#Phenotypic, biochemical and #genetic analysis of #KPC-41, a KPC-3 variant conferring #resistance to #ceftazidime-avibactam and exhibiting reduced #carbapenemase activity (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Phenotypic, biochemical and genetic analysis of KPC-41, a KPC-3 variant conferring resistance to ceftazidime-avibactam and exhibiting reduced carbapenemase activity

Linda Mueller, Amandine Masseron, Guy Prod’Hom, Tatiana Galperine, Gilbert Greub, Laurent Poirel, Patrice Nordmann

DOI: 10.1128/AAC.01111-19

 

ABSTRACT

A novel KPC variant, KPC-41, was identified in a Klebsiella pneumoniae clinical isolate from Switzerland. This ß-lactamase possessed a three amino-acid insertion (Pro-Asn-Lys) located between amino acids 269 and 270 compared to the KPC-3 amino acid sequence. Cloning and expression of the blaKPC-41 gene in Escherichia coli, followed by determination of MIC values and kinetic parameters, showed that KPC-41, compared to KPC-3, has an increased affinity to ceftazidime and a decreased sensitivity to avibactam, leading to resistance to ceftazidime-avibactam once produced in K. pneumoniae. Furthermore, KPC-41 exhibited a drastic decrease of its carbapenemase activity. This report highlights that a diversity of KPC variants conferring resistance to ceftazidime-avibactam already circulate in Europe.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; Klebsiella pneumoniae; Ceftazidime; Avibactam.

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An in situ high-throughput screen identifies #inhibitors of intracellular #Burkholderia pseudomallei with therapeutic efficacy (Proc Natl Acad Sci USA, abstract)

[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy

Philip L. Bulterys, Isabelle J. Toesca, Michael H. Norris, Jeffrey P. Maloy, Sorel T. Fitz-Gibbon, Bryan France, Babak Toffig, Marco Morselli, Nawarat Somprasong, Matteo Pellegrini, Herbert P. Schweizer, Apichai Tuanyok, Robert Damoiseaux, Christopher T. French, and Jeff F. Miller

PNAS first published August 22, 2019 / DOI: https://doi.org/10.1073/pnas.1906388116

Contributed by Jeff F. Miller, July 13, 2019 (sent for review April 15, 2019; reviewed by Yunn-Hwen Gan and John J. Mekalanos)

 

Significance

Burkholderia pseudomallei, the etiologic agent of melioidosis, is an environmental organism that inhabits tropical soils and kills an estimated 90,000 people each year. Caused by an intracellular and often drug-resistant pathogen, melioidosis is notoriously difficult to treat, with mortality rates approaching 50% in some settings despite appropriate diagnosis and clinical management. Using a high-throughput, cell-based phenotypic screen we have discovered 2 antibiotic candidates with improved in vivo efficacy compared to the current standard of care: a fluoroquinolone analog, burkfloxacin, and an FDA-approved antifungal drug, flucytosine. As a widely used antifungal with a well-known safety profile, the potential to repurpose flucytosine for treating melioidosis may represent a rapid route to clinical translation.

 

Abstract

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm. Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell–cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia. In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

Burkholderia pseudomallei  – melioidosis – type 6 secretion system (T6SS) – small molecule – drug discovery

Keywords: Antibiotics; Burkholderia pseudomallei; Melioidosis; Burkfloxacin; Flucytosine.

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Emergence of #ceftazidime / #avibactam #resistance in #KPC-3-producing #Klebsiella pneumoniae in vivo (J Antimicrob Chemother., abstract)

[Source: Journal of Antimicrobial Chemotherapy, full page: (LINK). Abstract, edited.]

Emergence of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae in vivo

Stephan Göttig, Denia Frank, Eleonora Mungo, Anika Nolte, Michael Hogardt, Silke Besier,Thomas A Wichelhaus

Journal of Antimicrobial Chemotherapy, dkz330, https://doi.org/10.1093/jac/dkz330

Published: 31 July 2019

 

Abstract

Objectives

The β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is active against KPC-producing Enterobacterales. Herein, we present molecular and phenotypic characterization of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae that emerged in vivo and in vitro.

Methods

Sequence analysis of blaKPC-3 was performed from clinical and in vitro-generated ceftazidime/avibactam-resistant K. pneumoniae isolates. Time–kill kinetics and the Galleria mellonella infection model were applied to evaluate the activity of ceftazidime/avibactam and imipenem alone and in combination.

Results

The ceftazidime/avibactam-resistant clinical K. pneumoniae isolate revealed the amino acid change D179Y in KPC-3. Sixteen novel mutational changes in KPC-3 among in vitro-selected ceftazidime/avibactam-resistant isolates were described. Time–kill kinetics showed the emergence of a resistant subpopulation under selection pressure with either imipenem or ceftazidime/avibactam. However, combined selection pressure with imipenem plus ceftazidime/avibactam prevented the development of resistance and resulted in bactericidal activity. Concordantly, the G. mellonella infection model revealed that monotherapy with ceftazidime/avibactam is prone to select for resistance in vivo and that combination therapy with imipenem results in significantly better survival.

Conclusions

Ceftazidime/avibactam is a valuable antibiotic against MDR and carbapenem-resistant Enterobacterales. Based on time–kill kinetics as well as an in vivoinfection model we postulate a combination therapy of ceftazidime/avibactam and imipenem as a strategy to prevent the development of ceftazidime/avibactam resistance in KPC-producing Enterobacterales in vivo.

