Biochemical and molecular characterization of the novobiocin and rifampicin resistant Aeromonas hydrophila vaccine strain AL09-71N+R compared to its virulent parent strain AL09-71.

To understand the fitness cost of novobiocin- and rifampicin-resistance in an attenuated Aeromonas hydrophiila vaccine strain AL09-71 N+R compared to its virulent parent strain AL09-71, colony size, cell size, cell proliferation rate, chemotactic response, and the ability to invade catfish gill cells of the two strains were compared. Our results revealed that: (1) the cell size and the colony size of AL09-71 N+R was significantly (P<0.05) smaller than that of AL09-71; (2) the proliferation rate of AL09-71 N+R was significantly (P<0.05) slower than that of AL09-71; (3) AL09-71 N+R had a significantly (P<0.05) lower chemotactic response to catfish mucus than that of AL09-71; 4) the ability of AL09-71 N+R to invade catfish gill cells was significantly (P<0.05) lower than that of AL09-71. To understand whether target site mutation might play a role in antibiotic resistance, novobiocin's target site DNA gyrase subunit B gyrB and rifampicin's target site RNA polymerase subunit B rpoB were sequenced from the two strains. Our results revealed the following five mutations: (1) two missense mutations (CGC to ATC resulting in arginine/R to serine/S; TAC to TGC resulting in tyrosine/Y to cysteine/C) between AL09-71 gyrB and AL09-71 N+R gyrB; (2) three missense mutations (GAC to AAC resulting in aspartic acid/D to asparagine/N; CTG to CCG resulting in leucine/L to proline/P; CTG to CCG resulting in leucine/L to proline/P) between AL09-71 rpoB and AL09-71 N+R rpoB. To determine whether any unique DNA sequences were present in AL09-71 but absent in AL09-71 N+R, PCR-select bacterial genome subtractive hybridization was performed. Of 96 clones selected from the subtractive genomic DNA library, 32 sequences were found. None of the 32 sequences was confirmed to be present in AL09-71 but absent in AL09-71 N+R. At the transcription level, 29 of the 32 genes were found to be expressed greater than 10-fold in AL09-71 N+R compared to that in AL09-71.