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 u...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.展开更多
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,...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.展开更多
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 int...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.展开更多
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 structur...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展开更多
Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced b...Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.展开更多
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...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.展开更多
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin prote...The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.展开更多
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 ...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.展开更多
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 i...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.展开更多
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,a...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.展开更多
Vibrio parahaemolyticus is the leading causal agent of human acute gas- troenteritis. Real-time accurate detection means is the key to prevention and control of its spread. This study provided a novel detection strate...Vibrio parahaemolyticus is the leading causal agent of human acute gas- troenteritis. Real-time accurate detection means is the key to prevention and control of its spread. This study provided a novel detection strategy for realizing rapid and specific determination of V. parahaemolyticus by labeling its monoclonal antibody （Ab） with quantum dots （QDs）. The results showed that the fluorescence of these QDs-Ab bioconjugates was quenched by graphene oxide （GO） to produce a bacteri- um capture probe. And the optimal quenched concentration of GO was 60 ng/ml. When the bacterium capture probe was exposed to the target, green color fluores- cence was turned on by releasing the QDs-Ab due to the antibody antigen combi- nation. The detection limit of V. parahaemolyticus was 104 CFU/ml based on 3 times signal-to-noise ratio. The specificity of the FRET sensor towards V. para- haemolyticus was examined by comparing with controls such as V. splendidus, V. alginolyticus, Edwardsiella tarda and Aeromonas hydrophila with the same condition. The controls couldn＇t cause obvious fluorescence alteration, while the target resulted in significant fluorescence enhancement. This strategy could be further used as a universal method for any bacterial determination by changing the conjugated antibod- ies in early disease diagnosis. Therefore, the sensor has good potential to expand its application to the early diagnosis and determination of bacteria.展开更多
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 bacter...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.展开更多
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer m...Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.展开更多
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 pl...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.展开更多
[ 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 ...[ 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.展开更多
This study aimed to evaluate the supplementation of the probiotic Pseudoalteromonas piscicida 1Ub to the biofloc system as an ecofriendly strategy for protecting white shrimp(Penaeus vannamei)from Vibrio parahaemolyti...This study aimed to evaluate the supplementation of the probiotic Pseudoalteromonas piscicida 1Ub to the biofloc system as an ecofriendly strategy for protecting white shrimp(Penaeus vannamei)from Vibrio parahaemolyticus infection.Shrimp with an average body weight of(0.50±0.09)g were reared in 30 glass jars with a working volume of 2.5 L at a density of 20 ind/L.Shrimp were reared for 5 d for each treatment,which included the biofloc system without and with 106 colony forming unit(CFU)per mL probiotic.The regular clear water system was used as control.All treatment groups were challenged with 103,105,and 107 CFU/mL V.parahaemolyticus.For the negative control,shrimp were reared without V.parahaemolyticus.The results showed that the density of V.parahaemolyticus cocultured with P.piscicida 1Ub decreased and the density of V.parahaemolyticus in rearing water and shrimp body in the probiotic-treated group was lower than that in the control group(P<0.05).The survival and immune response(total hemocyte count,phagocytic activity,respiratory burst,phenoloxidase,and superoxide dismutase)of shrimp in the probiotic group was higher than that in the positive control(P<0.05).Moreover,supplementing the biofloc system with the probiotic could protect shrimp hepatopancreas from damage caused by V.parahaemolyticus,regardless of bacterial density.Thus,the supplementation of the probiotic P.piscicida 1Ub in the biofloc system could significantly protect and increase the resistance of shrimp to V.parahaemolyticus infection.展开更多
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 diag...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.展开更多
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 w...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.展开更多
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 cont...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.展开更多
基金funded by grants from the National Key Research and Development Program of China[2018YFC1602201]Key Research and Development Program of Shaanxi Province[2021NY-158]+1 种基金the National Natural Science Foundation of China[31671780]the Agricultural Product Quality and Safety Risk Assessment Foundation of the Ministry of Agriculture and Rural Affairs of the People’s Republic of China[GJFP2020002,GJFP2020003].
文摘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.
基金Major Science and Technology Project of Hainan Province(No.ZDKJ202003)。
文摘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.
基金supported by the National Key Research and Development Plan of China[2018YFC1602500]the Natural Science Foundation of Tianjin[19JCZDJC39900]
文摘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.
基金supported by the National Natural Science Foundation of China [81601809]the Natural Science Foundation of Jiangsu Province [BK20160505]
文摘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
基金supported by National High Technology Research and Development Program of China grant(2006AA1003062006AA100307)
文摘Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.
基金The National Natural Science Foundation of China under contract No.30671596
文摘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.
基金Supported by the Dalian Municipal Government of China (No. 2007B11NC069)the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)the Grant of Dalian Fisheries University (No. SY2007005)
文摘The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.
基金supported by grants from the National Natural Science Foundation of China[Grant No.82072239]。
文摘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.
文摘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.
