BolA-like protein (IbaG) promotes biofilm formation and pathogenicity of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium widespread in temperate and tropical coastal waters; it is considered to be the most frequent cause of Vibrio-associated gastroenteritis in many countries. BolA-like proteins, which reportedly affect various growth and metabolic processes including flagellar synthesis in bacteria, are widely conserved from prokaryotes to eukaryotes. However, the effects exerted by BolA-like proteins on V. parahaemolyticus remain unclear, and thus require further investigation. In this study, our purpose was to investigate the role played by BolA-like protein (IbaG) in the pathogenicity of V. parahaemolyticus. We used homologous recombination to obtain the deletion strain ΔibaG and investigated the biological role of BolA family protein IbaG in V. parahaemolyticus. Our results showed that IbaG is a bacterial transcription factor that negatively modulates swimming capacity. Furthermore, overexpressing IbaG enhanced the capabilities of V. parahaemolyticus for swarming and biofilm formation. In addition, inactivation of ibaG in V. parahaemolyticus SH112 impaired its capacity for colonizing the heart, liver, spleen, and kidneys, and reduced visceral tissue damage, thereby leading to diminished virulence, compared with the wild-type strain. Finally, RNA-sequencing revealed 53 upregulated and 71 downregulated genes in the deletion strain ΔibaG. KEGG enrichment analysis showed that the two-component system, quorum sensing, bacterial secretion system, and numerous amino acid metabolism pathways had been altered due to the inactivation of ibaG. The results of this study indicated that IbaG exerts a considerable effect on gene regulation, motility, biofilm formation, and pathogenicity of V. parahaemolyticus. To the best of our knowledge, this is the first systematic study on the role played by IbaG in V. parahaemolyticus infections. Thus, our findings may lead to a better understanding of the metabolic processes involved in bacterial infections and provide a basis for the prevention and control of such infections.

Characterization and transmission of plasmid-mediated multidrug resistance in foodborne Vibrio parahaemolyticus.

Objectives: The purpose of this study was to determine the structural features and transferability of the multidrug-resistance (MDR) plasmid, and resistance phenotypes for the tested antimicrobials in foodborne Vibrio parahaemolyticus. Methods: Plasmids were isolated from a V. parahaemolyticus strain of seafood origin, then sequenced using the Illumina NovaSeq 6000 and PacBio Sequel II sequencing platforms to obtain the complete genome data. Characterization of the MDR plasmid pVP52-1, including determination of antimicrobial resistance genes (ARGs), plasmid incompatibility groups, and transferability, was carried out. Results: V. parahaemolyticus strain NJIFDCVp52 contained two circular chromosomes and two circular plasmids (pVP52-1 and pVP52-2). Plasmid typing indicated that pVP52-1 belonged to the incompatibility group IncA/C2 and the sequence type pST3. pVP52-1 carried 12 different ARGs, an IS110-composite transposon consisting of aac(6')-Ib-cr, qnrVC1, aac(6')-Ib, dfrA14, and the IS26-mphA-IS6100 unit flanked by inverted sequences of IS5075 and IS4321. pVP52-2 carried no ARGs. A plasmid elimination assay showed that only pVP52-1 and its ARGs were lost, the loss of resistance to several antimicrobials, causing a change from the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-trimethoprim/sulfamethoxazole resistance pattern to the ampicillin resistance pattern. In accordance, a conjugation transfer assay showed that only pVP52-1 and its ARGs were horizontally transferred, leading to increased antimicrobial resistance in Escherichia coli strain EC600, causing a change from the ampicillin-nalidixic acid resistance pattern to the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-nalidixic acid-chloramphenicol-tetracycline-trimethoprim/sulfamethoxazole-azithromycin resistance pattern. Further transferability experiments revealed that pVP52-1 could be transferred to other enterobacterial strains of E. coli and Salmonella. Discussion: This study emphasizes the urgent need for continued surveillance of resistance plasmids and changes in antimicrobial resistance profiles among the V. parahaemolyticus population.

