Transcriptome analysis of extended spectrum beta-lactamase (ESBL) producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) exposed to cefotaxime

The overall aim was to investigate reversal of antibiotic resistance by combining antibiotics with “helper drugs” able to restore susceptibility either by inhibiting the expression of the resistance gene (true reversal of resistance) or by synergistic mechanisms (false reversal). An objective to this study was to identify helper drug targets (genes) targeted by helper drugs to counteract resistance towards cefotaxime (CTX). Transcriptome analysis of extended spectrum β-lactamase (ESBL) producing Escherichia coli (UR40-ST131, CTX-M-15 and R7AC-ST297, CMY-2) and methicillin-resistant Staphylococcus aureus (MRSA, 55488-ST398, MecA) were conducted by exposing the cell broth to CTX. Strains were cultured as follow to analyze the effect of CTX. A single colony of UR40-ST131, R7AC-ST297 or 55488-ST398 was grown overnight in cation-adjusted Müller-Hinton broth, incubated and shaken at 37°C and 180 rpm. Each overnight culture was then diluted in fresh media (OD600=0.05) and grown to OD600=0.4 (10E8 CFU/mL). This pre-culture was then diluted a thousand fold to produce triplicate cultures (600 mL) in 5L conical flasks (OD600=0.001, 10E5 CFU/mL). Incubation was continued as above. At OD600=0.1 (10E7 CFU/mL) each culture was divided into two new 3L flasks with 250 mL in each. One was added 30 µg/mL CTX while the second formed an unexposed control. All cultures were further incubated as above, sampled every hour for optical density measurements and cell counts. Samples (3-6 mL) for RNA extraction were taken just before antimicrobial exposure and 30 and 90 min after, immediately mixed with 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 RNA extraction.