| Vibrios are a group of genetically and metabolically diverged Gram-negative motile bacteria which distribute in various habitats,especially in marine,brackish and freshwater ecosystems.The high abundance and versatile features makes Vibrios species play important roles in biogeochemical cycling of aquatic ecosystems.Vibrio is also one of the most common,widely distributed and most serious bacterial pathogens in aquaculture.With the development of aquaculture,the outbreak of vibriosis has become more frequent and the Vibrio transmission has become faster and faster,which has brought huge economic losses to the world aquaculture industry.Some Vibrio species,e.g.,V.alginolyticus,V.cholerae,V.cincinnatiensis,V.damsel,V.fluvialis,V.furnisii,V.harveyi,V.metschnikovii,V.mimicus,V.parahaemolyticus and V.vulnificus are known to be human pathogens.Due to the high abundance in marine and aquaculture,various seafood and reared fish and shellfish may be contaminated by Vibrios,consumption of those vibrios contaminated aquatic products can cause human gastroenteritis and lead to disease outbreaks.Epidemiological investigation results show that pathogenic Vibrio is the main pathogen of human diarrheal diseases in both coastal and along the Yangtze River area in Jiangsu province of China.Jiangsu province is neighboring with the Yellow Sea,being a typical temperate climate.The inner river,marine fishing and also the freshwater,marine aquaculture industries are developed in Jiangsu area.There are many varieties of aquatic products and flourishing aquatic product markets are common in Jiangsu province,leading to a high-incidence of Vibrio infection among freshwater produt and seafood consumers in this area.So investigation on the distribution,contamination level,pathogenicity,environmental adaptability and biological control methods of Vibrios in aquatic products originated from Jiangsu province will greatly contribute to the safety of aquatic products,and ultimately the health of people prefer to consuming above products in this aeea.1 Vibrio diversity in aquatic products oringinated from Jiangsu area1.1 High-throughput sequencing analysis of Vibrio diversity in aquatic productsIn the autumn of 2015(early November),90 samples of aquatic products were collected from the five supermarkets and fresh-live aquatic product markets in Yangzhou City.Four samples were divided into and designated as following,CSDS(freshwater culture products from supermarket),CSHS(marine culture products from supermarket),NMDS(freshwater culture products from fresh-live aquatic product market)and NMHS(marine culture products from fresh-live aquatic product market),which were diluted into 10%(v/v)with 3%sodium chloride alkaline peptone water(APW)and immediately spread onto TCBS(Thiosulfate citrate bile salts sucrose agar culture medium)plate to selectively culture vibrioes.The duplicate four samples was pre-cultured in APW overnight firstly and then spread on TCBS plate to selectively culture vibrioes.After overnight culture,the total DNA was extracted from all colonies on the plate and sequenced by 16S rDNA 454 pyrosequencing.The results showed that the colonies on TCBS derived from the four kinds of samples exhibited significant differences in the level of the gate,class,order,family,and genera of bacteria obtained with or without pre-culture.There are significant taxonomy diversities among TCBS colonies originated from freshwater culture product and marine culture product,or from supermarket and fresh-live aquatic product market mentioned above.The common genus of cultures of 4 samples without pre-culture belonged to:Vibrio?Streptococcus?Peptostreptococcus?Lactobacillus?Clostridium.Meanwhile,the common genus of cultures of 4 samples with APW pre-culture belonged to:Nitrospira?Streptococcus?Shewanella?Dokdonella?Arcobacter?Vibrio.1.2 Isolation,identification and pathogenicity of Vibrios in aquatic productsDuring 2011-2013(May-October each year),225 aquatic product samples were collected from parts of Jiangsu province,and 1850 vibrio strains were isolated using TCBS plates,identified by biochemical characterization,16S rDNA PCR amplification and sequencing.The results showed that vibrio contamination was common in aquatic products in collected samples,with great diversity in vibrio species.195 out of 225 samples were positive in vibrio detection.The positive rate of vibrio was 86.7%.Detected vibrio species covered:V.parahaemolyticus 76.0%?V.alginolyticus 79.0%?V.harveyi 