Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates

Edwardsiella tarda is responsible for hemorrhagic septicemia (edwardsiellos is) in fish and also causes diseases in higher vertebrates such as birds, r eptiles, and mammals, including humans. Interactions of E. tarda with blue gourami phagocytes were studied by light microscopy as well as by adherence , intracellular replication, and superoxide anion assays. Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bac teria could adhere to and survive and replicate within phagocytes, while on ly opsonized virulent strains replicated within the phagocytes. Furthermore , only avirulent E. tarda elicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish p hagocytes. TnphoA transposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA(+)) fusion mutants from a total of 182,000 transconjugants derived from E. tarda PPD130/91. Five of these mutants indu ced more ROI production in phagocytes than the wild-type strain. Two mutant s had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain. Sequence analysis revealed t hat three of these mutants had insertions at sequences having homology to P hoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens. These three independent mutations might have changed the c ell surface characteristics of the bacteria, which in turn induced phagocyt es to produce increased ROIs. Sequences from two other mutants had no homol ogy to known genes, indicating that they may be novel genes for antiphagocy tic killing. The present study showed that there are differences in the int eractions of virulent and avirulent E. tarda organisms with fish phagocytes and PhoA(+) fusion mutants that could be used successfully to identify vir ulence genes. The information elucidated here would help in the development of suitable strategies to combat the disease caused by E. tarda.