Comparative genome analysis and characterization of a MDR Klebsiella variicola.

Klebsiella variicola is an emerging pathogen responsible for causing blood-stream infections, urinary and respiratory tract related diseases in humans. In this report, we describe the genome sequence data and phenotypic characterization of K. variicola strain KV093 isolated from India. Comparative genome sequence analysis revealed the presence of genes linked with virulence, iron acquisition and transport, type 1 and type 3 pili, secretion systems including the capsular gene cluster. The plant-associated genes such as nitrogen fixation, growth and defense mechanisms against oxidative stress were also identified. On performing antibiotic susceptibility testing, growth inhibition, and stress challenge assays it was observed that the drug resistant K. variicola KV093 exhibited cross resistance to various antibiotics, antiseptics, including disinfectants. This report highlights the arsenal of virulence and antibiotic resistance determinants in K. variicola KV093, an effort emphasizing the current pressing need for regular surveillance of K. variicola strains especially in India.

Genome analysis of urease positive Serratia marcescens, co-producing SRT-2 and AAC(6')-Ic with multidrug efflux pumps for antimicrobial resistance.

In this study, we present the genome sequence of Serratia marcescens SM03, recovered from a human gut in India. The final assembly consists of 26 scaffolds (4620 coding DNA sequences, 5.08 Mb, 59.6% G + C ratio) and 79 tRNA genes. Analysis identified novel genes associated with lactose utilization, virulence, P-loop GTPases involved in urease production, CFA/I fimbriae apparatus and Yersinia - type CRISPR proteins. Antibiotic susceptibility testing indicated drug tolerant phenotype and inhibition assays demonstrated involvement of extrusion in resistance. Presence of enzymes SRT-2, AAC(6')-Ic, with additional Ybh transporter and EamA-like efflux pumps signifies the genetic plasticity observed in S. marcescens SM03.

Genome sequence and comparative analysis of Bacillus cereus BC04, reveals genetic diversity and alterations for antimicrobial resistance.

In this study, we delineated the genome sequence of a Bacillus cereus strain BC04 isolated from a stool sample in India. The draft genome is 5.1 Mb in size and consists of total 109 scaffolds, GC content is 35.2% with 5182 coding genes. The comparative analysis with other completely sequenced genomes highlights the unique presence of genomic islands, hemolysin, capsular synthetic protein, modifying enzymes accC7 and catA15, regulators of antibiotic resistance MarR and LysR with annotated functions related to virulence, stress response, and antimicrobial resistance. Overall, this study not only signifies the genetic diversity in gut isolate BC04 in particular, but also pinpoints the presence of unique genes possessed by B. cereus which can be pertinently exploited to design novel drugs and intervention strategies for the treatment of food borne diseases.

Correction: Occurrence of Diverse Antimicrobial Resistance Determinants in Genetically Unrelated Biocide Tolerant Klebsiella pneumoniae.

[This corrects the article DOI: 10.1371/journal.pone.0166730.].

Occurrence of Diverse Antimicrobial Resistance Determinants in Genetically Unrelated Biocide Tolerant Klebsiella pneumoniae.

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 ß-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.

Functional Characterization of AbeD, an RND-Type Membrane Transporter in Antimicrobial Resistance in Acinetobacter baumannii.

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. RESULTS: The 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. CONCLUSION: This study demonstrates the contributions of membrane transporter AbeD in bacterial physiology, stress response and antimicrobial resistance in A. baumannii for the first time.

AbuO, a TolC-like outer membrane protein of Acinetobacter baumannii, is involved in antimicrobial and oxidative stress resistance.

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 ΔabuO 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. The reverse transcription (RT)-PCR analysis indicated increased expression of efflux pumps (resistance nodulation cell division [RND] efflux pump acrD, 8-fold; SMR-type emrE homolog, 12-fold; and major facilitator superfamily [MFS]-type ampG homolog, 2.7-fold) and two-component response regulators (baeR, 4.67-fold; ompR, 10.43-fold) in the ΔabuO mutant together with downregulation of rstA (4.22-fold) and the pilin chaperone (9-fold). The isogenic mutant displayed lower virulence in a nematode model (P<0.01). Experimental evidence for the binding of MerR-type transcriptional regulator SoxR to radiolabeled abuO promoter suggests regulation of abuO by SoxR in A. baumannii.

Role of novel multidrug efflux pump involved in drug resistance in Klebsiella pneumoniae.

BACKGROUND: Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum ß-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance. PRINCIPAL FINDINGS: Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology. CONCLUSIONS AND SIGNIFICANCE: The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.

