Bhi agar

The strain of E. coli O157:H7 Sakai (19 (link), 20 (link)) used in this study was provided by Carlton Gyles (Guelph, Canada). A stock culture was maintained at −80 °C and was cultivated by plating onto brain-heart infusion (BHI) agar (Thermo Scientific, Waltham, MA, CM225) and incubating at 37 °C for 24 h. An isolated colony was inoculated into 25 ml of BHI broth and incubated at 37 °C for 20 h. The resulting broth culture was kept at 4 °C and used as a “working” culture within a week.

Homogenates of 50mg liver tissue were filtered using a cell strainer (100 μm pore size; Corning, Tewksbury, MA 01876 USA) and serial dilutions were plated onto a blood agar plate (Thermo Scientific^™ Oxoid^™, Fisher Scientific, Wesel, Germany), brain heart infusion (BHI) agar (Thermo Scientific™), and water-blue metachrome-yellow lactose agar (acc. to Merck KGaA, Darmstadt, Germany). Additionally, Schaedler agar (Becton Dickinson GmbH, Heidelberg, Germany) was used for anaerobic growth examination in an anaerobic jar system (AnaeroGen^™; Thermo Scientific^™ Oxoid^™, Fisher Scientific, Oxford, OX14 4SD, UK) after 72 h of incubation at 37 °C. For enrichment purposes, each sample was inoculated to nutrient broth no. 2 containing 10% bovine serum and broth were streaked onto blood, BHI and Schaedler agar plate after 24 h incubation at 37 °C. Plates were examined after 24 and 48 h, as well as for BHI and Schaedler agar after 72 h at 10% CO₂. Morphologically diverse colonies were sub-cultivated. Pure cultures were identified using MALDI-TOF MS (Biotyper Version V3.3.1.0, Bruker Daltonics, Bremen, Germany) by the direct smear method and information provided in the DB7311 database. The MALDI-TOF results were supported by the Gram stain method and morphological properties of the bacteria.

Actinobacillus pleuropneumoniae non-hemolytic and non-cytotoxic MBHPP147 was kindly provided by Ruud P.A.M. Segers (MSD Animal Health, Boxmeer, The Netherlands). This strain is a mutant of the serotype 1 reference strain S4074 producing non-active ApxI and ApxII toxins (AppΔapxICΔapxIIC) [41 (link)]. Bacteria was grown at 37°C in brain heart infusion broth (BHI; Oxoid Ltd., Basingstoke, Hampshire, England) or in BHI agar (Oxoid Ltd.) supplemented with 5 μg/mL or 15 μg/mL of β-nicotinamide adenine dinucleotide (β-NAD; Sigma-Aldrich, St Louis, MO, USA), respectively.
The PRRSV North American reference strain IAF-Klop was used in this study. This strain is a genotype II strain [42 (link), 43 (link)].

One thousand five hundred Lactobacillus isolates of food, human and animal origin were assessed for anti-bacterial activity against C. difficile EM304 using agar diffusion assays. Lactobacillus strains were grown overnight on MRS agar at 37°C anaerobically. C. difficile cells were grown to mid log phase in RCM and harvested at an optical density (OD_600nm) of 0.8 and washed twice in preconditioned (pre-boiled and cooled under anaerobic conditions) MRD (Oxoid Ltd, Basingstoke, England) and re-suspended in MRD before use. One hundred microliters of the washed C. difficile cells were mixed with 5 ml BHI agar (0.7% agar, BHI, Oxoid) and poured onto BHI agar. Lactobacillus colonies were stabbed from the MRS agar into the C. difficile lawn with an inoculating needle. Plates were incubated anaerobically for 24 h and assessed for zones of inhibition.
In addition L. gasseri APC 678 was assessed for bacteriocin activity against a range of target organisms, as previously described (Rea et al., 2010 (link)). The full list of target strains, together with their growth conditions are outlined in Supplementary Table 2.

Toluidine Blue DNA Agar was used for the detection of thermostable deoxyribonuclease activity. The agar was prepared according to the manufacturers protocol (HiMedia, India). Nuclease activity was detected using colony overlay as described by Lachica et al. (1970) [38 (link)] with minor modifications. L. lactis strain harboring nuc gene was spotted into the Brain Heart Infusion (BHI) agar (Oxoid, UK) and incubated overnight at 30 °C. Then, 15 ml of boiled TBD agar that has been cooled to 50 °C was poured onto the overnight cultured BHI agar. The overlaid agar was incubated at 37 °C for 4 h and bright pink halos observed.

The antibiotic susceptibility of A. kunkeei K18, K34 and K45 was assessed with the Epsilometer test (E-test) gradient technology (Biomerieux, Marcy-l’Etoile, France) to determine the minimum inhibitory concentration (MIC) of various antimicrobial agents. For comparative purposes, A. kunkeei DSM 12361 and L. rhamnosus GG ATCC 53103 were also tested. In the present study, chloramphenicol, clindamycin, ampicillin, gentamicin, tetracycline, streptomycin, kanamycin and erythromycin were used in a concentration range of 0.016–256 µg/mL. The antibiotics were selected on the basis of the EFSA document regarding bacteria of human importance, and the cut-off values are those indicated in the same document [24 ]. BHI agar (Oxoid Ltd., Hampshire, UK) plates were inoculated with the bacterial suspensions in a sterile saline solution. The bacterial cell density of suspensions was adjusted to match McFarland turbidity standard 0.5 using a spectrophotometer (Bio-spectrometer Basic, Eppendorf, Italy).
After drying the plates surfaces for 15 to 20 min, the E-test strips of tested antibiotics were applied directly onto the surface. The plates were incubated overnight under anaerobic conditions at 37 °C; then, the MIC values were read following the manufacturer’s instructions.