Mining and multifaceted applications of phage lysin for combatting Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a prevalent foodborne pathogen found in both water and seafood, poses substantial risks to public health. The conventional countermeasure, antibiotics, has exacerbated the issue of antibiotic resistance, increasing the difficulty of controlling this bacterium. Phage lysins, as naturally occurring active proteins, offer a safe and reliable strategy to mitigate the impact of V. parahaemolyticus on public health. However, there is currently a research gap concerning bacteriophage lysins specific to Vibrio species. To address this, our study innovatively and systematically evaluates 37 phage lysins sourced from the NCBI database, revealing a diverse array of conserved domains and notable variations in similarity among Vibrio phage lysins. Three lysins, including Lyz_V_pgrp, Lyz_V_prgp60, and Lyz_V_zlis, were successfully expressed and purified. Optimal enzymatic activity was observed at 45℃, 800 mM NaCl, and pH 8-10, with significant enhancements noted in the presence of 1 mM membrane permeabilizers such as EDTA or organic acids. These lysins demonstrated effective inhibition against 63 V. parahaemolyticus isolates from clinical, food, and environmental sources, including the reversal of partial resistance, synergistic interactions with antibiotics, and disruption of biofilms. Flow cytometry analyses revealed that the combination of Lyz_V_pgp60 and gentamicin markedly increased bacterial killing rates. Notably, Lyz_V_pgrp, Lyz_V_pgp60, and Lyz_V_zlis exhibited highly efficient biofilm hydrolysis, clearing over 90 % of preformed V. parahaemolyticus biofilms within 48 h. Moreover, these lysins significantly reduced bacterial loads in various food samples and environmental sources, with reductions averaging between 1.06 and 1.29 Log CFU/cm2 on surfaces such as stainless-steel and bamboo cutting boards and approximately 0.87 CFU/mL in lake water and sediment samples. These findings underscore the exceptional efficacy and versatile application potential of phage lysins, offering a promising avenue for controlling V. parahaemolyticus contamination in both food and environmental contexts.

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells.

V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75 mM and 15 °C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7 cells, with its optimal concentration determined to be 7.5 µg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p < 0.05) in NO levels. Additionally, the relative mRNA levels of IL-1ß, IL-6, and TNF-α in RAW264.7 cells were measured by qRT-PCR, showing a remarkable elevation (p < 0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7 cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.

Induction and comparative resuscitation of viable but nonculturable state on Vibrio parahaemolyticus serotypes O3:K6 and O1:K25.

Vibrio parahaemolyticus, an important food-borne pathogens found to be associated with seafoods and marine environs. It has been a topic of debate for many decades that most pathogens are known to enter a viable but nonculturable (VBNC) state under cold temperature and nutrient limited conditions. The present study examined the time required for the induction of VBNC state and the revival strategies of both the endemic O3:K6 and O1:K25 sporadic strains of V. parahaemolyticus. The results revealed that V. parahaemolyticus survived even after 55 days of incubation in nutrient starved media such as phosphate buffered saline (PBS) and Coastal Water (CW) and could be recovered by temperature upshift method, and compared the resuscitation using Dulbecco's Modified Eagle Medium (DMEM), sheep blood serum, chitin flakes with live Artemia salina, and the results suggests that chitin plays a significant role in regulating the VBNC state. It was also confirmed by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscope (SEM) analysis that VBNC cells can alter their morphology to coccoid forms in order to survive in most extreme nutrient limited environment. Further data on the promoting factors and the exact mechanism that resuscitate VBNC V. parahaemolyticus in cold natural environments and frozen foods are needed to perform a robust risk assessment.

Differential expression of microRNAs in giant freshwater prawn (Macrobrachium rosenbergii) during the infection of Vibrio parahaemolyticus.