13.0%?V,aestuarianus 7.5%?V.metschnikovii 6.3%?V.azureus 6.0%?V.Natriegens 3.6%?V.diazotrophicus 2.7%and V.owensii 2.0%.The isolation rate of V.parahaemolyticus and V.alginolyticus was significantly higher than that of other Vibrioes.Vibrio positive rate in marine products(92.4%)was significantly higher than that of freshwater culture products(82.4%),and the Vibrio positive rate in fish products(89.7%)was slightly higher than that of shrimp and shellfish samples(83.1%);Vibrio positive rate in samples collected from the coastal and river-rich area(88.2%)is also higher than that in those collected from the inland area(84.6%);Vibrio positive rate samples collected from fresh-live aquatic product market(96.0%)was significantly higher than that of samples from the supermarket(76.8%).The pathogenicity of the isolates was evaluaed by tdh,trh gene,Kanagawa test and intestinal effusion test in mice.13 tdh+and 10 trh+strains were detected,and accounted for 0.92%and 0.71%of V.parahaemolyticus isolates,respectively.Results of the Kanagawa test showed that 49.32%of vibrio isolates were hemolytic.Intestinal effusion test result showed that almost all the tested strains,especially the tdh+and trh+strains,experienced different degrees of intestinal effusion,and/or intestinal wall congestion.2 Biological characteristics and transcriptomic analysis of vibrio under stressful growth conditions2.1 Biological characteristics of vibrio under stressful growth conditionsVibrio parahaemolyticus(V.p,CICC 21617)and Vibrio alginolyticus(V.a,CICC 10484)were used to study the growth ability,cell morphology,biofilm formation and hemolysin activity under stressful conditions including extreme salinity,pH in culture medium and temperatures in culture environment.The stressful conditions for viability of Vp(OD600nm>0.3)were determined as following:0.9%salinity as a minimum,and 8%salinity as a maximum;pH 5.0 as a minimum,and pH 10 as a maximum;45? lasted for 5 min as a highest temperature,,and-20? kept for 2 h as a lowest temperature,.The stressful conditions for viability of V.a(OD600nm>0.3)were determined as following:0.5%salinity as a minimum,and 8%salinity as a maximum;pH 4.0 as a minimum,and pH 9 as a maximum;50? lasted for 5 min as a highest temperature,and-20? kept for 2 h as a lowest temperature.The tested strains were able to grow slowly in liquid medium with either a minimum or a maximum salinity,also a minimum or a maximum pH.However,the growth ability of different stressed strains exhibited quite different when they were re-cultured in conventional LBS broth(3.5%NaCl).Growth capacity of minimum or maximum pH,and lowest or highest temperature stressed strains was more significantly recovered than that of minimum or maximum salinity stressed strains.The results showed that the stressed strains were more prone to grow under the same stressful environments than the unstressed strains.The microscopic morphology of the stressed strains showed obvious changes on both cell wall and cell surface,and even cell contents leaked out from the seriously damaged cells.2 tested strains stressed with highest temperature or maximum pH and V.a with maximum salinity experienced most serious cell damage,and clots of cellular debris were observed in space between the cells.The biofilm formation ability of Vp and V.a was quite different,however,the adaptability trend of biofilm formation of stressed strains was consistent compared with those of unstressed strains.The biofilm formation ability(BFA)of the most stressed strain was elevated,and the minimum salinity stressed Vp strainharbored the highest BFA,followed by the minimum pH,highest temperature and maximum pH stressed strain;The BFA of the minimum or maximum pH,highest temperature,minimum salinity stressed V.a strain was also higher than that of unstressed strain.However,the BFA of maximum salinity and lowest temperature stressed strains was much lower than that of unstressed strains.The hemolysin activity of the stressed strains was reduced,especially under the stressful conditions,e,g.minimum salinity,minimum pH and highest temperature,and the difference was significant(P<0.05)compared with that of the unstressed strains.The extracellular protease activity of the stressed strains was also decreased,although the difference was not significant compared with that of the unstressed strain.In summary,the biological characteristics of the two tested strains under the stressful growth conditions were significantly altered