Bacterial growth conditions and identification
Creamish-yellow pigmented isolate CH2B was cultured from diseased freshwater tilapia (Oreochromis mossambicus) from a South African aquaculture facility. Isolate CH2B was presumptively identified as E. meningoseptica using the following tests: Gram stain, colony characteristics, and flexirubin pigment production [1]; and this identification was confirmed by 16S rRNA gene PCR and sequencing [9] (GenBank: EU598807).
E. meningoseptica isolate CH2B and type strain NCTC 10016T were maintained on enriched Anacker and Ordal's agar (EAOA) [10] at ambient temperature (21°C ± 2°C). For long-term storage, cultures were placed in 20% glycerol and enriched Anacker and Ordal's broth (EAOB) and stored at -80°C.
Biofilm formation and quantification
E. meningoseptica isolates CH2B and NCTC 10016T were cultured overnight in EAOB at room temperature (21°C ± 2°C) and centrifuged for 2 min at 12000 rpm. Cell pellets were washed and re-suspended in phosphate-buffered saline (PBS, pH 7.2) to a turbidity equivalent to a 0.5 M McFarland standard [11]. In order to determine bacterial microtitre plate adherence, wells of sterile 96-well U-bottomed polystyrene microtiter plates (Deltalabs S.L, Barcelona, Spain) were each filled with 90 μl EAOB/tryptic soy broth (TSB; Merck Chemicals, Gauteng, RSA) and inoculated with 10 μl of standardized cell suspensions, in triplicate [12]. Negative control wells containing only broth or PBS were included in each assay while a Vibrio mimicus isolate (VIB1; isolated from cultured trout) was used as a positive control. Plates were placed on a C1 platform shaker (New Brunswick Scientific, Edison, NJ, USA) and/or the benchtop to simulate dynamic and static conditions, respectively, and incubated aerobically at room temperature (21°C ± 2°C) and/or 37°C for 24 h, in either nutrient-poor EAOB/nutrient-rich TSB media. An optical density (OD) reading of each well was obtained at 595 nm using an automated microtiter-plate reader (Microplate Reader model 680, BioRad Laboratories Inc., Hercules, California). Tests were done in triplicate on three separate occasions and the results averaged [12].
Biofilm formation was classified as non-adherent, weakly-, moderately- or strongly-adherent. The cut-off OD (ODc) for the microtiter plate test was defined as three standard deviations above the mean OD of the negative control. Isolates were classified as follows: ODODC = non-adherent, ODC < OD(2 × ODC) = weakly adherent; (2 × ODC) < OD ≤ (4 × ODC) = moderately adherent and (4 × ODC) < OD = strongly adherent [12]. Statistical significance of differences (p < 0.05) due to altered variables (temperature, medium, agitation) in the microtiter adherence assays were determined using one way repeated measures analysis of variance (ANOVA; SigmaStat, V3.5, Systat Software, Inc., USA).
Bacterial adherence to hydrocarbon assay
Surface hydrophobicity was assessed using the bacterial adherence to hydrocarbons (BATH) assay, with xylene (BDH, VWR International, Leicestershire, UK) as the hydrocarbon of choice [11]. E. meningoseptica isolates CH2B and NCTC 10016T grown in EAOB at room temperature (21°C ± 2°C) were harvested during the exponential growth phase (18 h old cultures), washed three times and resuspended in sterile 0.1 M phosphate buffer (pH 7) to an OD of 0.8 at a wavelength of 550 nm (A0 of 108 CFU/ml). Samples (3 ml) of the bacterial suspension were placed in glass tubes with 400 μl of xylene, equilibrated in a water bath at 25°C for 10 min and vortexed [11, 13]. After a 15 min phase separation, the lower aqueous phase was removed and its OD550 determined (A1). Strains were considered strongly hydrophobic when values were >50%, moderately hydrophobic when values were in the range of 20-50%, and hydrophilic when values were <20% [14]. Each value represents the mean of experiments done in triplicate and on two separate occasions. PBS was used as a negative control and V. mimicus isolate VIB1 was used as a control [11].
