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.

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.

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.

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.

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-

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.

Photoinactivation of bacteriophage MS2,Tulane virus and Vibrio parahaemolyticus in oysters by microencapsulated rose bengal

Bivalve molluscan shellfish such as oysters are important vectors for the transmission of foodborne pathogens including both viruses and bacteria.Photoinactivation provides a cold-sterilization option against the contamination as excited photosensitizers could transfer electronic energy to oxygen molecules producing reactive oxygen species such as singlet oxygen,leading to oxidative damage and death of the pathogens.However,the efficacy of photoinactivation is very often compromised by the presence of food matrix due to the nonselective reactions of short-lived singlet oxygen with organic matter other than the target pathogens.In order to address this issue,we encapsulated a food-grade photosensitizer rose bengal(RB)in alginate microbeads.An extra coating of chitosan effectively prevented the release of RB from the microbeads in seawater,and more importantly,enhanced the selectivity of the photoinactivation via the electrostatic interactions between cationic chitosan and anionic charge of the virus particles(bacteriophage MS2 and Tulane virus)and the Gram-negative bacteria Vibrio parahaemolyticus(V parahaemolyticus).The treatment of oysters with microencapsulated RB resulted in significantly higher reductions of MS2 phage,Tulane virus and V parahaemolyticus than free RB and non-RB carrying microbeads(P<0.05)tested with both in vitro and in vivo experimental set-ups.This study demonstrated a new strategy in delivering comprehensively formulated biochemical sanitizers in bivalve shellfish through their natural filter-feeding activity and thereby enhancing the mitigation efficiency of foodborne pathogen contamination.

Comparison of gene expression responses of zebrafish larvae to Vibrio parahaemolyticus infection by static immersion and caudal vein microinjection

infection model for studying inflammation and the innate immune response.We investigated the genes and signaling pathways activated by static immersion and caudal vein microinjection infection using transcriptome profiling and reverse-transcription quantitative PCR(RT-qPCR)to compare the innate immune response in 3 days post-fertilization(dpf)zebrafish larvae infected by Vibrio parahaemolyticus Vp13 strain.The median lethal dose(LD50)values at 96 h following immersion and microinjection were 3.63×107 CFU/mL and 5.76×102 CFU/nL,respectively.An innate immune response was initiated after 2 h of incubation with the respective LD50 for each infection method.Six hundred and two genes in the immersion group and 359 genes in the microinjection group were activated and differentially expressed post-infection.Sixty-three Gene Ontology(GO)terms and four Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were significantly enriched in the immersion group,compared with only three GO terms and no KEGG pathways in the microinjection group.Two genes,tnfb and ccl20a.3,were significantly up-regulated in both groups.We speculated that immersion infection may affect initial dorsal determination,cytochromes,and fatty acid-binding proteins,as well as inflammation,while microinjection infection may mainly directly affect the immune response.Infection with doses>LD50(1.09×109 CFU/mL and 1.09×103 CFU/nL by immersion and microinjection,respectively)caused more significant up-regulation of il11a,tnfa,tnfb,il1b,ccl34a.4,ccl20a.3,irak3,cxcl18b,and ccl35.1,suggesting that in addition to the classical innate immunity genes tnfa,tnfb,il1b,and il6,the genes il11a,ccl34a.4,ccl20a.3,cxcl18b,and ccl35.1 were also important for defending against Vp13 infection.These findings highlight the genes involved in the responses of zebrafish to Vp13 infection via different routes and doses,and thus provide the basis for further analyses of immune response signaling pathways.

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。

养虾池中1株副溶血弧菌的分离和鉴定

该研究从凡纳滨对虾(Litopenaeus vannamei)养虾池的水体中分离得到一株致病菌,对其进行生理生化、16S rDNA鉴定,最终确定该致病菌株为副溶血性弧菌(Vibrio parahaemolyticus)。采用该菌进行凡纳滨对虾浸染实验,结果表明:该菌的致病能力较强,在1×10^(5)cfu/mL的浓度下,出现附肢发红、肝胰腺肿大的症状。

12种中药及其组方对3种水产致病菌的抑菌作用

为了筛选出对嗜水气单胞菌(Aeromonas hydrophila)、副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)有抗菌效果的安全药物,本实验采用琼脂扩散法和二倍稀释法测定了12种中药的抑菌效果,同时以“棋盘法+中药互配”研究了对上述致病菌具有较强协同抑制作用的药物组合。结果显示:12种中药对3种供试病原菌均具有不同程度的抑菌效果,丁香、罗汉果、山茱萸、五味子和八角的抑菌作用较强,其抑菌圈直径达到17~44 mm,最小抑菌浓度(MIC)<2.031 mg/mL,最小杀菌浓度(MBC)<4.062 mg/mL;其中,山茱萸的抑菌作用最显著,对嗜水气单胞菌、副溶血弧菌、溶藻弧菌的MIC分别为0.172、0.043、0.086 mg/mL。互配实验结果表明,10种不同的药物组合对嗜水气单胞菌的联合抑菌作用具有显著协同作用;而大部分药物组合对副溶血弧菌的抑菌效果表现为无相关作用,仅五味子和八角对副溶血弧菌的组合效果表现为协同作用。另外,丁香和罗汉果对副溶血弧菌的组合效果表现为拮抗作用。10种中药组合对溶藻弧菌的联合抑菌效果均表现为协同作用。