MicroRNAs (miRNAs) are a category of small non-coding RNAs regarded as vital regulatory factors in various biological processes, especially immune regulation. The differently expressed miRNAs in Macrobrachium rosenbergii after the challenge of Vibrio parahaemolyticus were identified using high-throughput sequencing. A total of 18 known as well as 12 novel miRNAs were markedly differently expressed during the bacterial infection. The results of the target gene prediction and enrichment analysis indicated that a total of 230 target genes involved in a large variety of signaling pathways and biological processes were mediated by the miRNAs identified in the current research. Additionally, the effects of novel-miR-56, a representative differentially expressed miRNA identified in the previous infection experiment, on the immune-related gene expression in M. rosenbergii were explored. The expression of the immune-related genes including Spätzle1(Spz1), Spz4, Toll-like receptor 1 (TLR1), TLR2, TLR3, immune deficiency (IMD), myeloid differentiation factor 88 (MyD88), anti-lipopolysaccharide factor 1 (ALF1), crustin1, as well as prophenoloxidase (proPO) was significantly repressed in the novel-miR-56-overexpressed prawns. The expression of these genes tested in the novel-miR-56-overexpressed M. rosenbergii was still signally lower than the control in the subsequent V. parahaemolyticus challenge, despite the gene expression in each treatment increased significantly after the infection. Additionally, the cumulative mortality of the agomiR-56-treated prawns was significantly higher than the other treatments post the bacterial challenge. These results suggested that novel-miR-56 might function as a negative regulator of the immune-related gene expression of M. rosenbergii in the innate immune defense against V. parahaemolyticus.

A positive loop between relish and cuticle proteins and their roles in regulating AMPs expression during bacterial infection in Eriocheir sinensis.

Cuticle proteins (CPs) are the vital components of the cuticle and chitin lining covering the digestive tract of crustaceans. In this study, four new CP genes (designated as EsCP3, EsCP4, EsCP5, and EsCP8) were initially cloned and identified from the Chinese mitten crab Eriocheir sinensis. EsCP3/4/5/8 included 375, 411, 381, and 570 bp open reading frame encoding 124, 136, 126, and 189 amino acid proteins, respectively. Except for EsCP8, EsCP3/4/5 all contained a Chitin_bind_4 domain. EsCP3/4/5/8 were clustered into different groups in the phylogenetic tree. Quantitative real-time PCR results indicated that four EsCP genes have different patterns of tissue distribution. Changes in the expression levels of these four EsCP genes were observed in the intestine of crabs under Vibrio parahaemolyticus challenge. RNA interference assay showed that the knockdown of EsCPs in the intestine could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. In addition, the knockdown of EsRelish in the intestine decreased the expression levels of these four EsCP genes. These results indicated that EsCPs were involved in regulating the expression of AMPs, and EsCPs were regulated by EsRelish.

Bile acids activate the antibacterial T6SS1 in the gut pathogen Vibrio parahaemolyticus.

The marine bacterium Vibrio parahaemolyticus is a major cause of seafood-borne gastroenteritis in humans and of acute hepatopancreatic necrosis disease in shrimp. Bile acids, produced by the host and modified into secondary bile acids by commensal bacteria in the gastrointestinal tract, induce the virulence factors leading to disease in humans and shrimp. Here, we show that secondary bile acids also activate this pathogen's type VI secretion system 1, a toxin delivery apparatus mediating interbacterial competition. This finding implies that Vibrio parahaemolyticus exploits secondary bile acids to activate its virulence factors and identify the presence of commensal bacteria that it needs to outcompete in order to colonize the host.IMPORTANCEBacterial pathogens often manipulate their host and cause disease by secreting toxic proteins. However, to successfully colonize a host, they must also remove commensal bacteria that reside in it and may compete with them over resources. Here, we find that the same host-derived molecules that activate the secreted virulence toxins in a gut bacterial pathogen, Vibrio parahaemolyticus, also activate an antibacterial toxin delivery system that targets such commensal bacteria. These findings suggest that a pathogen can use one cue to launch a coordinated, trans-kingdom attack that enables it to colonize a host.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp.

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

Bimetallic nanoparticles with sulfated galactan eliminate Vibrio parahaemolyticus in shrimp Penaeus vannamei.

