scholarly journals Sequential Time-Kill, a Simple Experimental Trick To Discriminate between Pharmacokinetics/Pharmacodynamics Models with Distinct Heterogeneous Subpopulations versus Homogenous Population with Adaptive Resistance

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
A. Chauzy ◽  
H. Ih ◽  
M. Jacobs ◽  
S. Marchand ◽  
N. Grégoire ◽  
...  
Keyword(s):  
Polymyxin B ◽  
Klebsiella Pneumonia ◽  
Wild Type ◽  
Colistin Resistance ◽  
Content Type ◽  
Adaptive Resistance ◽  
Consecutive Time ◽  
Time Kill ◽  
Isogenic Strains ◽  
Homogenous Population

ABSTRACT Experiments were conducted with polymyxin B and two Klebsiella pneumonia isogenic strains (the wild type, KP_WT, and its transconjugant carrying the mobile colistin resistance gene, KP_MCR-1) to demonstrate that conducting two consecutive time-kill experiments (sequential TK) represents a simple approach to discriminate between pharmacokinetics/pharmacodynamics models with two heterogeneous subpopulations or adaptive resistance.

scholarly journals Bactericidal Activity, Absence of Serum Effect, and Time-Kill Kinetics of Ceftazidime-Avibactam against β-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

2014 ◽  
Vol 58 (9) ◽  
pp. 5297-5305 ◽  
Author(s):  
Tiffany R. Keepers ◽  
Marcela Gomez ◽  
Chris Celeri ◽  
Wright W. Nichols ◽  
Kevin M. Krause
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Serum ◽  
Bactericidal Activity ◽  
Minimum Bactericidal Concentration ◽  
Wild Type ◽  
Content Type ◽  
Positive Isolate ◽  
New Treatment ◽  
Time Kill ◽  
Kinetics Of

ABSTRACTAvibactam, a non-β-lactam β-lactamase inhibitor with activity against extended-spectrum β-lactamases (ESBLs), KPC, AmpC, and some OXA enzymes, extends the antibacterial activity of ceftazidime against most ceftazidime-resistant organisms producing these enzymes. In this study, the bactericidal activity of ceftazidime-avibactam against 18Pseudomonas aeruginosaisolates and 15Enterobacteriaceaeisolates, including wild-type isolates and ESBL, KPC, and/or AmpC producers, was evaluated. Ceftazidime-avibactam MICs (0.016 to 32 μg/ml) were lower than those for ceftazidime alone (0.06 to ≥256 μg/ml) against all isolates except for 2P. aeruginosaisolates (1blaVIM-positive isolate and 1blaOXA-23-positive isolate). The minimum bactericidal concentration/MIC ratios of ceftazidime-avibactam were ≤4 for all isolates, indicating bactericidal activity. Human serum and human serum albumin had a minimal effect on ceftazidime-avibactam MICs. Ceftazidime-avibactam time-kill kinetics were evaluated at low MIC multiples and showed time-dependent reductions in the number of CFU/ml from 0 to 6 h for all strains tested. A ≥3-log10decrease in the number of CFU/ml was observed at 6 h for allEnterobacteriaceae, and a 2-log10reduction in the number of CFU/ml was observed at 6 h for 3 of the 6P. aeruginosaisolates. Regrowth was noted at 24 h for some of the isolates tested in time-kill assays. These data demonstrate the potent bactericidal activity of ceftazidime-avibactam and support the continued clinical development of ceftazidime-avibactam as a new treatment option for infections caused byEnterobacteriaceaeandP. aeruginosa, including isolates resistant to ceftazidime by mechanisms dependent on avibactam-sensitive β-lactamases.

scholarly journals Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Anna Olsson ◽  
Pikkei Wistrand-Yuen ◽  
Elisabet I. Nielsen ◽  
Lena E. Friberg ◽  
Linus Sandegren ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Time Lapse ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Positive Interactions ◽  
Synergistic Activity ◽  
Content Type ◽  
Time Lapse Microscopy ◽  
Time Kill ◽  
Antibiotic Combination

ABSTRACT Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (≤106 CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal β-lactams, ciprofloxacin, and amikacin. Genes encoding β-lactamases (e.g., blaPAO and blaOXA-50) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.

scholarly journals Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Shannon R. Coleman ◽  
Travis Blimkie ◽  
Reza Falsafi ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Iron Acquisition ◽  
Polymyxin B ◽  
Rna Seq ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

scholarly journals A Novel Deletion Mutation in pmrB Contributes to Concurrent Colistin Resistance in Carbapenem-Resistant Escherichia coli Sequence Type 405 of Clinical Origin

