Tryptone soya agar

MS2 bacteriophages (ATCC 15597-B1), from the family Leviviridae, have an isoelectric point between 3.5 and 3.9 [46 ] and are relatively small viruses at 20 to 25 nm in diameter. They were enumerated using the ISO standardised double-agar-layer method (ISO 10705-2; [46 ]), and the results were expressed as plaque-forming units (PFU) per 10 mL of the sample. For MS2 bacteriophages, the host Escherichia coli strain C-3000 (ATCC EC15597) was used for enumeration. Tryptone Soya Broth (Thermo Fischer Scientific™) was used for host strain growth and Tryptone Soya Agar (Thermo Fischer Scientific™) was used for solid media. The concentrations of agar in the top and bottom layers used were the same as those for standardised methods reported elsewhere (ISO 10705/2). Samples were filtered through 0.22 μm polyvinylidene difluoride membrane syringe filter units (Millipore™ (Burlington, VT, USA)/Thermo Fischer Scientific™). For a single assay, up to 5 mL of the sample was added to 1 mL of the exponentially growing host strain and 2.5 mL of semi-solid agar. The resulting suspension was mixed briefly and poured onto previously prepared bottom agar layers in 90 mm diameter Petri plates. Once solidified, the plates were inverted and incubated at 37 °C for 18 to 24 h. Clearly visible circular ’zones of lysis’ in a confluent lawn of host were recorded as PFU [47 (link)].

Strains of S. gallolyticus subsp. gallolyticus (Table A in S1 File) were grown in brain-heart infusion broth (BHI; Thermo Scientific, Waltham, USA) at 37°C and 220 rpm for overnight cultures. Bacterial cultures in the exponential growth phase were generated by inoculating 5 ml BHI medium with 100 μl overnight culture. The exponential growth phase was reached after 2.5 h at 37°C and 220 rpm. The bacterial titer was determined by serial dilutions in Dulbecco’s phosphate-buffered saline (DPBS) and plating 100 μl of an adequate concentration in triplicate on tryptone soya agar (Thermo Scientific, Waltham, USA). tryptone soya agar plates were incubated at 37°C for at least 24 h and the colonies produced were counted using an aCOLyte colony counter (Synbiosis, Cambridge, UK).

Information about the strains of L. monocytogenes used in the present study is summarized in Table 1. Bacterial stock cultures were kept at -80°C in Brain heart infusion (BHI) broth (Oxoid Limited, UK) with 20% glycerol added as cryogenic agent. Fresh cultures in their early stationary growth phase were prepared for each experiment by inoculating a loopful of the frozen culture in BHI and incubating at 37°C for 18 h. BHI was chosen as medium with optimal growth rates under controllable conditions based on the EURL technical guidance document (European Union Reference Laboratory for Listeria monocytogenes [EURL Lm], 2014 ). The resulting cell suspension of L. monocytogenes contained about 10⁹ colony forming units (CFU) mL^-1. Microbial counts were performed by spreading 0.1 mL of the appropriate serial dilution in peptone water (PW; Merck, DE) on the non-selective medium Tryptone Soya Agar supplemented with 0.6% Yeast Extract (TSA + YE; Oxoid Limited, UK), incubating at 37°C and colony counting after 24 h.

Antimicrobial activity of fungal cultures was tested against the Gram positive and Gram negative facultative pathogens Staphylococcus (S.) aureus (Rosenbach 1884, ATCC6538) and Pseudomonas (P.) aeruginosa (Migula 1900, ATCC9027) and the yeast pathogens Candida (C.) albicans (Robin Berkhout, ATCC10231); all are obtained from DSMZ, Germany. For detailed analysis in an enhanced screen, we used Klebsiella pneumonia ATCC13883, Escherichia coli MC1061, the yeast-like potential pathogenic fungus Exophiala (E.) dermatitidis (internal strain collection number 132), and three multiresistant clinical isolates: extended-spectrum betalactamase (ESBL) Klebsiella (K.) pneumonia (B100173), ESBL Escherichia (E.) coli (B300129), and methicillin-resistant S. aureus (MRSA) B337919 (kindly provided by Analyse Biolab GmbH, Austria). Initial cultivation was performed on tryptone soya agar (Oxoid, USA) for 18 h at 37°C. One single colony was inoculated in tryptone soya broth supplemented with 6 g/L yeast extract (TSBY, Oxoid, USA) and incubated at 37°C for 6 h for S. aureus and P. aeruginosa and for 10 h for C. albicans. All fungal strains used in this study were obtained from the fungal strain collection of the AIT (Austrian Institute of Technology, Fungal Genetics and Genomics Unit, Table 1). Identification of the fungi was performed according to Klaubauf et al. [28 (link)].

Antibacterial activity due to the photo-thermal effect of was investigated against Staphylococcus aureus ATCC 6538 (Gram+) and Escherichia coli ATCC 10356 (Gram−). One functionalized slide was cut in four sections of 10 × 10 mm in order to be completely irradiated by laser. A volume of 0.020 mL of bacterial suspension was deposited on two sections. For each pair of functionalized glass slides, one was irradiated for 15 min, whereas the other was not irradiated. After this time, the glass section covered with bacterial suspensions was suspended in 1 mL of sterile water, then it was taken away and water was used to make three dilutions in 3 different tubes, in which 5 mL of sterile water was contained: 1:100, 1:10,000, 1:100,000. From each tube, 1 mL of suspension was taken and then grown in Tryptone Soya Agar (Oxoid; Basingstoke, Hampshire, UK) to count viable cells. The decimal-log reduction rate (i.e., the “thermal microbicidal effect”) ME_T was calculated with the following formula

where N_C is the number of CFU/mL developed in contact with a nonirradiated modified control glass sample, and N_T the number of CFU/mL counted after exposure to modified glass samples and irradiation. Each experiment was repeated three times.