The active ingredients of the selected antibiotics were cefotaxime (CTX), ceftazidime (CAZ), cefoxitin (FOX) and ceftiofur (CEF). The isolate was further tested by the double Selleckchem PSI-7977 disk diffusion tests using cefotaxime (CTX), ceftazidime (CAZ), cefoxitin (FOX) in combination with amoxicillin/clavulanic
acid (AMC) (Becton Dickinson, Germany) and Oxoid Ltd., UK) [17]. The MICS were determined by micro broth dilution method for the cephalosporins that showed buy VX-765 complete or decreased inhibition zone diameter in the disk diffusion test. Performance and evaluation of the MIC determinations followed the recommendation of the CLSI [18]. Sequence analysis of the β-lactamases genes Oligonucleotide primers targeting TEM and SHV β-lactamases and sequencing of the PCR products was performed as described in our previous study
[5]. The search for the homologous sequence was conducted in the GenBank database using the Basic Local Alignment BLZ945 chemical structure Search Tool (BLAST) through the National Center for Biotechnology Information (NCBI) web site (http://www.ncbi.nlm.nih.gov/BLAST). Nucleotide substitutions were analyzed based on information available in http://www.lahey.org/studies/webt.htm Site directed mutagenesis of blaSHV-1 genes Wild type bla SHV-1 gene from K. pneumoniae was cloned in pET 200 cloning SSR128129E vector. This plasmid was used as template for generating bla SHV(L138P), bla SHV-33(P226S) and bla SHV-33(L138P) genes by site directed mutagenesis following the procedures described by Zheng et. al [8, 19]. Description of the primers used in the study are
listed in Table 1. All the PCR-amplified products were evaluated by agarose gel electrophoresis and the band with the expected size was extracted using QIAEX® II gel extraction kit (Qiagen, Hilden, Germany) and further treated with 10 U DpnI (New England, Hertfordshire, UK) and incubated at 37°C for 3 hrs. An aliquot of 2 μl of this PCR product was transformed into TOPO 10 competent cells and plated on Tryptic Soy Agar (TSA) (Difco Laboratories, Detroit, MI) agar plate containing 100 μg/ml of kanamycin. A total of 3 colonies were selected and their plasmids were extracted using mini-prep. Sequences of all these β-lactamases were confirmed twice by the nucleotide sequencing using T7 forward and reverse primers. Table 1 Primers used for detection of TEM and SHV β-lactamases and for site directed mutagenesis in this study Targets Primer Sequence (5′-3′) Product size(bp) Annealing temp Gene bank Accession no.