Molecular Serotype, Drug-resistance And Rapid Detection Of Streptococcus Agalactiae Isolated From Tilapia

In2009disease outbreaks happened in tilapia farms in Southern China. Highinfectivity(>50%) and high mortalities (>95%) were observed in some farms. Thedisease constrained the tilapia aquaculture development. By biochemistry assays andmolecular analysis of16S rRNA, the pathogen was identified as Streptococcusagalactiae. That bacteria distributes widely in the world and can cause many disease inhuman, cattle and fish. This paper presented our reseach work on isolation andidentification of S.agalactiae, their molecular serotyping, the establishment ofloop-mediated isothermal amplification method for rapid detection, the minimalinhibitory concentration about quinolones and amplification of the resistance genes.Themain results are as follows.1Molecular serotype and drug-resistance of Streptococcus agalactiae from tilapiaThe goal of the present study is to investigate the relationship between serotype ofStreptococcus agalactiae and its resistance to antibiotic. Twenty four bacteria strainswere isolated from infected tilapia in several farms of Hainan province. With the RapidID32strep technique, morphological, regular physiological and biochemical analysis,molecular analysis of16S rRNA gene, the isolates were identified as S. agalactiae.Polymerase chain reaction (PCR) assay of the capsular polysaccharide (cps) antigengene was employed to identify their molecular serotype (MS). The result show thatmolecular serotype of all of isolates were Ⅰa. Meanwhile, their sensitivities to31kindsof antimicrobial drugs were determined. The result showed that the isolates wereresistant to most aminoglycoside and quinolones, and highly sensitive to doxycycline.For most of antimicrobial drugs, the isolates in2010were more sensitive than theisolates in2011. The results showed that the drug resistant of isolates in2011increasedstrongly. All of24isolates had the same molecular serotype, but showed no relatedrelationship. It was suggested that there were no relationship between serotype and drugrestistance.2Development of loop-mediated isothermal amplification method for rapiddetection of Streptococcus agalactiae in tilapiaStreptococcus agalactiae is a major pathogen that causes sever economic losses intilapia aquaculture. A set of four specific primers was designed by targeting cfb gene. With Bst DNA polymerase, the target DNA can be clearly amplified for60min at65°Cin a simple water bath. The sensitivity of the LAMP assay for the detection of S.agalactiae is about20cells per reaction of pure cultures. LAMP products could bejudged with agar gel or naked eye after addition of SYBR Green I. There were nocross-reactions with other bacterial strains indicating high specificity of the LAMP. TheLAMP method was also applied to detect S. agalactiae infected tilapia tissueseffectively. The LAMP assay reported here indicates the potential usefulness of thetechnique as a valuable simple, rapid alternative procedure for the detection ofS.agalactiae during streptococcicosis monitoring of cultured fish.3The association study between quinolones drug resistance and resistance genes inS.agalactiae of TilapiaThe aim of this study was to investigate minimal inhibitory concentration of41Streptococcus agalactiae isolates to quinolones and the gyrA and parC mutation of thequinolone resistance determining regions (QRDR) in quinolones resistant isolates. Inthis study, microdilution method was used to determine the susceptibility ofS.agalactiae isolated from tilapia against4quinolones. The fragments of the gyrA geneand parC gene were amplified by PCR. The results showed that the resistance rates oflevofloxacin、fleroxacin、lomefloxacin and flumark were22.0%,75.6%,73.2%and100%, respectively. There were9isolates that were against all of4quinolones.Sequence analysising result indicated only6resistant strains showed amio acidmutation. There were3resistant strains had amutation points in gyrA and other3resistant strains showed amutation in parC. The others were not detected any mutationpoints. It indicated that quinolone resistance of S.agalactiae isolates were not onlyrelated to mutations of resistant gene but also including other resistance mechanisms.