凡纳滨对虾急性肝胰腺坏死病致病菌的分离鉴定、药敏特性及其组织病理学观察

为查明上海市奉贤区某对虾养殖场疑似患急性肝胰腺坏死病的凡纳滨对虾(Litopenaeus vannamei)的病原菌,本研究从患病凡纳滨对虾肝胰腺组织分离纯化到1株优势菌FHBX-1,并通过人工回归感染试验确定其致病性,利用VITEK-2全自动微生物鉴定仪和16S rRNA、热休克蛋白HSP60基因序列分析进行综合鉴定,并检测其毒力基因。同时采用K-B纸片扩散法检测病原菌的药物敏感性并通过石蜡切片观察患病凡纳滨对虾肝胰腺组织的病理特征。结果显示:菌株FHBX-1经生理生化特征测定、16S rRNA和热休克蛋白HSP60基因序列分析,综合鉴定为副溶血弧菌(Vibrio parahaemolyticus),且携带了急性肝胰腺坏死病相关的毒力基因pirA^(VP)、pirB^(VP),未携带副溶血弧菌临床主要毒力基因耐热直接溶血毒素tdh和相对耐热直接溶血毒素trh基因。人工回归感染试验结果显示,凡纳滨对虾在菌液浓度为1.0×10^(6) CFU/mL浸泡感染试验组,7 d内累计死亡率为80%,患病凡纳滨对虾具有肝胰腺颜色变白、萎缩变小,胃肠变空等症状,从病虾再次分离的优势细菌与原攻毒菌FHBX-1特性相同。组织病理学观察显示,患病凡纳滨对虾肝胰腺上皮细胞严重脱落,肝小管崩塌,呈典型的AHPND病理特征。药敏试验结果表明,菌株FHBX-1对氟苯尼考、多西环素、恩诺沙星等11种抗菌药物敏感,对新霉素、红霉素中度敏感,对链霉素、庆大霉素等5种抗菌药物耐药。本研究可为对虾急性肝胰腺坏死病的流行病学研究及药物防控提供基础依据。

广西凡纳滨对虾源副溶血弧菌3种毒力基因检测及tlh基因的蛋白表达

采用PCR技术检测分离自广西钦州、北海和防城港3市凡纳滨对虾样品中的67株副溶血弧菌(Vibrio parahaemolyticus)3种毒力基因tdh、trh和tlh的携带情况,并将tlh基因进行原核表达,SDS-PAGE和Western blotting鉴定分析。结果显示,67株副溶血弧菌均未扩增出tdh和trh基因,而tlh基因检出率为100.0%,所检副溶血弧菌的毒力基因型为tdh-trh-tlh+。成功构建了原核表达质粒pET-28a-tlh,将其转化至大肠杆菌BL21(DE3)感受态细胞后,经IPTG诱导表达,SDS-PAGE电泳检测得到大小约为53 kDa的产物,与预测值相符。经Western blotting鉴定,该重组表达产物能与抗6×His标签的单克隆抗体发生特异性反应。

中华绒螯蟹苗种生产中副溶血性弧菌病的诊断与防治

为了研究中华绒螯蟹副溶血性弧菌病的症状、诊断及防治方法,从发病蟹苗体上分离出病原体,提纯培养后经全自动微生物分析仪分析鉴定,再用三糖铁培养基培养确定为副溶血性弧菌,然后进行药酶试验。试验表明,其对氧哌嗪青霉素和呋喃西林高度敏感,对庆大霉素、新霉素等中度敏感,对复方新诺明、青霉素G等轻度敏感,而对链霉素、土霉素等则不敏感。在此基础上提出采用生态学方法进行预防,并采用中草药及抗生素进行治疗的防治措施。

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

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

Disinfection effect of adding slightly acidic electrolyzed water to artificial seawater under the condition of static hybrid

Mixed solution of slightly acidic electrolyzed water(SAEW)and artificial seawater was used to investigate the disinfection potential of SAEW in artificial seawater.Inoculated Vibrio parahaemolyticus(suspended in 3%sodium chloride alkaline peptone water and 0.85%sodium chloride water,respectively)was subjected to different mixed-SAEW and SAEW immersion treatments(5-20 mg/L available chlorine concentration(ACC)).In the presence of organic matter,4.07 logCFU/mL significant reduction(p<0.05)was achieved after treating with 20 mg/L mixed-SAEW for 15 min.There was 5.13 logCFU/mL reduction after treating with 15 mg/L SAEW for 15 min.For V.parahaemolyticus suspended in 0.85%sodium chloride solution,it was undetected after 30 s SAEW treatment(5 mg/L ACC)or 120 s mixed-SAEW treatment(10 mg/L ACC).At a ratio of SAEW and artificial seawater at 1:15(V/V),SAEW could inactivate V.parahaemolyticus to undetectable level in artificial seawater in one minute,which was comparable with UV treatment of 10 W.The results indicated high sanitization potential of SAEW against V.parahaemolyticus in aquaculture seawater.