pxAR Confers b-Lactam Resistance DNA methods Restriction digestion, ligation, transformation, and agarose gel electrophoresis were done according to standard protocols. Plasmids were prepared 
from E. coli using a QIAprep Spin miniprep kit from Qiagen according to the manufacturer’s protocol. Mobilization of plasmids into K. pneumoniae cells was performed as previously described. Genomic DNA of K. pneumoniae was extracted using the Gene Aid DNA purification kit according to the manufacturer’s instructions. DNA Vonoprazan price fragments used for cloning were extracted from agarose gels ” using a QIA quick gel extraction kit from Qiagen. PCR products were purified using a QIA quick PCR purification kit and, when cloned, sequenced to confirm the correct sequences. Primers used in the present study were custom-synthesized. Construction 16302825” of the cpxAR deletion mutant in K. pneumoniae strain NTUH-K2044 The MisT2 database shows the presence of.466 signaling proteins in the 5,472,672 bp genome sequence of the K1 serotype. The CpxAR operon is located starting from nucleotides 76799 bps to 78867 bps in the genome sequence of K. pneumoniae NTUH-K2044. To construct cpxAR knock out, a 700 bp internal fragment encompassing cpxA and cpxR of the operon was amplified by PCR using DcpxA/cpxR-F and DcpxA/cpxR-R primer from its genomic DNA. The PCR product was ligated into an EcoRI digested plasmid pUT-Km which was blunted by klenow reaction that contained the kanamycin resistance gene, transformed into E. coli S17-1l pir and the resulting recombinant plasmid harbouring the internal fragment of cpxAR was designated as pUT-Km/GR. The plasmid pUT-Km/GR was mobilized into recipient K. pneumoniae NTUH-K2044 from donor E. coli S17-1l pir. Briefly, K. pneumoniae was inoculated into 10 ml LB and was incubated for 23 h till OD600 nm reaches 0.2. For matings, recipient and donor culture were mixed in a ratio of 1:2 respectively, pelleted and spotted onto the centre of an LB agar plate. After 3 h of growth at 37uC the cells were plated on Klebsiella selective agar containing Kanamycin 100 mg/ml and 5 mg/ml chlorhexidine to select for colonies. It is expected that colonies that appear on the selective plate would be transconjugants that resulted from one DNA exchange event in which the whole suicidal plasmid gets incorporated in the K. pneumoniae genome. The disruption at cpxAR operon was confirmed with selected transconjugant by PCR and DNA sequencing using gene specific and genome flanking primers and deleted mutant was denoted as NTUH-K2044DcpxAR. Intact cpxAR genes were amplified along with its promoter using primer NT and primer CT and cloned into a pCRIITOPO-CAT plasmid. The selected recombinant plasmid harbouring the intact cpxAR operon was transformed into the cpxAR isogenic mutant strain by electroporation. The efflux pump inhibitors used in this study was carbonyl cyanide 3-chlorophenylhydrazone and reserpine. CCCP is an extremely effective proton motive force inhibitor and used in this study as an active efflux pump blocker. Efflux pump inhibitors had no intrinsic antibacterial activity against wild type strain at the concentration used in the experiments. Osmotic, bile, chlorhexidine challenge assays Various stress assays were performed as described previously. Briefly, K. pneumoniae NTUH-K2044 and NTUHK2044DcpxAR were grown to mid-exponential phase, cultures were spread plated onto LB agar plates containing different concentrations of NaCl, bile and chlorhexidine respectivel