基金supported by grants from the Natural Science Foundation of Chongqing(cstc2020jcyj-msxm X0685)the Science and Technology Research Program of Chongqing Municipal Education Commission(KJQN202001231)+1 种基金the National Natural Science Foundation of China(NSFC)(No.31201372)Science and Technology Project of Wanzhou in 2020。
文摘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.
基金Supported by Shandong Scientific and Technological Development Program(2014GHY115024)~~
文摘Vibrio parahaemolyticus is the leading causal agent of human acute gas- troenteritis. Real-time accurate detection means is the key to prevention and control of its spread. This study provided a novel detection strategy for realizing rapid and specific determination of V. parahaemolyticus by labeling its monoclonal antibody （Ab） with quantum dots （QDs）. The results showed that the fluorescence of these QDs-Ab bioconjugates was quenched by graphene oxide （GO） to produce a bacteri- um capture probe. And the optimal quenched concentration of GO was 60 ng/ml. When the bacterium capture probe was exposed to the target, green color fluores- cence was turned on by releasing the QDs-Ab due to the antibody antigen combi- nation. The detection limit of V. parahaemolyticus was 104 CFU/ml based on 3 times signal-to-noise ratio. The specificity of the FRET sensor towards V. para- haemolyticus was examined by comparing with controls such as V. splendidus, V. alginolyticus, Edwardsiella tarda and Aeromonas hydrophila with the same condition. The controls couldn＇t cause obvious fluorescence alteration, while the target resulted in significant fluorescence enhancement. This strategy could be further used as a universal method for any bacterial determination by changing the conjugated antibod- ies in early disease diagnosis. Therefore, the sensor has good potential to expand its application to the early diagnosis and determination of bacteria.
基金Supported by the National Basic Research Program of China(973 Program)(No.2012CB114405)the Special Foundation Under the Construction Program for the“Taishan Scholarship”of Shandong Province of Chinathe Program for Chinese Outstanding Talents in Agricultural Scientific Research
文摘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.
基金Supported by the Key Laboratory Foundation of the Educational Department of Liaoning Province (No. 2009S024)the Dalian Municipal Government of China (No. 2007B11NC069)the Grant of Dalian Ocean University (No. SY2007005)
文摘Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.
基金Supported by Technology Development Program of Scientific Research Institutes of Hainan Province(11-20410-0010,11-20410-0009)
文摘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.
基金funded by the Science and Technology Projects of the State Administration of Quality Supervision,Inspection and Quarantine(2010IK016)
文摘[ 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.
基金financially supported by the Ministry of Education,Culture,Research,and Technology of the Republic of Indonesia for funding the present study in the form of the Pendidikan Magister menuju Doktor untuk Sarjana Unggul(PMDSU)program scholarship(Grant number:200/SP2H/PMDSU/DRPM/2020).
文摘This study aimed to evaluate the supplementation of the probiotic Pseudoalteromonas piscicida 1Ub to the biofloc system as an ecofriendly strategy for protecting white shrimp(Penaeus vannamei)from Vibrio parahaemolyticus infection.Shrimp with an average body weight of(0.50±0.09)g were reared in 30 glass jars with a working volume of 2.5 L at a density of 20 ind/L.Shrimp were reared for 5 d for each treatment,which included the biofloc system without and with 106 colony forming unit(CFU)per mL probiotic.The regular clear water system was used as control.All treatment groups were challenged with 103,105,and 107 CFU/mL V.parahaemolyticus.For the negative control,shrimp were reared without V.parahaemolyticus.The results showed that the density of V.parahaemolyticus cocultured with P.piscicida 1Ub decreased and the density of V.parahaemolyticus in rearing water and shrimp body in the probiotic-treated group was lower than that in the control group(P<0.05).The survival and immune response(total hemocyte count,phagocytic activity,respiratory burst,phenoloxidase,and superoxide dismutase)of shrimp in the probiotic group was higher than that in the positive control(P<0.05).Moreover,supplementing the biofloc system with the probiotic could protect shrimp hepatopancreas from damage caused by V.parahaemolyticus,regardless of bacterial density.Thus,the supplementation of the probiotic P.piscicida 1Ub in the biofloc system could significantly protect and increase the resistance of shrimp to V.parahaemolyticus infection.
基金financial supports from National High Technology Research and Development Program of China(No.2007AA10Z430)National Natural Science Foundation of China(No.30700535)Program for New Century Excellent Talents in Fujian Province University,and Fok Ying Tong Education Foundation(No.111032)
文摘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.
基金supported by grants from the Ministry of Sci-ence and Technology of China(Nos.2012AA101601 and 2011DFA31220)the National Natural Science Foundation of China(Grant Nos.31171690,30972485,31000779 and U1031003)the Science and Technology Commission of Shanghai Municipality(Nos.10DZ0503500 and 10142201300).
文摘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.
基金supported by the Ministry of Education (MOE)academic research fund (AcRF)TIER 1 Project and the‘Study of important foodborne viruses from relevant foods in Singapore' (R-160-000-A79-114),Singapore。
文摘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.
