Virulence changes in Vibrio parahaemolyticus during the freezing of Penaeus chinensis

Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we used frozen Penaeus chinensis as examples,and the most virulent factors of V.parahaemolyticus,including amounts,viable but nonculturable(VBNC)status,toxins TDH and TRH,and virulence genes tdh and trh,were determined.Bacterial quantities were signifi cantly reduced during drain and sprinkling phases,but caused by different factors.By SYTO9 and PI staining showed that washing was the main reason for the bacterial reduction at the drain phase,while the strain entering VBNC state was another reason at sprinkling phase.Their hemolysis toxicity,produced by TDH and TRH,became stronger after inoculation on shrimp,and could be detected throughout the process.Moreover,tdh and trh also exhibited trends similar to that of the hemolysis toxicity test.tdh was almost to a two-fold expression level during ice-glazing phase,while trh only express at a low level,less than half of the expression level before inoculation.These results demonstrated that the strains were not dead during freezing process,but became VBNC cells,which still produced and accumulated toxins,especially TDH,the most virulent factor.

Monitoring and analysis of contamination of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou

Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,Vibrio parahaemolyticus and Vibrio alginolyticus were detected according to the National Food Safety Standard Food Microbiological Examination of Vibrio parahaemolyticus(GB 4789.7-2013),and they were identified by real-time fluorescence PCR.The detection of Vibrio parahaemolyticus and Vibrio alginolyticus in different kinds of seafood,different years and different quarters was analyzed.Results:A total of 119 seafood samples were collected.Among them,24 samples were positive with Vibrio parahaemolyticus,with a positive rate of 20.1%;46 samples were positive with Vibrio alginolyticus,with a positive rate of 38.7%.Among various types of seafood,shrimp have the highest positivity rate for Vibrio parahaemolyticus at 50%,while shellfish have the highest positivity rate for Vibrio alginolyticus at 48%.Comparing between monitoring years,the positive rate of Vibrio alginolyticus was the highest in 2021(76.7%),while the positive rate of Vibrio parahaemolyticus was the highest in 2022(25%).Comparing between different quarters,the positivity rate for Vibrio alginolyticus was found to be highest in the second quarter at 80%,while the positivity rate for Vibrio parahaemolyticus was highest in the fourth quarter at 33.3%.There were statistically significant differences(P<0.05)in the positivity rate for Vibrio alginolyticus in different years and quarters,as well as in the positivity rate for Vibrio parahaemolyticus in different types and quarters.Conclusion:Vibrio parahaemolyticus and Vibrio alginolyticus were found in seafood products in Haikou City from 2020 to 2022.It is recommended that relevant departments strengthen supervision to ensure the safety of seafood products consumed by the public.

Purification,characterization and production optimization of a vibriocin produced by mangrove associated vibrio parahaemolyticus

Objective:To identify a potential bacterium which produces antimicrobial peptide(vibriocin),and its purification,characterization and production optimization.The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem.Methods:The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis.The antibacterial activity was recognised by using agar well diffusion method.The vibriocin was purified using ammonium sulphate precipitation,butanol extraction,gel filtration chromatography,ion-exchange chromatography and subsequently,by HPLC.Molecular weight of the substance identified in SDS-PAGE.Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables.Results:The objective bacterium was identified as Vibrio parahaemolyticm.The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi.The peptide act stable in a wide range of pH,temperature.UV radiation,solvents and chemicals utilized.An overall^20%of vibriocin production was improved,and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin.Conclusion:The vibriocin identified here would he an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification

Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.

ToxR Is Required for Biofilm Formation and Motility of Vibrio Parahaemolyticus

Vibrio parahaemolyticus, the leading cause of seafood-borne gastroenteritis, has the ability to form biofilms on biotic and abiotic surfaces. Biofilm formation is a complicated process involving many specific structures and regulatory processes. The most significant of the structures and processes include polar and lateral flagella, mannose-sensitive hemagglutinin typeⅣpili, chitin-regulated pili,capsular polysaccharide (CPS), exopolysaccharide

Nonculturability of the pathogenic Vibrio parahaemolyticus in live culture of Grateloupia turuturu is associated with bacterial attachment to the algal thalli