For the modified salting aggregation test (SAT) assay, overnight EAOB cultures grown at room temperature (21°C ± 2°C) were harvested, washed twice and resuspended in PBS (pH 7.2). E. meningoseptica isolate CH2B was 'salted out' (aggregated) by combining 25 μl volumes, containing 2 × 109 bacteria, with 25 μl volumes of a series of methylene blue-containing ammonium sulphate [(NH4)2SO4] concentrations (0.2, 0.5, 1, 1.5, 2, 2.5, 3, and 4 M) on microscope slides [11]. The lowest final concentration of (NH4)2SO4 causing aggregation was recorded as the SAT value and classified as follows: < 0.1 M = highly hydrophobic, 0.1 M - 1.0 M = hydrophobic and >1.0 M = hydrophilic [15]. Experiments were done in triplicate on two separate occasions and respective (NH4)2SO4 concentrations were used as negative controls.
Autoaggregation and coaggregation assays
For the autoaggregation assay, E. meningoseptica isolates CH2B and NCTC 10016T were grown in 20 ml EAOB at room temperature (21°C ± 2°C), harvested after 36 h, washed and re-suspended in sterile distilled H2O to an OD of 0.3 at a wavelength of 660 nm. The percentage of autoaggregation was measured by transferring a 1 ml sample of bacterial suspension to a sterile plastic 2 ml cuvette and measuring the OD after 60 min using a DU 640 spectrophotometer (Beckman Coulter) at a wavelength of 660 nm [16]. The degree of autoaggregation was determined as the percent decrease of optical density after 60 min using the equation:
OD0 refers to the initial OD of the organism measured. Sixty min after OD0 was obtained, the cell suspension was centrifuged at 2000 rpm for 2 min. The OD of the supernatant was measured (OD60). Experiments were carried out in triplicate on two separate occasions [16].
E. meningoseptica isolates CH2B and NCTC 10016T were examined for their ability to coaggregate with each other as well as with the following bacterial partner strains: Aeromonas hydrophila, A. sobria, A. salmonicida, A. media, Acinetobacter spp., Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Flavobacterium johnsoniae-like spp. isolates YO12, YO19, YO51, YO60, YO64, Listeria monocytogenes NCTC 4885, L. innocua LMG 13568, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella enterica serovar Arizonae and Staphylococcus aureus ATCC 25923 [11].
For coaggregation assays, bacteria were grown in 20 ml EAOB or TSB, harvested after 36 h, washed and re-suspended in sterile distilled H2O to an OD of 0.3 at a wavelength of 660 nm. The degree of coaggregation was determined by OD readings of paired isolate suspensions (500 μl of each isolate). The cell mixture was centrifuged at 2000 rpm for 2 min and the OD of the supernatant (600 μl) was measured at a wavelength of 660 nm [16]. The quantitative coaggregation rate of paired isolates was calculated using the equation:
where ODTot value refers to the initial OD, taken immediately after the relevant strains were paired; and ODS refers to the OD of the supernatant, after the mixture was centrifuged after 60 min [16]. Experiments were carried out in triplicate on two separate occasions. Differences in coaggregation between E. meningoseptica CH2B and E. meningoseptica NCTC 10016T were determined using one way repeated measures analysis of variance (ANOVA; SigmaStat V3.5). Differences were considered significant if p < 0.05.
Reversal and inhibition of coaggregation
The effect of simple sugars, heat and protease treatment on isolate CH2B's ability to coaggregate with L. innocua LMG 13568 and L. monocytogenes NCTC 4885 was investigated.
The ability of simple sugars to reverse E. meningoseptica isolate CH2B coaggregation with Listeria spp. involved filter-sterilized solutions of lactose and galactose, respectively, being added to one of the coaggregating partners at final concentrations of 50 mM [17]. Mixtures were vortexed and tested for coaggregation using the coaggregation assay described above.
The ability of heat treatment to inhibit E. meningoseptica isolate CH2B coaggregation with Listeria spp. was conducted using the method of Kolenbrander et al. [18]. Cells were harvested from O/N EAOB/TSB cultures, washed three times and resuspended in de-ionized water. One of the coaggregating partners was then heated at 80°C for 30 min in a waterbath. Following heat treatment, the OD of each bacterial suspension was adjusted to 0.3 at a wavelength of 660 nm. Heat-treated and untreated cells were combined in reciprocal pairs and their capacity to coaggregate was assessed.