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ching-Hsun Wang ◽  
L. Kristopher Siu ◽  
Feng-Yee Chang ◽  
Yu-Kuo Tsai ◽  
Yi-Tsung Lin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Selective Pressure ◽  
Carbapenem Resistance ◽  
Sequence Type ◽  
Wild Type ◽  
Colistin Resistance ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Recombinant Dna Techniques

ABSTRACT We report the first clinical Escherichia coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrB that contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

scholarly journals Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Mei-Ling Han ◽  
Yan Zhu ◽  
Darren J. Creek ◽  
Yu-Wei Lin ◽  
Dovile Anderson ◽  
...  
Keyword(s):  
Lipid A ◽  
Therapeutic Option ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Wild Type ◽  
Metabolomics Data ◽  
Content Type ◽  
In The Wild ◽  
High Level ◽  
Acyl Coenzyme A

ABSTRACTMultidrug-resistantPseudomonas aeruginosapresents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance inP. aeruginosahas been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistantP. aeruginosastrains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-typeP. aeruginosastrain K ([PAK] polymyxin B MIC, 1 mg/liter) and its pairedpmrBmutant strains, PAKpmrB6and PAKpmrB12(polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) inspeE(encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6compared to that in PAKpmrB12. Our results indicate that spermidine may play an important role in high-level polymyxin resistance inP. aeruginosa. Interestingly, bothpmrBmutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12mutant exhibited much lower levels of phospholipids than the PAKpmrB6mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance inP. aeruginosaand highlights its impacts on bacterial metabolism.

scholarly journals Proteomic Investigation of the Signal Transduction Pathways Controlling Colistin Resistance in Klebsiella pneumoniae

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Ching Hei Phoebe Cheung ◽  
Punyawee Dulyayangkul ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Klebsiella Pneumoniae ◽  
Protein Production ◽  
Wild Type ◽  
Colistin Resistance ◽  
Reciprocal Effect ◽  
Content Type ◽  
Direct Activation ◽  
Proteomic Investigation ◽  
Two Component Systems ◽  
Operon Expression

ABSTRACT Colistin resistance in Klebsiella pneumoniae is predominantly caused by mutations that increase expression of the arn (also known as pbg or pmrF) operon. Expression is activated by the PhoPQ and PmrAB two-component systems. Constitutive PhoPQ activation occurs directly by mutation or following loss of MgrB. PhoPQ may also cross-activate PmrAB via the linker protein PmrD. Using proteomics, we show that MgrB loss causes a wider proteomic effect than direct PhoPQ activation, suggesting additional targets for MgrB. Different mgrB mutations cause different amounts of Arn protein production, which correlated with colistin MICs. Disruption of phoP in an mgrB mutant had a reciprocal effect to direct activation of PhoQ in a wild-type background, but the regulated proteins showed almost total overlap. Disruption of pmrD or pmrA slightly reduced Arn protein production in an mgrB mutant, but production was still high enough to confer colistin resistance; disruption of phoP conferred wild-type Arn production and colistin MIC. Activation of PhoPQ directly or through mgrB mutation did not significantly activate PmrAB or PmrC production, but direct activation of PmrAB by mutation was able to do this, and also activated Arn production and conferred colistin resistance. There was little overlap between the PmrAB and PhoPQ regulons. We conclude that under the conditions used for colistin susceptibility testing, PhoPQ-PmrD-PmrAB cross-regulation is not significant and that independent activation of PhoPQ or PmrAB is the main reason that Arn protein production increases above the threshold required for colistin resistance.

scholarly journals New Polymyxin B Dosing Strategies To Fortify Old Allies in the War against KPC-2-Producing Klebsiella pneumoniae

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Zackery P. Bulman ◽  
Michael J. Satlin ◽  
Liang Chen ◽  
Barry N. Kreiswirth ◽  
Beom Soo Shin ◽  
...  
Keyword(s):  
Single Dose ◽  
Klebsiella Pneumoniae ◽  
Polymyxin B ◽  
Infection Model ◽  
Triple Combination ◽  
Content Type ◽  
High Single Dose ◽  
Dosing Strategies ◽  
Time Required ◽  
Time Kill

ABSTRACT Pharmacodynamics of a polymyxin B, meropenem, and rifampin triple combination were examined against Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) ST258. In time-kill experiments against three KPC-Kp isolates, triple combination generated 8.14, 8.19, and 8.29 log10 CFU/ml reductions within 24 h. In the hollow-fiber infection model, the triple combination caused maximal killing of 5.16 log10 CFU/ml at 78 h and the time required for regrowth was more than doubled versus the 2-drug combinations. Remarkably, combinations with a high single-dose polymyxin B burst plus rifampin preserved KPC-Kp polymyxin susceptibility (MIC240 h = 0.5 mg/liter) versus the same combination with traditionally dosed polymyxin B, where resistance was amplified (MIC240 h = 32 mg/liter).

