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.