Influence of Glucose Concentrations on Biofilm Formation, Motility, Exoprotease Production, and Quorum Sensing in <i>Aeromonas</i> <i>hydrophila</i>

Aeromonas hydrophila recently has received increased attention because it is opportunistic and a primary human pathogen. A. hydrophila biofilm formation and its control are a major concern for food safety because biofilms are related to virulence. Therefore, we investigated biofilm formation, motility inhibition, quorum sensing, and exoprotease production of this opportunistic pathogen in response to various glucose concentrations from 0.05 to 2.5% (wt/vol). More than 0.05% glucose significantly impaired (P , 0.05) quorum sensing, biofilm formation, protease production, and swarming and swimming motility, whereas bacteria treated with 0.05% glucose had activity similar to that of the control (0% glucose). A stage shift biofilm assay revealed that the addition of glucose (2.5%) inhibited initial biofilm formation but not later stages. However, addition of quorum sensing molecules N-3-butanoyl-DL-homoserine lactone and N-3-hexanoyl homoserine lactone partially restored protease production, indicating that quorum sensing is controlled by glucose concentrations. Thus, glucose present in food or added as a preservative could regulate acyl-homoserine lactone quorum sensing molecules, which mediate biofilm formation and virulence in A. hydrophila. Aeromonas hydrophila is opportunistic and a primary pathogen of amphibians, reptiles, fish, and humans (1). A. hydrophila causes various human infections such as gastrointestinal tract syndromes, wound and soft tissue infections, and bloodborne dyscrasias (29). A. hydrophila recently has been categorized as an emerging foodborne pathogen (17), with food and/or water as possible sources of human infection (14). Biofilms are architecturally complex assemblies of microorganisms that form on biotic or abiotic surfaces or at interfaces. Biofilms are characterized by interactions between microorganisms embedded in a matrix of extracellular polymeric substances created by the microbial populations and exhibit altered phenotypes with respect to growth rate and gene transcription (7). Like many other microorganisms, A. hydrophila can form biofilms in laboratory settings on stainless steel (23), glass (40), and vegetables (8). A. hydrophila possesses two distinct types of flagella, polar and lateral, for swimming and swarming, respectively. Both flagellar systems play vital roles in pathogenesis (34) and optimal biofilm formation (18). A. hydrophila secretes metalloprotease, zinc protease, and serine protease, which play a vital role in invasiveness and generation of infection (5). Bacterial genes and their respective phenotypes are regulated in a population-dependent manner with the aid of small diffusible signal molecules through a phenomenon referred to as quorum sensing (QS) (36). Gram-negative bacteria produce acyl-homoserine lactone (AHL) QS systems to regulate a variety of physiological processes, including virulence. A. hydrophila produces N-3-butanoylDL-homoserine lactone (C4-HSL) synthase, encoded by ahyI, and N-3-hexanoyl homoserine lactone (C6-HSL) synthase, encoded by ahyR, at a ratio of 70:1 (35). Modulation of virulence, exoprotease production, T3SS and T6SS protein expression, and biofilm development by QS has been previously reported (15, 23, 36). Khajanchi et al. (14) also reported that AHLs and cytotoxic activity levels were higher in clinical strains than in strains isolated from water, establishing the importance of QS in human infection. Because biofilms are directly related to virulence (31) and sessile cells are resistant to antibiotics and various disinfectants (24), a better approach is to inhibit biofilm formation rather than attempt to kill bacteria already in established biofilms (32). Blockage of QS is a new phenomenon, and many antibacterial agents have been identified recently to combat disease in this manner (2). Furukawa et al. (10) reported that sugar fatty acid esters inhibited biofilm formation by foodborne pathogens. Similarly, EDTA (3), bacteriocins, and competition for nutrients (31) inhibited biofilm formation by Listeria monocytogenes. * Author for correspondence. Tel: 82-031-670-4831; Fax: 82-031-675-4853; E-mail: sangdoha@cau.ac. kr. 239 Journal of Food Protection, Vol. 76, No. 2, 2013, Pages 239–247 doi:10.4315/0362-028X.JFP-12-321 Copyright G, International Association for Food Protection D ow naded rom hp://m eridianenpress.com /jfp/article-pdf/76/2689/0362-028x_jfp-12-321.pdf by gest on 21 June 2020 Glucose also has been reported as having antibiofilm effects on Escherichia coli (13) and Bacillus cereus (33). Chestnut honey (38) and vanillin (30) also have been reported as having antibiofilm and anti-QS effects on A. hydrophila. The present study was conducted to examine the effect of various glucose concentrations on biofilm formation, motility, exoprotease production, and QS in A. hydrophila. MATERIALS AND METHODS Bacterial strains, culture media, and growth conditions. The strains used for the study were A. hydrophila strains KCTC 2358 (isolated from a can of milk with a fishy odor), KCTC 11533 (isolated from surface water), and KCCM 32586 (a clinical isolate). The bioreporter strain Chromobacterium violaceum CV026 (Animal, Plant and Fisheries Quarantine and Inspection Agency, Seoul, Korea) also was included. A 20% (wt/vol) glucose (Merck, Darmstadt, Germany) solution was prepared by filter sterilization with 0.22-mm-pore-size filters (Millipore Corporation, Billerica, MA). Nutrient broth (NB; Difco, BD, Sparks, MD) was used for all the experiments except the violacein assay, for which we used Luria-Bertani (LB) medium (Difco, BD). Before each experiment, the cultures were activated by transferring from the 280uC freezer on nutrient agar plates to 30uC for overnight incubation. A single colony from the plate was inoculated in 5 ml of NB and incubated overnight at 30uC for 48 h without shaking and then diluted 1:50 in NB or LB containing 0, 0.05, 0.25, 0.5, 1.0, and 2.5% (wt/vol) glucose. Bacteria were grown at 30uC unless otherwise indicated. Quantitative biofilm formation assay. The quantitative biofilm assay was performed according to the procedure described by O’Toole (28) with minor modifications. The cultures were grown in NB for 48 h without shaking and then diluted (1:50) in NB containing various concentrations of glucose, and 100-ml aliquots were placed in each well of a 96-well polystyrene microtiter plate (BD, Franklin Lakes, NJ). The microtiter plate was incubated at 30uC for 72 h without shaking. After incubation, the absorbance was determined as an indicator of cell growth at 600 nm with a microplate reader (Spectra Max 190, Molecular Devices, Sunnyvale, CA). The plate was washed in a small tub of water after the bacterial cultures were discarded, air dried overnight, stained with 125 ml of 0.1% (wt/vol) crystal violet (CV) by incubating for 30 min at room temperature, and dried again overnight. The CV was solubilized with 125 ml of 95% (vol/vol) ethanol at room temperature for 10 min, and the absorbance was read at 595 nm with a microplate reader. Biofilm formation was determined using equation 1, normalized to planktonic growth according to Teh et al. (37):