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.Principal FindingsIn 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 CpxRKP to promoter region of ompC KP in a concentration dependent manner.Conclusions and SignificanceThe 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.

Although Acinetobacter baumannii is well accepted as a nosocomial pathogen, only a few of the outer membrane proteins (OMPs) have been functionally characterized. In this study, we demonstrate the biological functions of AbuO, a homolog of TolC from Escherichia coli. Inactivation of abuO led to increased sensitivity to high osmolarity and oxidative stress challenge. The ⌬abuO mutant displayed increased susceptibility to antibiotics, such as amikacin, carbenicillin, ceftriaxone, meropenem, streptomycin, and tigecycline, and hospital-based disinfectants, such as benzalkonium chloride and chlorhexidine Outer membrane proteins (OMPs) are known to have a pivotal role in bacterial physiology, such as adherence, invasion, and serum resistance, maintenance of cell structure, and binding a variety of substances, including passive and active transport (1). The archetypical OMP, TolC, has been considered to be a multifunctional protein due to its involvement in cell membrane integrity, acid tolerance, expulsion of metabolites, export of siderophores that are required in iron acquisition, export of plasmid and chromosomally encoded toxins, such as hemolysin, colicin V, and microcins, and virulence, as evident from studies in Enterobacter, Borrelia, Salmonella, Vibrio, Legionella, Francisella,. In E. coli, TolC is promiscuous because it supports the functioning of multidrug resistance efflux pumps, such as AcrD, AcrEF, and MdtABC (the resistance nodulation cell division [RND] superfamily) (11-13), EmrAB and EmrKY (the major facilitator superfamily [MFS]), and MacAB (the ATP-binding cassette [ABC] superfamily) (14-17). Although the functions of TolC homologs in many Gram-negative bacteria, such as E. coli, Vibrio vulnificus, Stenotrophomonas maltophilia, Enterobacter cloacae, and Yersinia pestis (5, 18-21), have been elucidated, the biological functions of TolC in an important human pathogen, Acinetobacter baumannii, have remained enigmatic so far.Multidrug-resistant (MDR) Acinetobacter strains kill up to 50% of infected patients, despite treatment with last resort drugs, and the resistance rates of such strains continue to escalate globally (22, 23). Significant increases in the number of A. baumannii strains that are resistant to carbapenems, cephalosporins, aminoglycosides, and fluoroquinolones with a diverse antibiotic resistome have been reported from hospitals in the United States and other countries (24-27). In our previous study, we demonstrated the role of antibiotic resistance genes and efflux pumps in mediating antimicrobial resistance in A. baumannii isolates from Ohio (28, 29). To date, few OMPs have been implicated in carbapenem resistance when their expression is reduced: CarO, Omp33 to -36, and an OprD homolog (30-32).In a continuation of our efforts toward understanding the origin/network of multidrug resistance in Acinetobacter, in this study, using genetic and molecular approaches, we demonstrate the role of a putative OMP (a homolog of TolC, designated AbuO) in bacterial stress physiology in general and a...

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.

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP-PCR) and multi-locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA-carbapenemases, metallo-b-lactamases (MBLs) and efflux pumps. REP-PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103 B . Second most prevalent ST belonged to clonal complex (CC) 92 B which is also referred to as international clone II. Most of the isolates were multi-drug resistant, being susceptible only to polymyxin-B and newer quinolones. Class D b-lactamases such as bla OXA-51-like (100%), bla OXA-23-like (56.8%) and bla OXA-24-like (14.8%) were found to be predominant, followed by a class B b-lactamase, namely bla IMP-1 (40.7%); none of the isolates had bla OXA-58 like, bla NDM-1 or bla SIM-1 . Genes of efflux-pump adeABC were predominant, most of isolates being biofilm producers that were PCR-positive for autoinducer synthase gene (>94%). Carbapenem non-susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA-type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.

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).