Background The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC KP , to mediate antimicrobial resistance in K. pneumoniae . Principal Findings In this study, functional characterization of the putative porin OmpC KP (denoted kpnO ) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, Δ kpnO in a clinical isolate of K. pneumoniae . Estimation of uronic acid content confirmed that Δ kpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The Δ kpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by ∼1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO , a phoB KP isogenic mutant was constructed. The phoB KP mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB KP mutant compared to wild type. A regulatory role of PhoB KP for the expression of kpnO was further supported by the specific binding of PhoB KP to the putative promoter of kpnO . Conclusions and Significance Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.
Background Acinetobacter baumannii is becoming an increasing menace in health care settings especially in the intensive care units due to its ability to withstand adverse environmental conditions and exhibit innate resistance to different classes of antibiotics. Here we describe the biological contributions of abeD, a novel membrane transporter in bacterial stress response and antimicrobial resistance in A. baumannii.ResultsThe abeD mutant displayed ~ 3.37 fold decreased survival and >5-fold reduced growth in hostile osmotic (0.25 M; NaCl) and oxidative (2.631 μM–6.574 μM; H2O2) stress conditions respectively. The abeD inactivated cells displayed increased susceptibility to ceftriaxone, gentamicin, rifampicin and tobramycin (~ 4.0 fold). The mutant displayed increased sensitivity to the hospital-based disinfectant benzalkonium chloride (~3.18-fold). In Caenorhabditis elegans model, the abeD mutant exhibited (P<0.01) lower virulence capability. Binding of SoxR on the regulatory fragments of abeD provide strong evidence for the involvement of SoxR system in regulating the expression of abeD in A. baumannii.ConclusionThis study demonstrates the contributions of membrane transporter AbeD in bacterial physiology, stress response and antimicrobial resistance in A. baumannii for the first time.
a b s t r a c tKlebsiella pneumoniae MGH78578 contains $500 uncharacterized signaling proteins and in this study, we characterized the biological functions of a novel eukaryotic-like serine/threonine kinase; ESTK (KpnK). Studies demonstrated that KpnK undergoes autophosphorylation within the pH range 7.0-7.5 at 37°C in a time-and concentration-dependent manner, with Mn 2+ as its cofactor. TheDkpnK mutant exhibited higher sensitivity to gastrointestinal and oxidative stresses. Deletion of kpnK resulted in a two to threefold increased susceptibility towards imipenem, cefepime, ceftriaxone and ceftazidime. Our study has provided overall evidence for the involvement of ESTK in regulating bacterial physiology, stress response and drug resistance. This report has unmasked the occurrence of Ser/Thr kinase mediated signaling for the first time in K. pneumoniae. Structured summary of protein interactions:KpnK phosphorylates KpnK by protein kinase assay (View interaction).
Nosocomial infections due to Klebsiella pneumoniae is a significant problem in health care settings worldwide. In this study, we examined the antimicrobial susceptibility, genetic profiles and mechanisms of antibiotic resistance in K. pneumoniae isolates of Indian origin. To our knowledge this is the first report demonstrating the high prevalence of β-lactamases, aminoglycoside modifying enzymes, quinolone resistance genes besides demonstrating the involvement of active efflux in K. pneumoniae Indian isolates. This study has enabled us to correlate the phenotypic and genotypic characteristics in K. pneumoniae, providing an important base for continued monitoring and epidemiological studies of this emerging nosocomial pathogen in Indian hospitals.
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