Trypticase soy agar

The MICs of wortmannin and butyrate were determined using a standard broth microdilution assay as recommended by the Clinical and Laboratory Standards Institute (National Committee for Clinical Laboratory Standards, 2003 ) as we previously described (Xiao et al., 2006 (link), 2009 (link); Bommineni et al., 2007 (link)). Briefly, Salmonella enterica subsp. enterica serovar Enteritidis (ATCC 13076) and Escherichia coli (ATCC 25922) were streaked onto trypticase soy agar (Fisher Scientific) plates, followed by subculture of 2–3 individual colonies in trypticase soy broth (Fisher Scientific) with shaking at 37°C for 3 h to reach the mid-log phase. Bacteria were then diluted to 5 × 10⁵ CFU/ml in Mueller Hinton Broth (Fisher Scientific). After dispensing 90 μl/well in a 96-well tissue culture plate, 10 μl of wortmannin were added in duplicate to final concentrations of 5, 10, 20, 40, 80, 160, and 320 μM with or without 4 mM sodium butyrate. MIC was determined as the lowest concentration of the compound or compound combination that gave no visible bacterial growth after overnight incubation at 37°C.

Since the Gram-positive Staphylococcus aureus is the most commonly isolated organism in SSI it was chosen to determine the MIC. Therefore Culti-Loops™ Staphylococcus aureus subsp. aureus ATCC™ 29213™ (isolated from a lesion) and Culti-Loops™ Staphylococcus aureus subsp. aureus ATCC^® 6538P™ (isolated from a wound) (Fisher scientific GmbH, Schwerte, Germany) were solved in 2 mL NaCl solution. Before MIC experiments, an overnight culture was performed on Trypticase™ Soy Agar (Fisher scientific GmbH, Schwerte, Germany) at 37 °C. For the MIC testing individual colonies from the stock culture were picked up, diluted in 1 mL NaCl and compared to a 0.5 McFarland Standard (Carl Roth, Karlsruhe, Germany). A smear preparation was made on Mueller Hinton Agar and a vancomycin test stripe with a concentration gradient of 256–0.016 µg/mL (Oxoid Deutschland GmbH, Wesel, Germany) were prepared on the middle. After 24 h of incubation at 37 °C the MIC were determined. Since the vancomycin concentrations determined in the MIC-tests are very small and as the environment of the body is more complex (blood flow, enzymes etc.) than in a petri dish, we decided to start our cell experiments with a 10× higher concentration of 0.01 mg/mL. As the highest concentration in our in vitro experiments, we chose a concentration 2000 times higher than the MIC (2 mg/mL).

Hexafluoroisopropanol (HFIP) was purchased from Oakwood Chemicals, Estill, SC, USA. Tributylammonium chloride (TBAC), tert-butanol, potassium carbonate, bromohexane, iodomethane, Tween 20, N-3-dimethylaminopropyl-N-ethylcarbondiimide (EDC), N-hydroxy succinimide (NHS), 2-N-morpholino ethanesulfonic acid (MES), hexanes and fluorescent latex beads (L5155, L9904, L5530, L9654, L9529) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Trypticase soy broth, trypticase soy agar, phosphate buffered saline (PBS), and dey engley broth were purchased from Fisher, Waltham, MA, USA. E. coli (8739) and S. aureus (6538) were obtained from ATCC, Manassas, VA, USA. Vantocil 100 (B-1018-04) was purchased from Lonza, Allendale, NJ, USA. Lupasol WF (25,000 g/mol) was purchased from BASF, Florham Park, NJ, USA.

Haemolysis was evaluated by plating the strains on Columbia Agar Base (OXOID, Basingstoke, Hampshire, UK) supplemented with 5% defibrinated horse blood (Pro Animali Company, Wroclaw, Poland). The plates were incubated at 37 °C for 24 h in aerobic conditions. A positive result was indicated by the formation of haemolytic (clear) zones around the colonies. E. faecalis ATCC29212 (LGC Standards, Łomianki, Poland) was used as a positive control.
Gelatinase production was detected by inoculating the Enterococcus strains onto Trypticase Soy Agar (OXOID, Basingstoke, Hampshire, United Kingdom) containing 3% gelatine (Avantor Performance Materials, Gliwice, Poland). The appearance of a clear halo around the colonies after incubation at 37 °C for 24 h in aerobic conditions followed by refrigeration at 4 °C for 30 min was considered to be a positive indication of gelatinase production. E. faecalis ATCC29212 (LGC Standards, Łomianki, Poland) was used as a positive control.

Swabs of the oral cavity were taken at time of sacrifice and placed into sterile phosphate buffered saline (PBS). Dilutions were plated on trypticase soy agar (Oxoid, UK) and incubated at 37℃ for 24 h to detect non-selective growth of aerobic bacterial species. To detect anaerobic growth, oral swab suspensions were plated on Wilkins-Chalgrens agar (Oxoid, UK) and placed in a hypoxic chamber at 37℃ for 72 h. Colony-forming units (CFUs) were determined by manually counting bacterial colonies.

Bacterial strains used were clinical isolates of MSSA (n = 100) and MRSA (n = 100) isolated from St George’s Hospital, London. These isolates were collected from blood cultures, tissue fluid, or routine screening on skin of the patients in the South West London area. Most of these strains were isolated from bacteremia. Some were isolated as organ or skin colonization. Genotyping of these strains were performed previously (Hu et al., 2015 (link)). The isolates were grown in nutrient broth (Oxoid) or on trypticase soy agar (Oxoid) plates.

Prior to testing, each isolate was subcultured by steaking the thawed stock culture on trypticase soy agar (Oxoid Ltd., Basingstoke, United Kingdom) containing 5% horse blood and incubating for 48 h at 37°C. All isolates were screened phenotypically by Gram staining, latex agglutination (using latex particles coated with monoclonal antibodies specific for the 200-kDa exopolysaccharide of B. pseudomallei) (Duval et al., 2014 (link)), immunofluorescence assay (Olympus, Tokyo, Japan), and standard biochemical tests (arginine decarboxylase test, sulfide indole motility agar, triple sugar iron agar, citrate, and urea hydrolysis test). Isolates were further confirmed as B. pseudomallei, by the hospital labs, using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).
All strains were streaked on Columbia agar containing 5% sheep blood to test for antibiotic susceptibility. Fresh new colonies of each strain were suspended in 0.45% saline to make turbidity equivalent to 0.5 McFarland standard (~1.5 × 10⁷ CFU/ml), and this solution was used for antibiotic susceptibility testing using the broth microdilution method. The quality control strain used was Escherichia coli ATCC 25922. The results of antibiotic susceptibility testing were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) (Hindler and Richter, 2016 ).