Isolation and identification of Citrobacter spp. from the intestine of Procambarus clarkia

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Isolation and identification of Citrobacter spp. from the intestine of Procambarus clarkia

 Citrobacter have caused a wide spectrum of infectious diseases in aquaculture animals and human. In previous study, pathogenic Citrobacter spp. was frequently isolated from hepatopancreas of diseased Procambarus clarkii. It suggested that the intestine might be the reservoirs of Citrobacter spp. Intestinal flora were isolated from P. clarkii with Salmonella shigella agar. 126 isolates were performed with enterobacterial repetitive intergenic consensus (ERIC) PCR. 95 strains of seven ERIC-PCR electrophoretic genotypes were identified as Citrobacter spp. by 16S rRNA gene sequence analysis and the urease gene detection of the representative strains. Eight representative strains were defined by biochemical characteristics and multilocus sequence analysis of genes fusA, leuS, rpoB and pyrG. Isolates 425C1, 731C1 and 519C4 were determined as C. freundii, C. werkmanii and a new species of C. freundii complex, respectively. Isolates 425C3, 519C1, 519C3, 519C41 and 519C53 were identified as C. braakii. C. freundii strain 425C1 and C. werkmanii strain 731C1 were verified pathogenic bacteria to P. clarkii and Kunming mice in varying degrees. To our knowledge, this is the first report of pathogenic C. werkmanii.

C.freundii had frequently caused infectious diseases of aquaculture animals. Here, a large number of Citrobacter strains were isolated from the intestine of seemingly healthy crayfish with SS agar in central eastern China and the representative strains of seven Citrobacter ERIC-PCR genotypes were subsequently identified as C. freundii, C. braakii, C. werkmanii and a new species of C. freundii complex by means of biochemical characteristics and multilocus sequence analysis. Two representative strains, C. freundii 425C1 and C. werkmanii731C1, were confirmed as pathogens of P. clarkii and mice by challenge experiments. Furthermore, the techniques of SS agar and ERIC-PCR would be useful tools for isolating Citrobacter spp. from complex environment.

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