Bimetallic (Au/Ag) nanoparticles (BNPs) have shown enhanced antibacterial activity compared to their monometallic counterparts. Sulfated galactans (SG) are a naturally occurring polymer commonly found in red seaweed Gracilaria fisheri. They are biocompatible and biodegradable and environmentally friendly. In this study, we utilized SG in combination with BNPs to develop composite materials that potentially enhance antibacterial activity against shrimp pathogens Vibrio parahaemolyticus and Vibrio harveyi, compared to BNPs or SG alone. BNPs were coated with sulfated galactan (SGBNPs) and characterized using UV-vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, zeta potential, and transmission electron microscopy (TEM). UV-vis spectroscopy analysis revealed that the surface plasmon peaks of BNPs and SGBNPs appeared at 530 nm and 532 nm, respectively. Zeta potential measurements showed that SGBNPs had a negative charge of -32.4 mV, while the BNPs solution had a positive charge of 38.7 mV. TEM images demonstrated the spherical morphology of both BNPs and SGBNPs with narrow size distributions (3-10 nm). Analysis of the FTIR spectra indicated that SG maintained its backbone structure in SGBNPs, but some functional groups were altered. Notably, SGBNPs showed superior antimicrobial and antibiofilm activities against V. parahaemolyticus and V. harveyi compared to SG and BNPs. Furthermore, treatment with SGBNPs significantly down-regulated the expression of virulence-related genes (toxR, cpsQ, and mfpA) for V. parahaemolyticus 3HP compared to the respective control, bacteria treated with BNPs or SG. Diets supplemented with SGBNPs, BNPs, or SG showed no detrimental impact on the growth of shrimp Penaeus vannamei. Shrimp fed with SGBNPs-supplemented feed showed significantly higher survival rates than those fed with BNPs-supplemented feed when infected with 3HP after being on the supplemented feed for seven days and a subsequent number of fifteen days. These findings collectively demonstrate the benefit of using SG capped Au-Ag BNPs as an antibacterial agent for the prevention and control of Vibrio sp. Infection in shrimp while reducing the risk of environmental contamination.

Serodiversity, antibiotic resistance, and virulence genes of Vibrio parahaemolyticus in oysters collected in coastal areas of northwestern Mexico between 2012 and 2020.

This study aimed to determine the prevalence of V. parahaemolyticus in oysters from the northwestern coast of Mexico and to identify the serotypes, virulence factors, and antibiotic resistance of the strains. Oyster samples were collected from 2012 to 2020 from the northwest coast of Mexico; biochemical and molecular methods were used to identify V. parahaemolyticus from oysters; antiserum reaction to determine V. parahaemolyticus serotypes, and PCR assays were performed to identify pathogenic (tdh and/or trh) or pandemic (toxRS/new, and/or orf8) strains and antibiotic resistance testing. A total of 441 oyster samples were collected and tested for V. parahaemolyticus. Forty-seven percent of oyster samples were positive for V. parahaemolyticus. Ten different O serogroups and 72 serovars were identified, predominantly serotype O1:KUT with 22.2% and OUT:KUT with 17.3%. Twenty new serotypes that had not been previously reported in our region were identified. We detected 4.3% of pathogenic clones but no pandemic strains. About 73.5% of strains were resistant to at least one antibiotic, mainly ampicillin and ciprofloxacin; 25% were multi-drug resistant. In conclusion, the pathogenic strains in oysters and antibiotic resistance are of public health concern, as the potential for outbreaks throughout northwestern Mexico is well established.

Elevated temperature and decreased salinity impacts on exogenous Vibrio parahaemolyticus infection of eastern oyster, Crassostrea virginica.

Anthropogenic carbon emissions have resulted in drastic oceanic changes, including increased acidity, increased temperature, and decreased salinity. Anthropogenic carbon emissions have resulted in drastic oceanic changes, including increased acidity, increased temperature, and decreased salinity. Few studies have directly assessed the compounded impact of alterations to oceanic conditions on oyster physiology and the relation to the presence of V. parahaemolyticus. This project investigated the relationship between projected climate scenarios and their influence on both eastern oyster, Crassostrea virginica, and the aquatic bacteria, Vibrio parahaemolyticus. Specifically, we examined whether an increase in water temperature and/or decrease in salinity would impair oyster resistance to V. parahaemolyticus, a human food and waterborne pathogen. Using a culture-dependent approach, our data revealed that the alterations in environmental conditions did not significantly impact the numbers of V. parahaemolyticus numbers within oyster hemolymph or tissues. However, we did observe a dramatic increase in the total amount of bacteria and pathogenic native Vibrio species, Vibrio aestuarianus and Vibrio harveyi. Despite detecting V. parahaemolyticus in most tissues at 7 days post-challenge, oysters were able to reduce bacterial levels below our limit of detection by 28 days of exposure. Furthermore, in our second experimental trial exploring single vs. multiple inoculation of bacteria, we observed that oysters were either able to reduce total bacterial levels to pre-treatment burdens (i.e., below our limit of detection) or die. This study demonstrates that the synergistic effects of elevated temperature and decreased salinity do not inhibit oysters from preventing the long-term colonization of exogenous V. parahaemolyticus. However, our data do show these environmental stressors impact oyster physiology and the native microbiota. This can lead to the proliferation of opportunistic pathogens, which could have impacts on oyster population numbers and ecosystem and human health.

