| BackgroundVibrio parahaemolyticus is a halophilic gram-negative bacterium which is widely distributed in the warm climate of estuaries,oceans,coastal areas and in aquatic products.The bacterium not only causes damage to aquaculture,but can also infect people through contaminated food,leading to infections,outbreaks and even epidemics.Vibrio parahaemolyticus is a major foodborne pathogen in coastal areas of China,and the public health risk of Vibrio parahaemolyticus is likely to increase with increased aquaculture and consumption,as well as climate and environmental changes.Surveillance and analysis of Vibrio parahaemolyticus,as well as genomic epidemiological studies of the pathogen,can deepen the understanding of its genetic variation,distribution and spread patterns,and can provide support for the tracing and prevention and control of Vibrio parahaemolyticus infections.The treatment of Vibrio parahaemolyticus infections requires the use of antibiotics,which not only leads to the development of bacterial resistance,but also poses a threat to environmental safety and population health when antibiotics remain in the environment.Therefore,there is a need to find green and safe biocontrol agents to remove and control Vibrio parahaemolyticus contamination in the environment and food.Phages are natural bacterial inhibitors with the advantages of universality,specificity,and safety.However,there is also competition and co-evolution between strains and phages.Phage isolation is needed to target Vibrio parahaemolyticus currently carried and transmitted by seafood,and to screen for phages with high clearance capacity against,prevalent strains.In addition,genomic data information on phage is still scarce,so more studies need to be carried out to further improve the phage genome database for resource accumulation,characterization studies,screening of highly enzymatic phage and even genetic modification for enhanced lysis capacity.Shellfish in nature inhabit coastal and estuarine environments,and edible shellfish,one of the major cultured seafood,often contain a complex microbiota including a variety of pathogenic and non-pathogenic Vibrio species,which could potentially serve as a model for studying Vibrio parahaemolyticus colonisation and phage clearance of seafood-contaminated Vibrio parahaemolyticus.Objectives1.To analyse the microbiological characteristics of Vibrio parahaemolyticus monitored in four coastal provinces in China in recent years,to analyse the virulence and resistance characteristics,the prevalent dominant serotypes,the dominant clonal groups and their spatial and temporal distribution by using genomics,and to assess the risk of Vibrio parahaemolyticus outbreaks and epidemics.2.Optimising the method of phage enrichment in water to improve the efficiency of phage isolation and macrovirus sequencing analysis.It was also used to enrich for the isolation of Vibrio parahaemolyticus phages from seafood.3.To isolate and obtain Vibrio parahaemolyticus virulent phages,carry out biological and genomic profiling,determine their lytic capacity against pathogenic strains of Vibrio parahaemolyticus,screen for phages with high lytic capacity and assess their potential as biological agents for the removal of Vibrio parahaemolyticus.4.Preliminary modeling of Vibrio parahaemolyticus infection of clams in shellfish for assessing the effectiveness of using phages to remove Vibrio parahaemolyticus contamination from clams and providing a method for applying phages to reduce Vibrio parahaemolyticus contamination in seafood.Methods1.Microbiological characteristics and genomic epidemiological analysis of Vibrio parahaemolyticus in four coastal provinces.The genomic data of Vibrio parahaemolyticus collected,sequenced and uploaded in Shandong,Zhejiang,Guangdong and Fujian provinces from 2019-2022 were collated and analysed,and all strains were identified for serotypes,virulence genes and drug resistance genes.The results were used to determine the pathogenicity and prevalence of Vibrio parahaemolyticus.The phylogenetic analysis was combined with epidemiological data to analyse the spatial and temporal distribution of Vibrio parahaemolyticus.2.Establishment of a method for phage enrichment in water bodiesBased on the principle of iron flocculation,Klebsiella pneumoniae phage was used as the experimental material to determine the optimal iron ion concentration and suitable membrane filter to establish the enrichment method.Fluorescence staining observation,determination of fluorescence value and real-time fluorescence PCR assay were used to evaluate the enrichment effect of different sources and concentrations of phage samples,hospital wastewater samples were collected for phage enrichment