compared with those of the unstressed strains.2.2 Transcriptomic analysis of minimum salinity-stressed vibrio strainsIn order to understand the response mechanism of vibrio to extreme salinity stressful conditions,transcriptomic analysis of stressed strains was performed using RNA-seq technology.Vp(CICC 21617)was cultured for a single passage or serially passaged for 20 generations in LB broth with 0.5%NaCl.The same strain cultured in conventional LB broth(3.5%NaCl)was used as a control.The sequencing results showed that there was a significant difference in the transcriptom covered 1240 genes in stressed strain within a single passage under a minimum salinity stressful condition compared with those of the control strain,including 637 up-regulated genes and 603 down-regulated genes,meanwhile,the strain serially passaged for 20 generations also showed a significant difference in the transcriptom involved in 1418 genes,including 715 up-regulated genes and 703 down-regulated genes compared with those of the control strain.These differentially expressed genes are mainly involved in biological processes such as amino acid metabolism,energy metabolism,transport and binding,cell processes,and membrane translocations of the tested strain.Based on the KEGG pathway analysis,the differentially expressed genes were enriched into 342 signaling pathways in the single passaged strain.The pathways involved in abundant genes including amino acid metabolic pathway,energy metabolism pathway,and transporting-binding pathway.Compared with the control strain,the differentially expressed genes were enriched into 296 signaling pathways for the strain serially passaged for 20 generations,and the pathways involved genes were similar with those of the single passaged strain.3 Isolation and identification of Vibrio bacteriophages in aquatic products and their bacterial suppression effects on tested Vibrios3.1 Isolation and biological characteristics of Vibrio bacteriophagesWith the safety risks increasing raised by pathogenic Vibrio in aquatic products,it is urgent to develop a natural biological agents to suppress the growth of pathogenic Vibrio in aquatic products.5 of vibrio reference strains(V.alginolyticus CICC 17749,V parahaemolyticus CICC 21617,V.mimicus CICC 21613,V cholerae CICC 23794,V mimicus CICC 10383)were used as host to isolate phage.27 phages were isolated from aquatic product sewage,and 3 phages were lytic phage with cross-species of Vibrios.Phages designaed as Vmp 03 and Vpp 07 were able to lyse tested strains belonged to 6 vibrio species,and phage Vap 04 could lyse tested strains belonged to 4 vibrio species.Microscopic observation showed that Vmp 03 and Vpp 07 phages belonged to Podoviridae,and Vap 04 belonged to Myoviridae.The optimal multiply of infection(MOI)of phage Vmp 03,Vpp 07 and Vap 04 was 0.001,0.01 and 0.01,respectively;the incubation period was 20 min,30 min and 20 min,respectively,and the lytic period was 50 min,50 min and 60 min,respectively.The burst size of phage Vmp 03,Vpp 07 and Vap 04 was 60 PFU/cell,15.8 PFU/cell,and 7.9 PFU/cell respectively.The extreme temperature for phage Vmp 03,Vpp 07 and Vap 04 to keep their lysis activity within 1 h was 70 ?,60?,70 0C,respectively,and the pH range within 1 h was 4.0?10.0,6.0?10.0,4.0?12.0,respectively.The biological characteristics and lysis activity of these phages implied their potential application value.3.2 The genomic of Vmp3 bacteriophageBy whole genomic sequencing,the genomic of Vmp3 bacteriophage consisted of 43,479 base pairs,with the coding region up to 38403 base pairs representing 88%of the whole geomic.In whole genomic scale,Vmp3 bacteriophage harbored 65 open reading frames,with the percentage of G+C was about 49%.The genomic of Vpp07 bacteriophage was composed of 43,479 base pairs,summing up the coding region to 32592 base pairs representing 23%of the whole geomic.In whole genomic level,Vpp07 bacteriophage exhibited 35 open reading frames,with the percentage of G+C was about 42%.3.3 Targeted bacteriostasis of selected bacteriophagesThe bacteriostasis effect of phage Vmp 03,Vpp 07 and Vap 04 on 3 Vibrio strains(V mimicus 21613,V.parahaemolyticus CICC 21617 and V alginolyticus J 1-4)was determined in either aLBS medium or a boiled fish juice model.The results showed that phage Vmp 03,Vpp 07 and Vap 04 were able to effectively inhibit the growth of tested strains in both LBS medium and fish juice model. |
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