Expression regulation of type III secretion system 2 in Vibrio parahaemolyticus by catabolite activator protein.

Vibrio parahaemolyticus has two sets of type III secretion systems that are major pathogenic factors: T3SS1 (cytotoxicity) and T3SS2 (enterotoxicity). V. parahaemolyticus mainly colonizes the distal small intestine after oral infection and may be exposed to carbon-limiting stress due to the lack of readily available carbohydrates in this environment. Catabolite activator protein (CAP), a transcription factor involved in carbon-limiting metabolism in many Gram-negative bacteria, is well known to be involved in the regulation of the expression of many virulence factors. In this study, we determined the effects of CAP on the expression of T3SSs in this bacterium. Based on a lactate dehydrogenase-based cytotoxicity assay, CAP was found to have a greater contribution to the expression of T3SS2-dependent cytotoxicity than to that of T3SS1. Reverse transcription quantitative PCR revealed decreased expression of many T3SS2-related genes, including vpa1348, in the cap gene deletion mutant compared to the parent strain. CAP was demonstrated to bind near the T-rich elements within the vpa1348 promoter region in an electrophoretic mobility shift assay and DNase I footprinting. CAP also enhanced the expression of vpa1348 in a ß-galactosidase reporter assay. Collectively, these results suggest that CAP is involved in T3SS2-mediated virulence by regulating the expression of vpa1348 in V. parahaemolyticus.

Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

The Impact of Vp-Porin, an Outer Membrane Protein, on the Biological Characteristics and Virulence of Vibrio Parahaemolyticus.

Porins are crucial proteins located in the outer membrane that directly influence antimicrobial resistance mechanisms and virulence in bacteria. In this study, a porin gene (Vp-porin) was cloned in V. parahaemolyticus, and the function of Vp-Porin in biological characteristics and virulence was investigated. The results of sequence analysis showed that Vp-Porin is highly conserved in Vibrio spp., and the predicted 3D structure showed it could form a 20-strand transmembrane ß-barrel domian. Membrane permeabilization provides evidence that the membrane integrity of ∆Vp-porin was damaged and the sensitivity to tetracycline, polymyxin B, rifampicin and cephalothin of ∆Vp-porin obviously increased. In addition, loss of Vp-porin damaged motility due to downregulated flagellar synthesis. In addition, ∆Vp-porin exhibited attenuated cytotoxicity to Tetrahymena. The relative survival rate of Tetrahymena infection with ∆Vp-porin was 86%, which is much higher than that with WT (49%). Taken together, the results of this study indicate that Vp-Porin in V. parahaemolyticus plays various roles in biological characteristics in membrane integrity, antimicrobial resistance and motility and contributes to virulence.

Risk Factors and Effects of Climate Lag on Vibrio Parahaemolyticus Infection in Eastern Coastal Cities of China: A Study Based on Hangzhou City.

Bacterial foodborne diseases caused by Vibrio parahaemolyticus pose persistent challenges to coastal cities in China. In this study, we employed multiple logistic regression analysis and distributed lag non-linear models (DLNM) to investigate the epidemiological characteristics and associated risk factors of vibriosis in the metropolitan area of Hangzhou from 2014 to 2018. Analysis of foodborne cases indicated that certain demographics and occupational factors, including age between 16 and 44 years; houseworkers or unemployed individuals; preference for aquatic and meat products; and dining in collective canteens or catering services contribute to an increased likelihood of V. parahaemolyticus infection. Moreover, a higher per capita GDP and exposure to high temperatures were identified as risk factors for vibriosis. This study highlights the significance of the daily mean temperature as a meteorological factor influencing V. parahaemolyticus infection, with varying lag effects observed depending on temperature conditions. At low temperatures, the risk of infection occurs after a lag of 21 days, whereas at high temperatures, the risk is highest on the same day, while the second infection risk period occurs after a lag of 21 days. These findings provide a spatiotemporal perspective of the risk analysis of foodborne diseases, with a daily timescale and street spatial scale, which contributes to the development of public health strategies and food safety protocols in coastal cities.

