted to determine the characteristic differences of other strains found all over the world.

Prevalence, virulence, antimicrobial resistance, and molecular characterization of fluoroquinolone resistance of Vibrio parahaemolyticus from different types of food samples in China

Vibrio parahaemolyticus is the leading cause of foodborne bacterial poisoning in China. The aim of this research is to conduct a study on the prevalence, virulence, and antimicrobial resistance of V. parahaemolyticus from different types of food samples in 12 different cities of China. Since fluoroquinolones are the major choice of treatment for V. parahaemolyticus infections, the genetic basis for fluoroquinolone resistance in V. parahaemolyticus were also investigated. V. parahaemolyticus was detected in 163 of the 784 food samples collected from 12 different cities in China, resulting in a prevalence of 20.79%. The prevalence of V. parahaemolyticus in ready-to-eat (RTE) food (4.96%) was much lower than those of shrimp (32.62%) and fish (22.00%). Virulence gene screening showed that 44 (27.00%) V. parahaemolyticus strains carried at least one virulence gene. Four isolates from shrimp and three isolates from fish contained both the virulence genes tdh and trh. In addition, the trh was firstly detected in one isolate collected from RTE food. All isolates exhibited relatively high resistance rates to ampicillin (82.21%), gentamicin (19.63%), and tetracycline (14.11%), while <10% of strains were resistant to ciprofloxacin (4.91%), levofloxacin (4.91%), and tetracycline (4.29%). Eight fluoroquinolone-resistant V. parahaemolyticus were selected to determine the molecular basis for fluoroquinolone resistance. These eight isolates belonged to three different types according to enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). A Ser83Ile substitution in GyrA was deteted in seven fluoroquinolone-resistant strains, except V209 which harbored a Ser83Phe substitution in GyrA. Moreover, A Ser85Leu substitution in ParC was found in five isolates (V52, V53, V61, V163, and V209). Plasmid-mediated quinolone resistance (PMQR) genes were detected in all eight fluoroquinolone-resistant V. parahaemolyticus strains. This is the first report of Ser83Phe substitution in GyrA, qnrD and qnrS1 in V. parahaemolyticus. The information generated in this study will provide valuable information for risk assessment of V. parahaemolyticus infections and future control of antibiotic-resistant V. parahaemolyticus species in China.

Silver nanoparticles against acute hepatopancreatic necrosis disease (AHPND) in shrimp and their depuration kinetics

Ag-based nanoparticles (NPs) were successfully produced through green synthesis using an aqueous extract from the cultivated seaweed Ulva clathrata as the reducing and stabilizing agent. The biosynthesized NPs had spherical to polymorphic shapes with an average size of 9.5 nm. Microstructural and compositional studies revealed that these particles contained face-centred cubic crystallites of metallic Ag and AgCl. Characteristic peaks in the Fourier-transform infrared spectrum (FTIR) and Raman studies revealed the presence of functional bioactive metabolites from the seaweed extract, such as proteins, polysaccharides, and polyphenols, which are responsible for forming and stabilizing Ag/AgCl NPs. The biosynthesized Ag/AgCl NPs exhibited an important in vitro antibacterial effect against three Vibrio parahaemolyticus strains isolated from farmed shrimp affected with acute hepatopancreatic necrosis disease (AHPND) in northwestern Mexico. Litopenaeus vannamei shrimp were exposed for 7 days to feeds supplemented with Ag/AgCl NPs at 10, 100, 1000, or 10,000 ppm (Ag nominal dietary concentrations). Dietary NP supplement did not affect shrimp survival, growth, or feed conversion ratio, but high concentrations (1000 and 10,000 ppm) decreased the hepatosomatic index significantly. The short-term consumption of Ag/AgCl NPs produced a significant dose-dependent bioaccumulation of Ag in the hepatopancreas and to a lesser extent in the cuticle, while bioaccumulation in the muscle was not significant. The depuration study confirmed a fast Ag assimilation in shrimp’s hepatopancreas and showed a fast depuration rate in the hepatopancreas as well.

Synergistic Antimicrobial Effect of a Seaweed-Probiotic Blend Against Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus.

The outbreak of acute hepatopancreatic necrosis disease (AHPND) has caused great economic losses to the shrimp culture sector. However, the use of antibiotics to fight this disease has resulted in negative impacts on human health and the environment. Thus, the use of natural alternatives to antibiotics may be a better solution. In this study, four Bacillus species obtained from the guts of shrimps (Fenneropenaeus penicillatus and Penaeus monodon) showed antimicrobial activity against the AHPND-causing Vibrio parahaemolyticus strain 3HP using the cross-streaking and agar spot methods. Two of the Bacillus isolates, B2 and BT, also showed good probiotic properties, exhibiting tolerance to bile, good adhesion to shrimp mucus, non-hemolytic, susceptibility to antibiotics and being safe towards hosts. Moreover, a seaweed-probiotic blend (a combination of Bacillus B2 and 20 mg/ml of the red seaweed Gracilaria sp.) exhibited synergistic in vitro inhibition against V. parahaemolyticus strain 3HP, with an observed inhibition zone of 5.0 mm. The broth co-culture experiment results further indicated that the seaweed-probiotic blend inhibited V. parahaemolyticus through competitive exclusion. The in vivo challenge trials also confirmed that this seaweed-probiotic blend significantly reduced the mortality of shrimps post-challenge with the AHPND-causing V. parahaemolyticus strain 3HP (p < 0.05) compared to the negative control (mortality rate = 13.88% vs 72.19%). Thus, this seaweed-probiotic blend may serve as an alternative to antibiotics in controlling the outbreak of AHPND.