Epidemiological Investigation Of Vibrio In Shellfish In Shandong Province From 2021 To 2022 And Analysis Of Drug Resistance And Virulence Genes Of Isolated Strains

Shandong Province has unique marine fishery resources.The coastal waters are rich in seafood such as fish,shrimp,shellfish,algae and ginseng.Among them,there are more than100 species of shallow sea shellfish.The main economic shellfish are oysters,snails,mussels,scallops,clams,clams,razors and so on.In recent years,the cultivation of shallow sea shellfish and tidal flat shellfish has also faced severe breeding problems while creating huge output value.Among them,it is worth noting that the frequent outbreaks of shellfish vibriosis have seriously affected the healthy development of shellfish aquaculture in our province.In this study,540 shellfish samples were collected from shallow sea shellfish and beach shellfish in Yantai,Weifang,Binzhou,Weihai,Qingdao,Rizhao and Dongying coastal cities in Shandong Province from 2021 to 2022,and 526 Vibrio and 82 other bacteria were isolated from them.The isolation of Vibrio was mainly carried out by TCBS agarose medium enrichment and purification culture.The identification of Vibrio strains was carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry,supplemented by PCR identification.The biochemical identification of Vibrio was carried out for complex strains and some high-frequency isolated strains.The virulence gene detection,hemolytic test and drug sensitivity test of Vibrio parahaemolyticus,Vibrio harveyi and Vibrio alginolyticus were carried out for the hemolytic,virulence and drug resistance of Vibrio.The results of this vibrio epidemiology survey are as follows :From 2021 to 2022,nearly 20 species of shellfish were collected from seven cities along the coast of Shandong Province,including bay scallops,yellow clams,variegated clams,mussels,oysters,razor clams,clams,pointed tail snails,flat jade snails,stone snails,blister snails,Xishibei,corn snails,bamboo razor clams,clams,horseshoe snails,mud clams,snails,and sea ascaris.A total of 526 Vibrio strains belonging to 19 species were isolated and identified,mainly including:Vibrio parahaemolyticus、Vibrio tasmaniensis、Vibrio harveyi、Vibrio splendidus、Vibrio alginolyticus、Vibrio aestuarianus、Vibrio anguillarum、Vibrio rotiferianus、Vibrio natriegens、Vibrio mediterranei、Vibrio brasiliensis、Vibrio pelagius、Vibrio cyclitrophicus、Vibrio kanaloae、Vibrio pomeroyi、Vibrio chagasii、Vibrio fortis、Vibrio toranzoniae、Vibrio gigantis.Among them,11 strains of Vibrio parahaemolyticus,16 strains of Vibrio harveyi,46 strains of Vibrio alginolyticus,98 strains of Vibrio tasmania and 185 strains of Vibrio gigas were isolated in two years.The isolation rates of V.parahaemolyticus in clams,clams,yellow clams,oysters,mussels and flat snails were 9.09 %,45.45 %,18.18 %,9.09 %,9.09 % and9.09 %,respectively.The isolation rates of Vibrio harveyi in Scapharca subcrenata,Corbicula fluminea,Neverita didyma,Snail and Oyster were 6.25 %,37.50 %,31.25 %,12.50 % and12.50 %,respectively.The isolation rates of V.alginolyticus in bay scallops,yellow clams,clams,oysters,mussels,razor clams,Scapharca subcrenata,pointed tail snails,flat snails,stone snails and clams were 4.35 %,6.52 %,19.57 %,8.70 %,8.70 %,8.70 %,6.52 %,13.04 %,6.52 %,4.35 % and 8.70 %,respectively.The susceptibility test of 271 strains of Vibrio including Vibrio parahaemolyticus,Vibrio harveyi,Vibrio alginolyticus was carried out.Cefotaxime,ampicillin,cefuroxime,clindamycin,spectinomycin,kanamycin,amikacin,neomycin,enrofloxacin,polymyxin B,tetracycline,doxycycline,minocycline,compound sulfamethoxazole,florfenicol,aztreonam and other 9 categories of 16 antibiotics were selected.The results showed that the resistance rates of ampicillin and tetracycline were 76.01 % and 29.15 %,respectively.The antibiotic susceptibility tablets of aminoglycosides had good antibacterial effect,and the drug sensitivity of spectinomycin,amikacin and kanamycin reached more than 96 %.The drug sensitivity rates of clindamycin,cotrimoxazole,florfenicol and aztreonam were also above 90 %.Vibrio parahaemolyticus was moderately sensitive to cefuroxime,clindamycin,amikacin,neomycin,kanamycin,polymyxin B,doxycycline and minocycline.The moderate sensitivity rates were59.78 %,66.79 %,93.36 %,68.63 %,73.06 %,59.41,60.52 % and 48.71 %,respectively.In this test,the main virulence gene of Vibrio parahaemolyticus,alkaline serine protease-related gene Asp A,was detected in 28.3 %,transmembrane transcriptional regulatory protein-related genes tox S and tox R,were detected in 54.35 % and 39.13 %,respectively.The flagellin-related gene Fla A,the detection rate was 34.78 %,the iron regulatory gene fur,the detection rate was 56.52 %,and the type III secretion system-related gene vsc B,the detection rate was 19.57 %;the detection rate of alkaline serine protease-related gene Asp A,the main virulence-related gene of Vibrio alginolyticus,was28.3 %.The detection rates of tox S and tox R related genes of transmembrane transcriptional regulatory proteins were 54.35 % and 39.13 %,respectively.The detection rate of flagellin-related gene Fla A was 34.78 %,the detection rate of iron regulatory gene fur was56.52 %,and the detection rate of type III secretion system-related gene vsc B was 19.57 %.The detection rates of tox R,tox S,lux R,fla A,vhp A,vhh A,vhh B and pap6 were 62.50 %,37.50 %,25.00 %,31.25 %,31.25 %,31.25 %,56.25 %,43.75 % and 37.50 %,respectively.The investigation of the distribution of virulence genes and the detection of virulence genes of the three main pathogenic Vibrios provided relevant data for the identification of virulence strains.The epidemiological investigation of Vibrios in 2021-2022 showed that only the isolation and detection rates of Vibrio parahaemolyticus,Vibrio harveyi and Vibrio alginolyticus were observed.In the aquaculture sea areas of Weifang and Yantai,it is necessary to formulate a more standardized aquaculture model in shellfish aquaculture production activities,refine the division of labor,and implement the provisions of aquaculture management.In summary,through the epidemiological investigation of shellfish vibriosis in Shandong Province in 2021 and 2022,the epidemic situation of shellfish vibriosis was mastered,and the carrying situation of shellfish vibriosis was preliminarily explored.The relationship between vibrio strains and shellfish,between aquaculture sea areas in various cities and shellfish vibrio was preliminarily revealed,and the drug resistance results of vibrio were isolated,which provided data for the analysis of drug resistance changes of shellfish vibriosis in recent years.The distribution of the main virulence genes of the isolated vibrios was analyzed,which provided a basis for the identification of virulent strains and the screening of pathogenic strains,provided a scientific basis for the prevention of large-scale outbreaks of vibriosis,and had certain production guidance and reference significance.