Trypticase soy agar 2

To assess the potential hemolytic activities of the different A. baumannii isolates, we spotted 5 μL of an overnight (O/N) culture of bacteria previously grown in LB medium for 16 h at 37°C under constant agitation (175 rpm) on 4 different blood agar plates: (i) Columbia agar with 5% horse blood, (ii) Columbia agar with 5% sheep blood, (iii) Trypticase soy agar II with 5% horse blood, and (iv) Trypticase soy agar II with 5% sheep blood, all purchased from BD (Becton, Dickinson and Company, Franklin Lakes, NJ). To test for secreted protease activity, we used the same approaches as described above and spotted the 5 μL of bacteria on LB agar plates containing 2% skim milk powder for microbiology (Sigma-Aldrich/Merck KGaA, Darmstadt, Germany). Plates were incubated at 25°C and monitored for hemolytic and protease activities after 1, 2, and 6 days of incubation.

To allow for better comparability in the framework of the present study, both the culture using whole milk as well as cultures of milk sediment, which represents the routine diagnostic procedure for the ZOBA laboratory, were performed. For sedimentation, milk tubes were centrifuged 10 min at 3000 rpm and the supernatant discarded. One loop full of whole milk and one loop full of milk sediment (each about 6μL) were streaked separately on a blood agar plate (Trypticase Soy Agar II with 5% Sheep Blood, BD) and on a Brolac agar plate (ThermoFisher, Oxoid AG, Pratteln, Switzerland) each. The results of the whole milk culture were taken as the standard culture to allow better comparability. The results of the milk sediment of the laboratory ZOBA were used only for one part of the BLCA of Strept. uberis in this study (see Table 3).
In both laboratories, blood agar plates were incubated at 37°C in a 5% CO₂ atmosphere and Brolac plates were incubated aerobically. After 16 to 24 h colonies were identified with Maldi-Tof MS (MALDI Biotyper, Bruker, Fällanden, Switzerland).

The following bacterial strains were used: E. coli DH5, S. aureus (ATCC 25923), S. epidermidis (CNS) (ATCC 12228) and S. agalactiae (GBS) (ATCC 27956). Bacterial stocks were plated on blood agar plates (BD™ Trypticase™ Soy Agar II with 5% Sheep Blood) o/n at 37°C and 5% CO₂. Bacterial suspensions were made with a single colony and OD600 was measured in duplicate with Versamax™ Microplate reader (Molecular Devices). OD600 was set on 0.6 (equivalent to 5 x 10⁸ CFU/mL). Specific dilutions were plated on LB agar or Blood agar and incubated overnight at 37°C. Colonies were counted the next day and suspensions were aliquoted and stored at -80°C.

Isogenic DAP-S and DNS pairs A6300/A6298, R6837/R6838 and JH1/JH4, JH5 of S. aureus were streaked on Trypticase™ Soy Agar II with 5% sheep blood (BD Biosciences) using sterile loops and incubated overnight at 37°C with 5% CO₂. The colonies were resuspended in 10mL of Muller Hinton Broth (BD BBL^TM) and the optical density (OD₆₀₀) was adjusted at ~0.01. The cultures were grown in a shaking incubator at 37°C. Aliquots were collected at 1 hour intervals for a period of 8 hours. The aliquots were diluted 10-fold and plated on sheep blood agar plates to quantify bacterial numbers. The plates were incubated at 37°C overnight. The colonies were counted and the results were expressed as Log₁₀ CFU/mL.

Streptococcus pneumoniae ATCC 6303 (a type 3 strain) was obtained from the American Type Culture Collection (ATCC). For in vivo challenges, frozen bacterial stocks of S. pneumoniae were streaked out on Trypticase Soy Agar II (TSA-II, BD Biosciences) plates containing 5% sheep blood. A single colony was inoculated into 5 ml of Todd-Hewitt broth with 0.5% added yeast extract (THY; both from Sigma Aldrich), and grown at 37°C with 5% CO₂ without shaking until bacteria reached an OD of 0.45–0.55 at 600 nm, representing ~10⁸ CFU/ml. Bacteria were spun down and resuspended in Dulbecco's Phosphate Buffered Saline (DPBS, Fisher Scientific), then diluted to the appropriate intended inoculum (7 × 10⁴-1.5 × 10⁶ CFU per mouse). 20 μl of bacterial suspension was inoculated per nare (40 μl total per mouse). The inoculum was verified after serial dilution in DPBS and plating on TSA-II plates with 5% sheep blood, incubated overnight at 37°C with 5% CO₂.