The invasive red alga Grateloupia turuturu Yamada could turn Vibrio parahaemolyticus into nonculturable state in live algal culture.In order to elucidate the mechanism of such an effect,a series of culture experiments were performed in this investigation based on three hypothesized causes,namely bacterial attachment,production of reactive oxygen species （ROS） and the discharge of water soluble secondary metabolic compounds.The results reveal that attachment to the thallus surface of G.turuturu was the major reason for the decrease of V.parahaemolyticus in seawater.Further investigations show that V.parahaemolyticus attachment to the surface of algal thallus in live cultures of seaweeds was a common phenomenon.However,the disappearance of the culturability of V.parahaemolyticus occurred only on the thallus of G.turuturu over 72 h among all six algal species tested.Electron microscopic scanning shows that most of V.parahaemolyticus attached to G.turuturu changed from the initial normal bacilli to coccoid-shape after 72 h.The enclosure experiments by enclosing the algal thallus in tubes demonstrate that the nonculturability of V.parahaemolyticus in the water of live culture of G.turuturu occurred after the physical contact of the V.parahaemolyticus to the alga.The capacity of G.turuturu in affecting the culturability of V.parahaemolyticus was not influenced after inhibition of photosynthesis by treatment of 3（3,4dichlorophenyl）-1 ,1dimethyl urea （DCMU） at non-lethal levels.Production of reactive oxygen species after addition of live culture of bacteria was excluded by on-line analyzing the oxidation of dichlorohydrofluorescein （DCFH） to dichlorofluorescein （DCF） in the presence of peroxidase on a VersaFluor fluorometer.

H-NS Represses Biofilm Formation and c-di-GMP Synthesis in Vibrio parahaemolyticus

Objective This study aimed to investigate the regulation of histone-like nucleoid structuring protein(H-NS)on biofilm formation and cyclic diguanylate(c-di-GMP)synthesis in Vibrio parahaemolyticus RIMD2210633.Methods Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining,quantification of c-di-GMP,quantitative real-time polymerase chain reaction,LacZ fusion,and electrophoretic-mobility shift assay.Results The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V.parahaemolyticus RIMD2210633.H-NS can bind the upstream promoter-proximal DNA regions of scrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 to repress their transcription.These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism.Conclusion One of the mechanisms by which H-NS represses the biofilm formation by V.parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.

Impact of Vibrio parahaemolyticus and white spot syndrome virus(WSSV)co-infection on survival of penaeid shrimp Litopenaeus vannamei

White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P <0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.

Spontaneous small bowel perforation secondary to Vibrio parahaemolyticus infection:A case report

BACKGROUND Vibrio pararhaemolyticus(V.parahaemolyticus),a pathogen that commonly causes gastroenteritis,could potentially lead to a pandemic in Asia.Its pathogenesis and molecular mechanisms vary,and the severity of illness can be diverse,ranging from mild gastroenteritis,requiring only supportive care,to sepsis.CASE SUMMARY We outline a case of a 71-year-old female who experienced an acute onset of severe abdominal tenderness after two days of vomiting and diarrhea prior to her emergency department visit.A small bowel perforation was diagnosed using computed tomography.The ascites cultured revealed infection due to V.parahaemolyticus CONCLUSION Our case is the first reported case of V.parahaemolyticus-induced gastroenteritis resulting in small bowel perforation.

Deletion of the waaf gene affects O antigen synthesis and pathogenicity in Vibrio parahaemolyticus from shellfish

Vibrio parahaemolyticus is the main cause of foodborne gastroenteritis,which is widely distributed in shellfish and other seafood.Most V.parahaemolyticus are nonpathogenic,and only a few types,such as serotype O3:K6,are pathogenic,which is also the most prevalent strain in Asia.However,the relationship between this serotype and pathogenicity has yet to be established.The waaf gene is located in the O antigen synthesis gene cluster.Thus,we constructed a waaf gene deletion mutant(i.e.,Δwaaf)of wild-type(WT)which isolated from shellfish serotype O3:K6 via chitin-mediated transformation technology.We then constructed theΔwaaf complementary strain(i.e.,C-△waaf)via the Escherichia coli S17λpir strain by conjugation.The basic physiological characteristics,adhesion to Caco2 cells,and pathogenicity of the WT,△waaf,and C-△waaf strains were compared.Growth curves showed no remarkable differences between the WT andΔwaaf strains.However,theΔwaaf strain non-reactive to O3 antisera and other 12 O-group antisera of V.parahaemolyticus.Moreover,the number of flgella and extracellular polysaccharides decreased,the adhesion decreased,and the pathogenicity weakened.These characteristics of the C-△waaf strain were similar to those of the WT strain These results indicated that the waaf gene is vital to the serotype in V.parahaemolyticus,and changes in O antigen could affect the pathogenicity of this bacterium.This study will be helpful to understand the pathogenic mechanism of V.parahaemolyticus.

