Only a single bacterial isolate per patient was evaluated. MICs for ceftazidime, cefepime, aztreonam, imipenem, meropenem, gentamicin, amikacin and ciprofloxacin were determined by agar dilution and interpreted according to Clinical Laboratory Standards Institute [20, 21]. P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 strains were used as quality

GSK1904529A in vivo control strains. Pulsed Field Gel Electrophoresis Genomic DNA of isolates was prepared in agarose blocks and digested with the restriction enzyme SpeI (New England, Beverly, MA). Electrophoresis was performed on CHEF-DR III (BioRad, Richmond, CA), with the following conditions: 0.5 × TBE, 1% agarose, 13°C, 200 V, for 24 h with switch time ramped from 5 to 90 s. The band patterns selleck kinase inhibitor were interpreted as previously recommended [22]. Screening for carbapenemase producers and detection of β-lactamases-encoding genes Investigation of carbapenemase activity in crude extracts was performed by UV spectrophotometric assays. Briefly, a full 10 μl loop of the test organism was inoculated into 500 μl of phosphate buffer 100 mM (pH 7.0) and disrupted by sonication. The cells were removed by centrifugation and the supernatants were used for further

experiments. Protein quantification in the crude extracts was performed using the Bradford stain. Hydrolytic activity of crude extracts was determined against 100 μM imipenem and 100 μM meropenem in 100 mM phosphate buffer (pH 7.0). Measurements were carried out at a 297 nm wavelength. Positive control included SPM-1-producing P. aeruginosa 48-1997A [23]. Carbapenem hydrolysis inhibition was performed by incubating the crude extract with 25 mM EDTA during 15

min, previously to the assay with imipenem and meropenem. Detection MBL-encoding genes was performed for all carbapenem-resistant isolates by multiplex PCR, as previously described [24]. The presence of ESBL-encoding genes bla TEM, bla SHV, bla CTX-M, bla GES, bla VEB and bla PER was investigated by PCR, as previously reported [12, 25]. Quantitative RT-PCR (RT-qPCR) Transcriptional levels of mexB, mexD, mexF, mexY, Lenvatinib molecular weight ampC and oprD were determined with Mastercycler Realplex2 (Eppendorf, Hamburg, Germany). In brief, total RNA was extracted using the RNase Mini Kit, following the manufacturer recommendations (Qiagen, Hilden, Germany). Five micrograms of total RNA was submitted to cDNA synthesis using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, USA). Quantitative RT-PCR was performed with Platinum SYBR Green Supermix (Invitrogen, Carlsbad, USA), using specific primers for mexB, mexD, mexF, mexY, ampC and oprD as previously described [26–29] or designed for this study using the GeneFisher online software http://​bibiserv.​techfak.​uni-bielefeld.​de/​genefisher/​old.​html (Table 3). Amplification was carried out in triplicate from cDNA preparations.