Bacterial isolates. The QREC strains were originally collected as part of the NORM-VET program in 2014 (4), and originated from broiler caecal samples (n = 85) and retail chicken meat (n = 73).
All strains were stored at −80°C in brain heart infusion broth (BHI; Difco, BD, Franklin Lakes, NJ, USA) supplemented with 15 % glycerol (Merck KGaA, Darmstadt, Germany) and recovered on blood agar (sheep blood) at 37.0 ± 1.0°C overnight. The bacterial cultures were then transferred into Luria-Bertani broth (LB; Merck) and incubated statically overnight at 37.0 ± 1.0°C. LB without NaCl (LBwo/NaCl; Bacto-tryptone 10 g/liter, yeast extract 5 g/liter) was used as the test broth in the biofilm assays.
Determination of phylotypes. All isolates were subjected to phylotyping using multiplex PCR as described previously (31). The isolates were classified into phylotype A, B1, B2 or D. An isolate belonging to the B2 group, (E. coli2003500827) (32) producing amplicons with all four primer sets, was included as positive control in each PCR run and milli-Q water was used as negative control.
Biofilm production on polystyrene (microtiter plate assay). Biofilm production on polystyrene was measured in the microtiter plate assay and performed as described previously (14) using 96-well NuncTM NunclonTM microtiter plates (Nunc A/S, Roskilde, Denmark). In short, 30 μL of an overnight culture were added to 100 μL LBwo/NaCl in three parallel wells of a microtiter plate. The microtiter plates were incubated statically for two days at 20.0 ± 1.0 °C. After incubation, OD595 of the planktonic solution was measured. Then the wells were washed with tap water, stained with 1 % crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at room temperature, and washed at least three times with tap water to remove excess dye. The remaining dye was dissolved in ethanol-acetone (70:30, vol/vol) for 10 minutes at room temperature, and the optical density at 595 nm (OD595) (Multiscan MS; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was measured. The assay was performed four times for all strains. In each assay, the results were calculated by subtracting the median OD595 of the three parallel control wells (test broth only) from the median OD595 of the three parallel sample wells. Finally, the mean OD505 value of all four assays was calculated for each strain. OD595 values higher than three standard deviations of the negative controls were classified as positive for biofilm production. The results of different groups were compared using a Mann Whitney test (if nothing else is specified), and p-values ≤ 0.05 were considered statistically significant.
Biofilm morphotyping. Biofilm morphotyping was performed as previously described methods with slight modifications (33). In brief; 1 µL of overnight culture was inoculated onto CR agar plates, i.e. LB agar without NaCl containing 40 μg/mL Congo Red (Merck) and 20 μg/mL Coomassie brilliant blue (Sigma-Aldrich, St. Louis, MO). After inoculation, the CR agar plates were incubated at 20.0 ± 1.0 °C. All plates were visually examined after two, six and eight days of incubation, and the morphotypes were categorized as: RDAR - indicating expression of curli fimbriae and cellulose, PDAR - indicating expression of cellulose but not fimbriae, BDAR - indicating expression of fimbriae but not cellulose, and SAW - indicating expression of neither cellulose nor fimbriae.
Mixed biofilm production on glass slides. Six strains, two of each of the morphotypes RDAR, BDAR and PDAR, were used for these experiments (Table 5). Twelve different pairs of the combinations RDAR + BDAR, RDAR + PDAR and BDAR + PDAR were tested (Table S1). For each pair, overnight cultures of the two strains were mixed in the ratios 30:70, 50:50 and 70:30. From each mixture, as well as from the overnight cultures of the single strains, 200 µL were inoculated into sterile centrifuge tubes (Sarstedt AG & Co KG, Nürnbrecht, Germany) containing 5 mL LBwo/NaCl. An autoclaved microscope slide (76 by 26 mm; (Menzel GmbH + CoKG, Braunschweig, Germany) was placed in each tube. The tubes were incubated at 20.0 ± 1.0°C for two days. During incubation, biofilm was formed on both sides of the microscope slides at the liquid-air interface. After incubation, total cfu was enumerated in the planktonic phase in the growth medium by serial dilutions and spreading on CR agar plates. To enumerate the bacteria in the biofilms, these were washed three times in sterile saline to remove loosely adhered bacteria. Thereafter, the biofilms were removed by scraping with a sterile cell scraper (BD Falcon, Bedford, MA, USA) and transferred to sterile reagent tubes containing 5 mL sterile saline and 20 glass beads (3 mm; Assistent, Glaswarenfabrik Karl Hecht GmbH & Co KG, Bavaria, Germany). The tubes were vortexed at 2000 rpm for one minute and the solutions was serial diluted in sterile saline, spread on CR agar plates. All plates were incubated at 37.0 ± 1.0 °C for 24 h. After incubation, the number of cfu of each strain was counted. The strains were differentiated by their morphotype. All experiments were performed twice. A two-tailed Students’ t test were used in statistical analyses on these resulta, and p-values ≤ 0.05 were considered statistically significant.
Mixed biofilm production on CR agar plates. The same strains, pairs and ratios as in the glass slide assay were used in the CR agar plate assay. When inoculating single strains, 1 µL of overnight culture was used. For each pair of strains, 1 µL from each mixture of overnight cultures was inoculated. All inoculations were made in parallel. The CR agar plates were incubated at 20.0 ± 1.0 °C for ten days, and the plates were visually examined every day the first four days, and every third day thereafter. All experiments were performed twice.