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.

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.

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.

宁德市南美白对虾副溶血弧菌的分离与药敏试验

为给宁德市南美白对虾(Penaeus vannmei)养殖病害提供精准用药依据,从7个海水养殖场南美白对虾体内分离病原菌,通过形态学观察、生理生化和16s rRNA鉴定,确定其分类地位,并进行药物敏感试验。结果表明,分离的9株病原菌在TCBS琼脂培养基中菌落均呈蓝-绿色,通过革兰氏染色镜检可见为革兰氏阴性菌,呈棒状、弧状、卵圆状,无芽孢。在氧化酶反应、葡萄糖、赖氨酸、鸟氨酸、甘露醇、6%盐胨水、8%盐胨水、动力等8项试验中,结果呈阳性,确定为副溶血弧菌。9株副溶血弧菌对青霉素、新霉素、四环素等3种药物耐受比例较高,对恩诺沙星、呋喃唑酮、万古霉素等6种药物均高度敏感。

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。

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-

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。

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。

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.