and isolation,and further analysis of the enrichment effect of the method on phage in natural actual water samples.3.Phage isolation,biological characterization,and genomic analysisSamples of venerupis philippinarum,scapharca subcrenata,scallops,oysters and crabs were collected and phages of Vibrio parahaemolyticus were isolated and purified using an Agar double layer plate method.Determine the basic characteristics of these phages such as the range of infected hosts,Optimal Multiplicity Of Infection and the one-step growth curve.The phage genome sequence was determined and functional gene prediction was performed.The terminase large subunits and major capsid proteins were selected for phage genomic classification and phylogenetic analysis.4.Phage clearance of Vibrio parahaemolyticus from venerupis philippinarum(1)Determination of the effectiveness of phage removal of Vibrio parahaemolyticus biofilmsThe biofilm of 123 strains of Vibrio parahaemolyticus was stained by gentian violet and lysed using dimethyl sulfoxide.The OD600 before staining and OD570 after lysing were determined and the biofilm formation rate of Vibrio parahaemolyticus was calculated based on the OD570/OD600.Phage was added to the biofilm and the OD500 and OD570 were measured to observe the effect of phage on biofilm removal within 24 hours.(2)Determination of Vibrio parahaemolyticus colonization in venerupis philippinarum and the effect of phage clearancePrimers for conserved Vibrio parahaemolyticus genes were designed and the bioluminescent plasmids were modified by PCR amplification,vector plasmid extraction,double digestion,ligation and transformation.Bioluminescent Vibrio parahaemolyticus was constructed by splice transfer and its bioluminescence intensity was measured.The venerupis philippinarum were decolonised and incubated in a culture medium containing bioluminescent bacteria to observe colonisation;after colonisation was found,specific phages were added to observe and determine the effect of phage clearance.Results1.Epidemiological analysis of the Vibrio parahaemolyticus genomeThe 633 strains of Virio parahaemolyticus collected from 2019-2022 were analysed,including 613 clinical isolates,5 environmental isolates and 15 food isolates.Eight serogroups and 17 serotypes were identified by genomic analysis,with the largest number of O4:K4 serotypes(298),followed by O3:K6(112)and O4:KUT(92).the dominant serotype in zhejiang province in 2019 was O3:K6,shifting to O4:K4 in 2020-2022.the dominant serotype in shandong province in 2020-2022 was O4.The predominant serotype in Guangdong Province is O4K4 in 2019-2021,with a large number of Vibrio parahaemolyticus serotypes of O3:K6 in 2022.Vibrio parahaemolyticus infections were mainly found in people aged 18-65 years,with a higher proportion of males than females in the other three provinces,except for Zhejiang Province,where the proportion of infected males and females was close.Phylogenetic analysis showed that Vibrio parahaemolyticus serotype O3:K6 and Vibrio parahaemolyticus serotype O4:K4 were closely related.serotype O4:KUT was located on two different clonal groups,one of which was the new dominant clonal group,and serotype O4:K8 was more closely related to the dominant clonal group.Virulence gene analysis showed that 153 of the 633 Vibrio parahaemolyticus strains contained genomic islands of VPaI-3 to VPaI-6.These strains were mainly serotypes O3:K6 and O4:K4,all of which had the genomic signature of pandemic strains.The secretory system TTSS1 and T6SS2 gene clusters were carried at a high rate(573/633),and 573 Vibrio parahaemolyticus strains contained both secretory systems.Resistance gene analysis showed that only six of the 633 Vibrio parahaemolyticus strains carried three or more resistance genes.70%(441/633)of the strains carried two resistance genes(tet(35),blaCARB-22),with one strain of Vibrio parahaemolyticus serotype 03:K6 carrying three resistance genes(tet(35),blaCARB-22,blaTEM-116)and one strain of Vibrio parahaemolyticus serotype O4:K4 1008_GD21VP041 containing nine resistance genes,blaTEM-116),and O4:K4 serotype Vibrio parahaemolyticus 1008_GD21 VP041 contained nine resistance genes.Pathogenicity analysis showed that both food and environmental strains were nonpathogenic and did not carry the tdh and trh genes.81%(499/613)of the 613 clinical strains were pathogenic and none of the major serotype O3:K6,O4:K4 and O4:KUT isolates carried the trh gene.75%(476/633)of the strains had pandemic strain 75%(476/633)of the strains had pandemic characteristics,while the food and environmental sources were all nonepidemic.2.Establishment of an enrichment method for FeCl3-PVDF membrane filtersBased on the principle of iron flocculation,a