title: Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044.
abstract: BACKGROUND
  Klebsiella pneumoniae is a gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress.
  
  
  RESULTS
  In the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR(KP) to promoter region of ompC(KP) in a concentration dependent manner.
  
  
  CONCLUSIONS
  The Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the CpxAR signaling system and diversity of the antibiotic resistome in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.
authors.affiliation: Council of Scientific Industrial Research-Institute of Microbial Technology, Sector 39 A, Chandigarh, India.
authors.forename: Vijaya Bharathi
authors.initials: VB
authors.lastname: Srinivasan
authors.forename: Vasanth
authors.initials: V
authors.lastname: Vaidyanathan
authors.forename: Amitabha
authors.initials: A
authors.lastname: Mondal
authors.forename: Govindan
authors.initials: G
authors.lastname: Rajamohan
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000633:toxicity; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D000077723:Cefepime; D002511:Cephalosporins / Q000633:toxicity*; D002701:Chloramphenicol / Q000633:toxicity*; D019295:Computational Biology; D004269:DNA, Bacterial / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D007710:Klebsiella Infections / Q000188:drug therapy* / Q000235:genetics / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000254:growth & development / Q000472:pathogenicity; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D009997:Osmotic Pressure; D018384:Oxidative Stress; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D018440:beta-Lactam Resistance / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002511:Cephalosporins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; C085504:CpxR protein, Bacteria; D002701:Chloramphenicol; D000077723:Cefepime; D011494:Protein Kinases; C092472:CpxA protein, bacteria