KpnEF, a new member of the Klebsiella pneumoniae cell envelope stress response regulon, is an SMR-type efflux pump involved in broad-spectrum antimicrobial resistance.

Klebsiella pneumoniae has been frequently associated with nosocomial infections. Efflux systems are ubiquitous transporters that also function in drug resistance. Genome analysis of K. pneumoniae strain NTUH-K2044 revealed the presence of ∼15 putative drug efflux systems. We discuss here for the first time the characterization of a putative SMR-type efflux pump, an ebrAB homolog (denoted here as kpnEF) with respect to Klebsiella physiology and the multidrug-resistant phenotype. Analysis of hypermucoviscosity revealed direct involvement of kpnEF in capsule synthesis. The ΔkpnEF mutant displayed higher sensitivity to hyperosmotic (∼2.8-fold) and high bile (∼4.0-fold) concentrations. Mutation in kpnEF resulted in increased susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin; mutated strains changed from being resistant to being susceptible, and the resistance was restored upon complementation. The ΔkpnEF mutant displayed enhanced sensitivity toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes, including clinically relevant disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The prevalence of kpnEF in clinical strains broadens the diversity of antibiotic resistance in K. pneumoniae. Experimental evidence of CpxR binding to the efflux pump promoter and quantification of its expression in a cpxAR mutant background demonstrated kpnEF to be a member of the Cpx regulon. This study helps to elucidate the unprecedented biological functions of the SMR-type efflux pump in Klebsiella spp.

Role of oxyRKP, a novel LysR-family transcriptional regulator, in antimicrobial resistance and virulence in Klebsiella pneumoniae.

Klebsiella pneumoniae is a Gram-negative bacillus that causes serious infections in immunocompromised human hosts and exhibits significant multidrug resistance. In this study, we identified a novel lysR-family regulator (designated oxyR(KP)) in the genome of K. pneumoniae NTUH-K2044 whose functions have remained enigmatic so far. Functional characterization of the putative lysR regulator oxyR(KP) with respect to cellular physiology and antimicrobial susceptibility was performed by generating an isogenic mutant, ΔoxyR(KP) in a hypervirulent clinical isolate of K. pneumoniae. The K. pneumoniae oxyR(KP) mutant was sensitive to hyperosmotic and bile conditions. Disruption of oxyR(KP) increased the susceptibility of K. pneumoniae to oxidative (0.78947 mM hydrogen peroxide) and nitrosative (30 mM acidified nitrite) stress by ~1.4-fold and ~10-fold, respectively. Loss of the Klebsiella regulator led to a decrease in the minimum inhibitory concentrations for chloramphenicol (10-fold), erythromycin (6-fold), nalidixic acid (>50-fold) and trimethoprim (10-fold), which could be restored following complementation. The relative change in expression of resistance-nodulation-cell division super family (RND) efflux gene acrB was decreased by approximately fivefold in the oxyR(KP) mutant as evidenced by qRT-PCR. In a Caenorhabditis elegans model, the oxyR(KP) mutant exhibited significantly (P<0.01) lower virulence. Overall, results detailed in this report reflect the pleiotropic role of the oxyR(KP) signalling system and diversity of the resistance determinants in hypervirulent K1 serotype K. pneumoniae NTUH-K2044.

Functional characterization of a novel Mn2+ dependent protein serine/threonine kinase KpnK, produced by Klebsiella pneumoniae strain MGH78578.

Klebsiella 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. The ΔkpnK 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.

Functional characterization of a novel outer membrane porin KpnO, regulated by PhoBR two-component system in Klebsiella pneumoniae NTUH-K2044.

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, ΔkpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ΔkpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The ΔkpnO 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.

Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044.

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 ß-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 FINDINGS: 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 ß-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 AND SIGNIFICANCE: 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.

Molecular cloning and functional characterization of two novel membrane fusion proteins in conferring antimicrobial resistance in Acinetobacter baumannii.

OBJECTIVES: The aim of this study was to elucidate the role of two novel membrane fusion proteins (MFPs) in the susceptibility of Acinetobacter baumannii to antimicrobial agents. METHODS: The genome sequence of A. baumannii ATCC 17978 contains two open reading frames (ORFs) annotated as AdeT in the NCBI genome database. Both the putative efflux genes display >30% similarity to known MFPs. The antimicrobial susceptibility profiles of Escherichia coli KAM32 cells carrying the genes were monitored by the broth dilution method. Different efflux pump inhibitors were used for fluorimetric efflux assays. The functions of putative ORFs were confirmed in A. baumannii by insertional inactivation and complementation. RESULTS: E. coli cells carrying the ORFs had decreased susceptibility to antibiotics, disinfectants, dyes and detergents, with enhanced efflux activity. Inactivation of the ORFs and further characterization in A. baumannii confirmed its role in antimicrobial resistance by active efflux. CONCLUSIONS: This report describes the functions of novel resistance determinants, members of the MFP family, for the first time in A. baumannii.

