Characterization of factors involved in and affecting biofilm formation by Aeromonas spp. Isolates.
Aeromonas spp. isolates, which are fish and opportunistic human pathogens, form biofilms, however the factors involved in and affecting biofilm formation have not been fully elucidated. Biofilm formation is affected by motility, cell surface characteristics, and/or metabolism, thus it is important to identify factors potentially contributing to initial attachment and/or biofilm formation and their correlation with biofilm formation by Aeromonas spp. isolates. With knowledge of the stages of biofilm formation, mechanisms involved in biofilm formation and its physiology, various strategies may be applied to control aeromonad biofilms. Factors potentially involved in initial attachment and/or biofilm formation were investigated for 99 Aeromonas isolates obtained from seawater and cultured fish. Aeromonad biofilm formation was assessed using microtiter plate assays under varying physicochemical conditions. The disk diffusion method was used to determine the antimicrobial susceptibility profiles of isolates, for comparison to clinical and aquaculture isolates reported in other studies. The MICs and MBICs for antimicrobial agents (azithromycin, ceftazidime, ciprofloxacin, gentamicin and tetracycline) of planktonic cells and biofilm cells, respectively, were investigated using the broth microdilution and modified microtiter plate assays. The effect of sub-MIC (0.5 × MIC) and supra-MIC (2 × MIC) exposures on biofilm-forming cells was also determined using microtiter plate assays. The presence of efflux pump-mediated resistance in 45 Aeromonas spp. isolates was determined using the disk diffusion assay incorporating efflux pump inhibitors (EPIs) [carbonyl cyanide 3-chlorophenylhydrazone (CCCP), phenylalanine arginine β-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP)]. Modified microtite plate assays were used to determine the effect of EPIs [CCCP, PAβN, and NMP], matrix-degrading DNase I and quorum-sensing inhibitors (QSIs; vanillin, 2(5H)-furanone, S-adenosylhomocysteine and cinnamaldehyde) on initial attachment and mature biofilm. Majority of isolates were motile by swimming and swarming and displayed caseinase, gelatinase, and DNase activities, as well as an A-layer phenotype. Majority of isolates displayed high levels of autoaggregation and were hydrophilic. Isolates showed varying levels of adherence, but majority were strongly adherent in nutrient-rich media at 30 ºC. Motility appeared to be a significant characteristic for biofilm formation. Majority of Aeromonas isolates spp. showed high levels of resistance to β-lactams, trimethoprim and sulphamethoxazole, and were susceptible to augmentin, piperacillin-tazobactam, aztreonam, 2nd and 3rd generation cephalosporins, carbapenems, macrolides, fluoroquinolones and aminoglycosides . High levels of resistance towards ceftazidime (MIC > 32 μg/ml) were observed for isolates, while levels of resistance towards remaining antimicrobial agents tested (tetracycline, azithromycin, ciprofloxacin, and gentamicin) were ≤ 32 μg/ml. There was a ≥16-fold increase in MBICs (4096 μg/ml) compared to the MICs for all the antimicrobial agents. The sub-MIC, MIC, and supra-MIC exposures of all antimicrobial agents had an inhibitory effect on both initial attachment and pre-formed biofilms by Aeromonas spp. isolates. Majority of isolates were more susceptible to tetracycline, norfloxacin, and azithromycin due to CCCP and NMP inhibition of the efflux pumps eliminating these antimicrobial agents. Susceptibility to erythromycin was observed for 51% and 47% of isolates, respectively, due to NMP and PAβN inhibition of the efflux pump/s eliminating erythromycin. In the microtiter plate assays, CCCP, NMP and PABN exposures resulted in significant reduction of biofilm formation by majority of Aeromonas spp. isolates in both initial attachment and mature biofilm assays, with CCCP being more effective. DNase I was more effective in reducing mature biofilm, causing reduction for 60% of isolates, compared to its effect on initial attachment. QSIs were also more effective in reducing mature biofilm compared to inhibiting initial attachment. Although increased biofilm dispersal was observed with all QSIs, vanillin and 2(5H)-furanone were more effective compared to S-adenosylhomocysteine and cinnamaldehyde. Based on data obtained in this study, antimicrobial agents, EPIs and QSIs can be used as potential biofilm-inhibiting compounds in aquaculture to control aeromonad infections and may not only prevent disease outbreaks but they could also increase the effectiveness of existing therapeutic agents.