Phytochemical analysis and antibacterial activities of Eleutherine bulbosa(Mill.) Urb. extract against Vibrio parahaemolyticus

Objective: To analyze compounds in Eleutherine bulbosa(E. bulbosa)(Mill.) Urb. extract and to determine its antibacterial capability against Vibrio parahaemolyticus(V. parahaemolyticus).Methods: E. bulbosa bulb extract was preprared using 96% ethanol by the maceration method. Phytochemical investigation of E. bulbosa extract was analyzed using GC-MS, spectrophotometry and titrimetry methods. The zone of inhibition was identified by the diffusion agar method. The minimum inhibitory concentration and minimum bactericidal concentration were determined using the plate count method. The inhibitory rate against V. parahaemolyticus was determined by the microdilution method. Cellular leakage was evaluated by spectrophotometry and cellular damage was observed by scanning electron microscopy. Results: GC-MS analysis showed the high compound of the E. bulbosa extract was securixanthone E(7-hydroxy-1,2-dimethoxyxanthone). The compound groups also included fatty acid esters, isoquinolines, naphthalenes, and phenolics. The total phenolic content was(2.50 ± 0.00) mg/g, flavonoid(6.61 ± 0.00) mg/g, and tannin(0.03 ± 0.00)%. The greatest zone of inhibition and inhibitory rate were(11.83 ± 0.06) mm and(91.32 ± 2.76)%, respectively, at 10 mg/m L. The minimum inhibitory concentration was 0.156 mg/m L, while the minimum bactericidal concentration was 10 mg/m L. The E. bulbosa extract caused leakage and cellular damage to V. parahaemolyticus. Conclusions: The E. bulbosa extract possesses inhibitory activities against V. parahaemolyticus and causes cellular leakage and damage.

Preliminary Study on Immuno-gold Chromatography Assay for Rapid Detection of Vibrio parahaemolyticus

[ Objective] Vibrio parahaemolyticus was chosen as the material to make rabbit anti- V. parahaemolyticus polyclonal antibody, the immuno-gold chromatography assay （IGCA） was used to develop immunogold labeling test strips for detecting V. parahaemolyticus. [Methods] Rabbit anti-V, parahaemolyticus IgG-2 was used to wrap nitrocellulose membrane detection line, goat anti-rabbit IgG was used to wrap control line, and im- muno-gold was used to label rabbit anti-V, parahaemolyticus IgG-1 to establish the immuno-gold chromatography assay （IGCA） for detection of V. parahaemolyticus. [Results] Test strip detection lines of positive results along with the control line were all red, test results could be got in only five to fifteen minutes, the minimum detectable amount of this method to V. parahaemolyticus was 3.60 x 104 cfu/ml, and cross reactions wouldnt occur when detecting common intestinal bacteria like Vibrio alginolyticus, vibrio cholerae, Vibrio damsela, Vibrio metschnikovii, Citrobacter freundii and salmonella etc. The test results were undifferentiated when the test strips were stored at 4 ~C for six months, stored at room temperature for three months or stored at 37 ~C for one month. [ Condusionl The established test strip assay was simple and rapid during operation, with high sensitivity, strong specificity and good stability, the test results were easy to be observed and judged, and this assay was very suitable for grassroots breeding department application.

Screening and Identification of a Bacterial Strain Antagonistic to Vibrio parahaemolyticus

In the present study, bacterial strains were isolated from the shrimp （Penaeus orientalis） ponds in different regions of Hainan Province, and tested for their antagonistic activity against Vibrio species on 2216E plates, using V. parahaemolyticus, V. harveyi and V. anguillarum as the indicator bacteria. As a result, an antagonistic strain was screened out and numbered 20160522Z-10 （referred to as Z-10）. Then, the antibacterial activity and minimum inhibitory concentration （MIG） of Z-10 against the indicator bacteria, and its antibacterial effect against other six Vibrio species were determined. The results proved that Z-10 was antagonistic to all the three indicator bacteria, and its MIC against V. parahaemolyticus, V. harveyi and V. anguillarum was 2.5× 10^4, 2.5× 10^5 and 2.5× 10^5 CFU/ml. Additionally, Z-10 had a certain antibacterial effect against other Vibrio species. Finally, the strain was identified as a Pseudoalteromonas sp. by 16S rDNA sequence analysis. The bacterium as a new probiotic, is expected to be used in disease prevention and control in marine aquaculture.

Pre-Enrichment of Estuarine and Fresh Water Environmental Samples with Sodium Chloride Yields in Better Recovery of <i>Vibrio parahaemolyticus</i>

Vibrio parahaemolyticus organisms cause acute gastroenteritis in humans. These bacteria are natural inhabitants of both marine and estuarine ecosystems. In the present study, we investigated the effectiveness of a non-selective enrichment of sediment samples with sodium chloride prior to selective enrichment with alkaline peptone water for a better recovery of V. parahaemolyticus. Sediment samples were collected with or without 1% NaCl from the river Buriganga, located besides Dhaka city and about 400 km away from the Bay of Bengal, and from the estuary of the river Karnaphuli which flows into the Bay of Bengal. Very small number of V. parahaemolyticus (<30 MPN/g) were detected in the sediments of both river and estuary, where NaCl was not added. On the other hand, the number of V. parahaemolyticus increased to more than 40 times (1500 MPN/g) in the river and 32 times (960 MPN/g) in the estuary where NaCl were added. River sediment sample contained the serotype O9:K41 of V. parahaemolyticus and the estuarine sample contained O3:K41 and O3:KUT Our results suggest that a pre-enrichment of environmental samples with 1% NaCl helps V. parahaemolyticus to survive for at least 7 days until they are enriched with alkaline peptone water, for better recovery.

