Identification of the molecular mechanism of synergy between doxycycline and carprofen by 2D-DIGE and RNA-seq. Staphylococcus pseudintermedius strain:E104 | isolate:E104

The overall aim was to investigate the molecular mechanism of synergy between doxycycline (DOX, an antibiotic) and carprofen (CPF,a non-antibiotic). A non-antibiotic is here defined as a pharmaceutical preparation targeting eukaryotic cells and used for management of non-infectious diseases. Proteomic and transcriptomic analyzes of methicillin-resistant Staphylococcus pseudintermedius (MRSP, E104-ST71, MecA) were conducted by exposing the cell broth to DOX, CPF or DOX+CPF. A single colony of E104-ST71 was grown overnight in cation-adjusted Müller-Hinton broth (CAMHB), incubated and shaken at 37°C and 180 rpm. Overnight culture was diluted to an OD600 of 0.05 in CAMHB and re-grown to OD600 0.4 (10E8 CFU/mL). Main culture was initiated by diluting pre-culture 1:1000 (10E5 CFU/mL) in three replicate flasks (5 L, 1050 mL in each). At an OD600 of 0.1 (10E7 CFU/mL) every replicate culture was split into set of four flasks (1 L, 250 mL in each). Three cultures in each set where exposed to DOX (0.5 µg/mL), CPF (32 µg/mL) or a combination of both compounds and further incubated with an untreated control as above. Samples were taken every hour for optical density measurements and cell counts. 45 mL aliquots of each culture were collected after 90 min of exposure, washed once with saline and pelleted. Cultivation and exposure procedures were reproduced for RNA extraction. 6-9 mL aliquots of each culture were collected just before antimicrobial exposure and 30 and 90 min after, immediately suspended in RNA protect solution (twice the amount of culture), incubated for 5 min and centrifuged at 4000 rpm for 10 min at room temperature 20˚C. Pellets were stored at -20°C prior to protein and RNA extraction.