Isolation And Identification Of Aeromonas From Nanjing And Typing Of A.Hydrophila Strains

Aeromonas species which are ubiquitous in aquatic ecosystems,are frequently isolated from fresh and estuarine water,surface waters,sewage,healthy or diseased fish,food products,animal and human faeces and able to cause infections in humans and fish.Aeromonas ssp.are known to be opportunistic pathogens for fish,and in conditions of stress,such as an increase in water temperature,poor water quality,etc.They are a major cause for epidemic outbreaks.In order to determine the disease-causing bacteria at one fish farm of Nanjing,tissue samples from diseased fish were collected and the pathogenic bacteria were identified by means of bacterial culture,biochemical characterizations and gyrB gene sequencing.One strain was isolated and named LK13-25.The results of biochemical tests showed that although the bacterial strain has many similarities compared with A.hydrophila,the differentiation from A.hydrophila can be obtained by the fact that LK13-25 did not utilize L-lactate.And then the strain was subjected to sequencing of the gyrB gene for species identification,and then demonstrated that the strain shared 99%homology with the A.aquariorum strain MDC 47.The corresponding gyrB phylogenetic analysis including representative strains from some known Aeromonas species showed that A.aquariorum LK13-25 was very closely related to the A.hydrophila ssp.dhakensis.The detection result of virulence factors indicated that A.aquariorum LK13-25 strain carried five virulent genes,including act,ahp,epr,hcp and alt genes,which encoded the cytotoxin,serine proteases,extracellular protease,hemolysin co-mediate protein and cytotonic enterotoxins,respectively.A.aquariorum LK13-25 also showed strong hemolytic and proteolytic activities.The median lethal dose(LD50)with 1.02×103 CFU/ml in zebrafish indicated that A.aquariorum LK13-25 is a highly virulent strain.A.aquariorum was a new species originally isolated from ornamental fish in 2008,and widely distributed in clinical and environmental specimens.Recent reports highlighted the importance of this species in cases of gastroenteritis,soft tissue infections,bacteremia and peritoneal dialysates in human,suggesting its pubic health signification.To our knowledge,this was the first report of A.aquariorum in China.To investigate the epidemiology of Aeromonas infection in Nanjing,163 samples from diseased or healthy fish and water were collected from freshwater fish farms in Nanjing and were identified by means of bacterial culture and gyrB gene sequencing.Six pairs of specific primers for act,alt,epr,ahp,ascV and hcp of Aeromonas,were used in PCR detection.The pathogenicity characterizations of the bacterial strains were analyzed by animal experiments.The result showed that 147 bacterial strains were identified to species,among which the main species were A.veronii and A.hydrophila with the detection rate of 46.3%and 34.7%,respectively.In virulence factors,in addition to ascV,the detection rates of the other five genes were all above 50%,up to 94.6%and down to 68.0%.The median lethal dose(LD50)in zebrafish indicated that the pathogenicity in A.hydrophila was significantly greater than other species.The results showed that Aeromonas were widely distributed in aquatic ecosystems in Nanjing,carrying different virulence factors,with potentially pathogenic significance,and need more attention particularly in A.hydrophila.Seventy-one strains of A.hydrophila were typed based on monosaccharide synthesis pathway genes rmlA,rmlC and rmlD based on the analysis for all known A.hydrophila O-antigen gene clusters.Finally,multilocus sequence typing(MLST)was used in some strains to compare two methods.The results showed that among 71 A.hydrophila strains,57 were consistent with the known O-antigen gene cluster types,in which the NJ-35 and ML09-119 O-antigen gene cluster types had relatively large proportion with the rate of 56.14%(32/57)and 33.33%(19/57),respectively.Compared with the result of MLST,it was found that except for JH-17 and CS-43,strains of the NJ-35 and ML09-119 O-antigen gene cluster types belong to ST251 and ST328,and the strains in the two types were proved to be virulent strains based on the pathogenicity test in zebrafish,indicating that maybe there exists a corelation between O-antigen monosaccharide biosynthesis genes and virulence in A.hydrophila.The results showed that NJ-35 type and ML09-119 type were the most popular O antigen gene cluster types in A.hydrophila in Jiangsu Province,and O-antigen gene clusters related genes typing could be used as a new molecule typing method in epidemiological investigation of A.hydrophila.In conclusion,in this study,Aeromonas infection was investigated in Nanjing and A.aquariorum was firstly reported in China.And a typing method based on O antigen gene cluster was established.The study will apply a good foundation for a better understanding of occurrence and spread of Aeromonas infection.