scholarly journals Serratia marcescensarn, a PhoP-Regulated Locus Necessary for Polymyxin B Resistance

2014 ◽  
Vol 58 (9) ◽  
pp. 5181-5190 ◽  
Author(s):  
Quei Yen Lin ◽  
Yi-Lin Tsai ◽  
Ming-Che Liu ◽  
Wei-Cheng Lin ◽  
Po-Ren Hsueh ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Challenge Test ◽  
Polymyxin B ◽  
Resistant Bacteria ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
In The Wild

ABSTRACTPolymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly againstSerratia marcescens. To investigate the underlying mechanisms, Tn5mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5inserted into thearnBandarnCgenes. In other bacteria,arnBandarnCbelong to the seven-genearnoperon, which is involved in lipopolysaccharide (LPS) modification. LPSs ofarnmutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility inS. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression ofphoPandarnin the wild-type strain but not in thephoPmutant. Complementation of thephoPmutant with the full-lengthphoPgene restored the PB MIC and induction by PB and low Mg2+levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to thearnpromoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+levels protectedS. marcescensfrom a PB challenge in the wild-type strain but not in thephoPmutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression ofugd, a gene required for LPS modification, inS. marcescensthrough a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression ofarnAupon exposure to PB than did susceptible isolates. This is the first report to describe the role ofS. marcescensarnin PB resistance and its modulation by PB and Mg2+through the PhoP protein.

scholarly journals Genetic Barcodes for Improved Environmental Tracking of an Anthrax Simulant

2012 ◽  
Vol 78 (23) ◽  
pp. 8272-8280 ◽  
Author(s):  
Patricia Buckley ◽  
Bryan Rivers ◽  
Sarah Katoski ◽  
Michael H. Kim ◽  
F. Joseph Kragl ◽  
...  
Keyword(s):  
Environmental Fate ◽  
Wild Type ◽  
Risk Models ◽  
Content Type ◽  
Genetic Signatures ◽  
Isogenic Strains ◽  
Type Strains ◽  
Long Term Studies

ABSTRACTThe development of realistic risk models that predict the dissemination, dispersion and persistence of potential biothreat agents have utilized nonpathogenic surrogate organisms such asBacillus atrophaeussubsp.globigiior commercial products such asBacillus thuringiensissubsp.kurstaki. Comparison of results from outdoor tests under different conditions requires the use of genetically identical strains; however, the requirement for isogenic strains limits the ability to compare other desirable properties, such as the behavior in the environment of the same strain prepared using different methods. Finally, current methods do not allow long-term studies of persistence or reaerosolization in test sites where simulants are heavily used or in areas whereB. thuringiensissubsp.kurstakiis applied as a biopesticide. To create a set of genetically heterogeneous yet phenotypically indistinguishable strains so that variables intrinsic to simulations (e.g., sample preparation) can be varied and the strains can be tested under otherwise identical conditions, we have developed a strategy of introducing small genetic signatures (“barcodes”) into neutral regions of the genome. The barcodes are stable over 300 generations and do not impactin vitrogrowth or sporulation. Each barcode contains common and specific tags that allow differentiation of marked strains from wild-type strains and from each other. Each tag is paired with specific real-time PCR assays that facilitate discrimination of barcoded strains from wild-type strains and from each other. These uniquely barcoded strains will be valuable tools for research into the environmental fate of released organisms by providing specific artificial detection signatures.

scholarly journals Activity of a Novel 1,3-Beta-D-Glucan Synthase Inhibitor, Ibrexafungerp (Formerly SCY-078), against Candida glabrata

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
M. Ghannoum ◽  
L. Long ◽  
N. Isham ◽  
C. Hager ◽  
R. Wilson ◽  
...  
Keyword(s):  
Candida Glabrata ◽  
Wild Type ◽  
Glucan Synthase ◽  
Content Type ◽  
Clinical Strains ◽  
Resistant Strains ◽  
Oral Availability ◽  
Synthase Inhibitor ◽  
Time Kill ◽  
Type Strains

ABSTRACT Ibrexafungerp (formerly SCY-078), a novel glucan synthase inhibitor with oral availability, was evaluated for activity against Candida glabrata. The susceptibility of clinical strains to ibrexafungerp was determined by microdilution and time-kill assays. The MIC range against wild-type strains was 1 to 2 μg/ml. Ibrexafungerp was also active against the majority of echinocandin-resistant strains. Time-kill studies showed 4- to 6-log-unit reductions in growth at 24 and 48 h with concentrations of 0.25 to 4 μg/ml.

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