e. the “”induction cultures”". Immediately after seeding, the colony forming units of these induction cultures were determined by plating serial dilutions on solid media. The induction cultures were incubated without or with the antibiotics ciprofloxacin, meropenem, fosfomycin, gentamicin, rifampicin, or chloramphenicol at the 4x, find more 1x, 0.25x, 0.064x, or 0.016x minimal inhibitory concentration (MIC) determined for STEC P5711 and P5765 for 24 hours at 37°C with vigorous shaking. Subsequently, cultures were centrifuged and supernatants were filtered through 0.45 μm signaling pathway filters (Millipore) and stored in aliquots

at -20°C. Quantitative RT-PCR for STX2 To determine the transcriptional induction of STX-encoding genes, 200 μl of the induction cultures were drawn two hours after start of the cultures. Total RNA was isolated (RNeasy Mini Kit, QIAGEN) and stored at −80°C. An 8 μl aliquot of each RNA extraction was transcribed into cDNA using random hexamers as

primer according to the manufacturer’s instructions (SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen). cDNA was stored at −20°C until further use. Quantitative PCR was set up using the hydrolysis probe assay for STX2 as described by Selleckchem Tubastatin A Sharma et al. for detection of STX2 genomic DNA [23]. Each cDNA was run in duplicate together with a dilution series of an STX2 plasmid standard on an LightCycler

480 realtime PCR machine with quantification software. Copy numbers of STX2 transcripts were calculated against the STX2 plasmid standard. Quantification of STX in STEC supernatants by EIA The contents of STX in the filtered supernatants of the bacterial cultures incubated with or without antibiotics were determined by a solid phase enzyme immunoassay (EIA) that detects both STX 1 and 2 (ProSpecT, REMEL, Lenexa, KS, USA). To assess the quantitative effect of antibiotics on the release of STX, 2-fold serial dilutions of the Orotidine 5′-phosphate decarboxylase supernatants were subjected to the EIA. The STX titer of a given supernatant was defined as the reciprocal dilution at which the optical density (OD) of the sample equaled the OD of the undiluted supernatant of the respective bacteria cultured without antibiotics. STX activity in STEC supernatants The toxin activity of STX in supernatants of bacterial cultures was determined by a Vero cell cytotoxicity assay modified from an assay of Gentry et al. [24]. Briefly, 100 μl of Vero cell suspensions were seeded in a 96-well plate at a density of 1.6 x 105 cells/ml and grown for 24 h at 37°C in 5% CO2 atmosphere. Subsequently, 100 μl of 10-fold serial dilutions of the filtered supernatants of bacterial cultures were added to the cultures.