method was developed for the enrichment of phage in natural water using PVDF membrane filters at a concentration of 50 mg/L of iron ions.The recoveries of phage from different concentrations and samples were 93%～100%;the observation under a multifunctional microscope showed that the phage in the enriched water samples were significantly increased,and the fluorescence value of the enriched water samples was about 13 times higher than that before enrichment by enzyme standardization;after enrichment of the actual water samples from the external drainage of the hospital,the positive separation rates of phage in the enriched and unenriched groups were 23%and 4%respectively,and the fluorescence value of the enriched group was 2～24 times higher than that of the unenriched group.The fluorescence values of the concentrated group were 2-24 times higher than those of the unenriched group.3.Isolation,biological characterization and bioinformatics analysis of six Vibrio parahaemolyticus phagesTwo strains of Vibrio mimicus phage(Vm72-P,Vm73-P),two strains of Vibrio algolyticus phage(Va40-P,Va76-P)and six strains of Vibrio parahaemolyticus phage(VP47-P,VP48-P,VP50-P,VP53-P,VP80-P,VP81-P)were isolated from the samples.The Optimal Multiplicity Of Infection,one-step growth curve,incubation period and outbreak period of the six Vibrio parahaemolyticus phage strains were characterized.The six phage strains had different pH ranges and temperature tolerances,with lysis rates ranging from 1%to 88%for the 139 Vibrio parahaemolyticus strains tested.The genome sequences of the six phage strains are all different,ranging from 41 kbp to 86 kbp in full length,all are double-stranded DNA and none contain tRNA structures or transmembrane proteins.4.Determination of the effectiveness of phage clearance of Vibrio parahaemolyticus(1)The biofilm formation rate of the 123 Vibrio parahaemolyticus strains tested was 54%in the test tubes,including 29%for the six O3:K6 serotype Vibrio parahaemolyticus strains,and the phage VP80-P had obvious scavenging effect on the biofilm formed by the strain in 24 hours.(2)Four O3:K6 serotype bioluminescent Vibrio parahaemolyticus strains were successfully constructed,with Vp1200(pBBR-Vp0780-luxCDBAE)having the highest luminescence intensity and maintaining it for a longer period of time.After 9 h of incubation in the infection solution,luminescence was detected on the internal soft tissues of the venerupis philippinarum,with no light intensity in the shell part,indicating that Vibrio parahaemolyticus colonized the soft tissues of the venerupis philippinarum and rarely colonized the shell part;the luminescence signal was basically undetectable in the internal soft tissues and shell of the venerupis philippinarum after 9 h of addition of phage,indicating that the colonized Vibrio parahaemolyticus could be ablated by phage lysis.Conclusions1.In 2019-2022,there is a common prevalent dominant serotype of Vibrio parahaemolyticus in four coastal provinces of China,with O4:K4,O4:KUT and O3:K6 being the most common types,with certain genomic differences in strains from different provinces in different years,and multiple dominant genomic clonal clusters were found,suggesting the existence of dispersal;most clinical isolates are endemic strains,carrying multiple virulence genes,but carrying The majority of clinical isolates were endemic strains carrying multiple virulence genes,but it was not common for them to carry resistance genes.We should continue to strengthen the isolation and genomic determination of Vibrio parahaemolyticus,and provide more accurate genomic surveillance tools and basis for decision making through comparative analysis for the detection of predominant Vibrio parahaemolyticus epidemic types,outbreaks and strain spread.2.The FeCl3-PVDF membrane filter method has been optimized for simple,efficient and rapid enrichment and separation of phages from aqueous bodies as well as from samples treated as liquids.3 Vibrio parahaemolyticus phages with a broad strain lysis spectrum,good tolerance to pH and temperature,some capable of lysing Vibrio parahaemolyticus in the biofilm state,with potential as Vibrio parahaemolyticus biological removal agents were obtained4.Vibrio parahaemolyticus was mainly colonized on the soft tissues of venerupis philippinarum,and basically not on the shells;a preliminary model of Vibrio parahaemolyticus colonized venerupis philippinarum was established,and practical tests observed that the phage VP80-P had a better removal effect on clams,which has a better application prospect of removing contaminated Vibrio parahaemolyticus from seafood as one of the means of bio-preventive control. |
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