关于嗜盐菌改名为副溶血性弧菌(vibrio parahaemolyticus)的建议

食品卫生细菌学检验方法座谈会于1973年11月6～15日于北京召开。与会全体同志鉴于国内外科学技术发展形势的需要,建议将嗜盐菌改名为副溶血性弧菌vibrio para-

水环境温度对戊二醛杀菌效果的影响

为了解水环境温度对戊二醛(glutaraldehyde,GA)杀菌效果的影响,在20℃、25℃和30℃三个温度条件下,定期检测了2株细菌(副溶血弧菌Vibrio parahaemolyticus、维氏气单胞菌Aeromonas veroni)在不同浓度戊二醛水溶液中的致死情况。检测结果显示,戊二醛对维氏气单胞菌的杀菌率与温度正相关,低药物浓度时(0.45 mg/L),30℃水体中的杀菌率在24 h达到90%以上,而较低温度(20℃)实现90%杀菌率需要27 h;当浓度升至1.8 mg/L时,30℃、5 h杀菌率可以达到90%,20℃时则需要延长至7 h;当更高药物浓度(18 mg/L)时,30℃、20 min可实现90%以上杀菌率,20℃时所需时间相应延长至30 min。戊二醛对副溶血弧菌灭菌效果的影响也呈现相似规律性,当药物浓度0.18 mg/L时,杀菌率分别在15 h(30℃)和18 h(20℃)达到90%以上;当药物浓度1.8 mg/L和18 mg/L时,30℃水体中分别于100 min和3 min达到90%以上的杀菌率;20℃时时间延长至180 min和5 min。该研究结果可为科学合理使用戊二醛提供参考。

Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray

In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA--DNA hybridization. Multiplex PCR was used to simultaneously amplify three diagnostic genes （tlh, tdh andfla） that serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were hybridized to primers immobilized on a microarray, and detected by chemiluminesce with avidin-conjugated alkaline phosphatase. With this method, forty-five samples were tested. Eight known virulent strains （tlh＋/tdh＋/fla＋） and four known avirulent strains （tlh＋/tdh /fla＋） of the V. parahaemolyticus were successfully detected, and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains （tlh-/tdh-/fla＋） of the Vibrio spp. In addition, all the other eighteen strains of non-Vibrio bacteria （tlh-/tdh /fla-） gave negative results. The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp. The results demonstrated that this was an efficient and robust method for identifying virulent strains of V. parahaemolyticus.

Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood

A real time PCR assay for the detection of Vibrio para-haemolyticus in seafood samples was developed using a novel specific target and a competitive internal ampli-fication control(IAC).The specificity of this assay was evaluated using 390 bacterial strains including V.parahaemolyticus,and other strains belonging to Vibrio and non-Vibrio species.The real time PCR assay un-ambiguously distinguished V.parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V.parahaemolyticus colonies.The assays of avoiding interference demonstrated that,even in the presence of 2.1μg genomic DNA or 107 CFU background bacteria,V.parahaemolyticus could still be accurately detected.In addition,the IAC was used to indicate false-negative results,and lower than 94 copies of IAC per reaction had no influence on the detection limit.Ninety-six sea-food samples were tested,of which 58(60.4%)were positive,including 3 false negative results.Conse-quently,the real time PCR assay is effective for the rapid detection of V.parahaemotyticus contaminants in seafood.

Prevalence of Antimicrobial Resistant of Vibrio parahaemolyticus Isolated from Diarrheal Patients——Six PLADs,China,2016−2020

Summary What is already known on this topic?Vibrio parahaemolyticus(V.parahaemolyticus)is frequently resistant to common antimicrobials such as ampicillin and generally highly susceptible to most clinically used antimicrobials.What is added by this report?V.parahaemolyticus were highly resistant to cefazolin and ampicillin:94.4% and 37.0%,respectively.

Electrochemical detection of a Vibrio parahaemolyticus sequence-specific gene based on a gold electrode modified with a single stranded probe

An electrochemical DNA biosensor for specific-sequences detection of Vibrio parahaemolyticus (VP) was fabricated.A singlestranded 20-mer oligonucleotide (ssDNA) and 6-mercapto-1-hexanol (MCH) were immobilized via a thiol linker on gold disk electrodes by self-assembling.The ssDNA underwent hybridization in a hybridization solution containing complementary or non-complementary or single base pair mismatched DNA sequences of VP.Examination of changes in response to these three target DNAs showed that the developed biosensor had a high selectivity and sensitivity.