Effect of plasma-activated water on planktonic and biofilm cells of Vibrio parahaemolyticus strains isolated from cutting board surfaces in retail seafood markets.

AIMS: This research aimed to analyze cutting board surfaces in seafood markets to find Vibrio parahaemolyticus, assess the isolates' ability to form biofilms, generate and evaluate characteristics of plasma-activated water (PAW), and compare the effect of PAW on planktonic and biofilm cells of the isolated V. parahaemolyticus strains. METHODS AND RESULTS: A total of 11 V. parahaemolyticus strains were isolated from 8.87% of the examined cutting boards. Biofilm-forming ability was evaluated for these isolates at temperatures of 10°C, 20°C, and 30°C using crystal violet staining. Four strains with the highest biofilm potential were selected for further analysis. The pH of the PAW used in the study was 3.41 ± 0.04, and the initial concentrations of hydrogen peroxide, nitrate, and nitrite were 108 ± 9.6, 742 ± 61, and 36.3 ± 2.9 µM, respectively. However, these concentrations decreased significantly within 3-4 days during storage at room temperature. PAW exhibited significant antimicrobial effects on V. parahaemolyticus planktonic cells, reducing viable bacteria up to 4.54 log CFU/ml within 20 min. PAW also reduced the number of biofilm cells on stainless steel (up to 3.55 log CFU/cm2) and high-density polyethylene (up to 3.06 log CFU/cm2) surfaces, although to a lesser extent than planktonic cells. CONCLUSIONS: PAW exhibited significant antibacterial activity against V. parahaemolyticus cells, although its antibacterial properties diminished over time. Furthermore, the antibacterial activity of PAW against biofilm cells of V. parahaemolyticus was less pronounced compared to the planktonic cells. Therefore, the actual effectiveness of PAW in seafood processing environments can be affected by biofilms that may form on various surfaces such as cutting boards if they are not cleaned properly.

In silico identification and functional study of long non-coding RNA involved in acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus infection in white shrimp, Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) caused by toxin-producing Vibrio parahaemolyticus (VpAHPND) has severely affected shrimp production. Long non-coding RNA (lncRNA), a regulatory non-coding RNA, which can play important function in shrimp disease responses. This study aimed to identify and investigate the role of lncRNA involved in VpAHPND infection in Pacific white shrimp, Litopenaeus vannamei. From a total of 368,736 de novo assembled transcripts, 67,559 were identified as putative lncRNAs, and only 72 putative lncRNAs showed differential expression between VpAHPND-infected and normal shrimp. The six candidate lncRNAs were validated for their expression profiles during VpAHPND infection and tissue distribution using RT-qPCR. The role of lnc2088 in response to VpAHPND infection was investigated through RNA interference. The result indicated that the suppression of lnc2088 expression led to an increase in shrimp mortality after VpAHPND infection. To explore the set of genes involved in lnc2088 knockdown, RNA sequencing was performed. A total of 275 differentially expressed transcripts were identified in the hepatopancreas of lnc2088 knockdown shrimp. The expression profiles of five candidate metabolic and immune-related genes were validated in lnc2088 knockdown and VpAHPND-infected shrimp. The result showed that the expression of ChiNAG was significantly increased, while that of NCBP1, WIPF2, and NFKB1 was significantly downregulated in ds2088-injected shrimp. Additionally, the expression of NFKB1, NCBP1 and WIPF2 was significantly increased, whereas that of ChiNAG and CUL5 were significantly decreased after infection with VpAHPND. Our work identified putative lncRNA profiles in L. vannamei in response to VpAHPND infection and investigated the role of lncRNA in shrimp immunity.

Mobile-CRISPRi protocol optimization for Vibrionaceae.

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl ß-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

Development of Polymerase Chain Reaction-High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni.

Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire's Disease in humans. L. pneumophila is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR-high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.