Molecular and functional characterization of a novel efflux pump, AmvA, mediating antimicrobial and disinfectant resistance in Acinetobacter baumannii.

OBJECTIVES: Acinetobacter baumannii has emerged as an important nosocomial pathogen in hospitalized patients, and causes a multitude of infections with significant morbidity and mortality. The aim of this study was to elucidate the role of a novel efflux pump in A. baumannii. METHODS: The open reading frame ABAYE1518, annotated as a putative Methyl Viologen resistance protein in the genome of strain A. baumannii AYE, exhibits >50% similarity with members of the major facilitator superfamily (MFS) multidrug efflux pumps. The antimicrobial susceptibility profiles of Escherichia coli KAM32 cells carrying the putative efflux pump were monitored by broth dilution method. Different efflux pump inhibitors were used for fluorimetric efflux assays. The functions of the putative efflux pump were confirmed in A. baumannii by insertional inactivation and complementation. Its expression in clinical isolates was analysed by reverse transcriptase-PCR. RESULTS: E. coli cells carrying the pump had decreased susceptibility to some antibiotics, disinfectants, dyes and detergents, with enhanced efflux activity. The pump was inactivated in a clinical isolate of A. baumannii AC0037 and further characterization confirmed its role in antimicrobial resistance by active efflux. We found increased expression of the pump in clinical isolates that also exhibited elevated tolerance to antibacterial agents. CONCLUSIONS: This report describes the functions of a novel resistance determinant, a member of the MFS efflux pumps, for the first time in A. baumannii.

Novel role of Acinetobacter baumannii RND efflux transporters in mediating decreased susceptibility to biocides.

OBJECTIVES: Biocides and dyes are commonly employed in hospital and laboratory settings. We investigated the biocide susceptibilities of a rapidly emerging pathogen, Acinetobacter baumannii, and the underlying molecular mechanisms, with a primary focus on resistance-nodulation-cell division (RND) efflux systems. METHODS: Biocide susceptibilities, efflux and in vitro inactivation profiles were monitored in the presence/absence of efflux pump inhibitors. The RND transporters encoded by adeB and adeJ were detected by PCR; null mutants were constructed in the native host. Expression of adeB and adeJ in clinical isolates was assayed by semi-quantitative RT-PCR. RESULTS: Susceptibility testing and phenotypic assays demonstrated the role of active efflux in mediating decreased susceptibility to biocides. Inactivation of either the adeB or adeJ transporter gene led to increased susceptibility to biocides. RT-PCR analysis exhibited increased adeB and adeJ expression in clinical isolates. CONCLUSIONS: This is the first study demonstrating the role of efflux pumps in mediating decreased susceptibility to disinfectants and other chemical substrates in A. baumannii.

Role of AbeS, a novel efflux pump of the SMR family of transporters, in resistance to antimicrobial agents in Acinetobacter baumannii.

In this study, a chromosomally encoded putative drug efflux pump of the SMR family, named AbeS, from a multidrug-resistant strain of Acinetobacter baumannii was characterized to elucidate its role in antimicrobial resistance. Expression of the cloned abeS gene in hypersensitive Escherichia coli host KAM32 resulted in decreased susceptibility to various classes of antimicrobial agents, detergents, and dyes. Deletion of the abeS gene in A. baumannii confirmed its role in conferring resistance to these compounds.

Mechanism of drug resistance in clonally related clinical isolates of Vibrio fluvialis isolated in Kolkata, India.

The molecular mechanisms of drug resistance in 19 strains of Vibrio fluvialis isolated from 1998 to 2002 in Kolkata, India, were investigated. Class 1 integrons were detected in eight strains, and four strains were found to carry SXT integrases. In the presence of carbonyl cyanide m-chlorophenylhydrazone or reserpine, all nalidixic acid- and ciprofloxacin-resistant strains became sensitive, suggesting that drug efflux plays a major role in quinolone resistance in V. fluvialis. It was further seen that strains which had MICs of > 25 microg/ml for nalidixic acid had a sense mutation (Ser to Ile) at position 83 of the quinolone resistance-determining region of gyrA. All except one of the integron- and SXT integrase-bearing strains belonged to the same ribotype.