Protease sensitivity of the polymers mediating coaggregation of isolate CH2B with Listeria spp. isolates was tested using a method described by Rickard et al. [19]. Cells were harvested from O/N EAOB/TSB cultures and resuspended in de-ionized water to an OD of 0.3 at a wavelength of 660 nm. Proteinase K was added to the standardized cell suspensions to a final concentration of 2 mg/ml. Incubation at 37°C for 2 h was followed by centrifugation and washing of the pelleted cells three times in de-ionized water. Cells were resuspended and the OD adjusted to 0.3 at 660 nm. Protease-treated and untreated cells were combined and their capacity to coaggregate determined.
Differences in coaggregation between untreated E. meningoseptica CH2B and treated bacteria (E. meningoseptica CH2B, L. innocua, and L. monocytogenes) were determined by paired t-tests (SigmaStat V3.5). Differences were considered significant if p < 0.05.
Induction of adherence
The standard microtiter plate adherence test [12] was modified to determine the ability of extracellular secretions of various aquaculture, food and/or human pathogens (Aeromonas hydrophila, A. salmonicida, A. sobria, Chryseobacterium spp. isolates CH8, CH15, CH23, CH25 and CH34, E. meningoseptica CH2B, E. coli, Edwardsiella tarda, F. johnsoniae-like isolate YO59, L. innocua, L. monocytogenes, Myroides odoratus MY1, P. aeruginosa, S. enterica serovar Arizonae, and V. mimicus VIB1) to induce enhanced adherence of E. meningoseptica CH2B.
Three-day old cultures of each of the above organisms were centrifuged at 2000 rpm for 10 min and supernatants were filter-sterilised using 0.2 μm filters, in order to obtain cell-free spent medium. E. meningoseptica CH2B cell pellets were washed and re-suspended in phosphate-buffered saline (PBS, pH 7.2) to a turbidity equivalent to a 0.5 M McFarland standard [11]. Ten μl of the standardized suspension was added to microtiter wells containing 100 μl TSB and 90 μl of the filtered supernatant. Controls included standardised isolate CH2B cell suspension added to TSB and respective filtered supernatants in TSB without isolate CH2B, in order to determine a change in adherence abilities and ensure that the change in adherence was due to induction, respectively. Microtiter plates were incubated at room temperature (21°C ± 2°C) for 48 h. An optical density (OD) reading of each well was obtained at 595 nm using an automated microtiter-plate reader (Microplate Reader model 680, BioRad Laboratories Inc., Hercules, California). Tests were done in triplicate on three separate occasions and the results averaged [12].
Characterization of biofilm formation using flow cell systems
Biofilm formation by E. menigoseptica isolate CH2B was investigated using continuous culture once-through eight channel flow-cell system, while the mixed-species biofilm flow cell study involved L. monocytogenes strain NCTC 4885 together with isolate CH2B. The eight-channel perspex flow cell (channel size 30 × 4.5 × 3 mm), the glass cover-slip covering (no. 1 thickness, 75 mm by 50 mm), and attached silicone tubing (1 × 1.6 mm × 3 mm × 5 m tubing; The Silicone Tube, RSA) was assembled as described previously [11]. Silicone tubing was connected to a reservoir containing 2 l of EAOB/TSB and the flow cell was filled with EAOB/TSB, with a flow rate of 0.25 ml/min being maintained using a multi-channel peristaltic pump (Model 205S, Watson-Marlow, UK) located upstream of the flow cell. A one ml volume of EAOB overnight cultures of isolate CH2B was inoculated into each channel, below the clamps sealing silicone tubes upstream of each channel, using sterile syringes. One ml mixed pure culture inoculations, consisted of 0.5 ml combinations of E. meningoseptica isolate CH2B and L. monocytogenes NCTC 4885. Stagnant conditions were maintained for the first hour to allow attachment, prior to inoculated channels being exposed to flowing EAOB/TSB at a constant flow rate of 0.25 ml/min. Flow cell systems were kept at room temperature (21°C ± 2) throughout the experiments. Each flow cell channel was investigated by transmitted light using a Nikon Eclipse E400 (Nikon, Japan) microscope at 600-fold magnification and after 24 h and 48 h, respectively, to visualize bacterial attachment to a glass surface and biofilm development. Images were documented with a CHU high-performance charge-coupled camera device (model 4912-5010/000).