Issue Section: ORIGINAL RESEARCH

© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Antibiotics; Drugs Resistance; Carbapenem; Ceftazidime; Avibactam; Imipenem; Klebsiella pneumoniae.

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Potentiation of #betalactam #antibiotics and β-lactam/β-lactamase inhibitor combinations against #MDR and #XDR #Pseudomonas aeruginosa using non-ribosomal #tobramycin–cyclam conjugates (J Antimicrob Chemother., abstract)

[Source: Journal of Antimicrobial Chemotherapy, full page: (LINK). Abstract, edited.]

Potentiation of β-lactam antibiotics and β-lactam/β-lactamase inhibitor combinations against MDR and XDR Pseudomonas aeruginosa using non-ribosomal tobramycin–cyclam conjugates

Temilolu Idowu, Derek Ammeter, Gilbert Arthur, George G Zhanel, Frank Schweizer

Journal of Antimicrobial Chemotherapy, dkz228, https://doi.org/10.1093/jac/dkz228

Published: 28 May 2019

 

Abstract

Objectives

To develop a multifunctional adjuvant molecule that can rescue β-lactam antibiotics and β-lactam/β-lactamase inhibitor combinations from resistance in carbapenem-resistant Pseudomonas aeruginosa clinical isolates.

Methods

Preparation of adjuvant was guided by structure–activity relationships, following standard protocols. Susceptibility and chequerboard studies were assessed using serial 2-fold dilution assays. Toxicity was evaluated against porcine erythrocytes, human embryonic kidney (HEK293) cells and liver carcinoma (HepG2) cells via MTS assay. Preliminary in vivo efficacy was evaluated using a Galleria mellonella infection model.

Results

Conjugation of tobramycin and cyclam abrogates the ribosomal effects of tobramycin but confers a potent adjuvant property that restores full antibiotic activity of meropenem and aztreonam against carbapenem-resistant P. aeruginosa. Therapeutic levels of susceptibility, as determined by CLSI susceptibility breakpoints, were attained in several MDR clinical isolates, and time–kill assays revealed a synergistic dose-dependent pharmacodynamic relationship. A triple combination of the adjuvant with ceftazidime/avibactam (approved), aztreonam/avibactam (Phase III) and meropenem/avibactam enhances the efficacies of β-lactam/β-lactamase inhibitors against recalcitrant strains, suggesting rapid access of the combination to their periplasmic targets. The newly developed adjuvants, and their combinations, were non-haemolytic and non-cytotoxic, and preliminary in vivo evaluation in G. mellonella suggests therapeutic potential for the double and triple combinations.

Conclusions

Non-ribosomal tobramycin–cyclam conjugate mitigates the effect of OprD/OprF porin loss in P. aeruginosa and potentiates β-lactam/β-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to β-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of β-lactam antibiotics.

Topic: antibiotics – pseudomonas aeruginosa – immunologic adjuvants – pharmaceutical adjuvants – aztreonam – ceftazidime – lactams – ribosomes – infection – tobramycin – meropenem – toxic effect – potentiation – avibactam – carbapenem resistance

Issue Section: ORIGINAL RESEARCH

© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Antibiotics; Drugs Resistance; Carbapenem; Beta-lactams; Pseudomonas aeruginosa; Tobramycin; Aztreonam; Avibactam; Ceftazidime.

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Reduced #ceftazidime and #ertapenem susceptibility due to production of #OXA-2 in #Klebsiella pneumoniae ST258 (J Antimicrob Chemother., abstract)

[Source: Journal of Antimicrobial Chemotherapy, full page: (LINK). Abstract, edited.]

Reduced ceftazidime and ertapenem susceptibility due to production of OXA-2 in Klebsiella pneumoniaeST258

Alina Iovleva, Roberta T Mettus, Christi L McElheny, Mustapha M Mustapha, Daria Van Tyne, Ryan K Shields, A William Pasculle, Vaughn S Cooper, Yohei Doi

Journal of Antimicrobial Chemotherapy, dkz183, https://doi.org/10.1093/jac/dkz183

Published: 24 May 2019

 

Abstract

Background

OXA-2 is a class D β-lactamase that confers resistance to penicillins, as well as narrow-spectrum cephalosporins. OXA-2 was recently reported to also possess carbapenem-hydrolysing activity. Here, we describe a KPC-2-encoding Klebsiella pneumoniae isolate that demonstrated reduced susceptibility to ceftazidime and ertapenem due to production of OXA-2.

Objectives

To elucidate the role of OXA-2 production in reduced ceftazidime and ertapenem susceptibility in a K. pneumoniae ST258 clinical isolate.

Methods

MICs were determined by the agar dilution method. WGS was conducted to identify and compare resistance genes between isolates. Expression of KPC-2 was quantified by quantitative RT–PCR and immunoblotting. OXA-2 was expressed in Escherichia coli TOP10, as well as in K. pneumoniae ATCC 13883, to define the relative contribution of OXA-2 in β-lactam resistance. Kinetic studies were conducted using purified OXA-2 enzyme.

Results

K. pneumoniae 1761 belonged to ST258 and carried both blaKPC-2 and blaOXA-2. However, expression of blaKPC-2 was substantially reduced due to an IS1294insertion in the promoter region. K. pneumoniae 1761, K. pneumoniae ATCC 13883 and E. coli TOP10 carrying blaOXA-2-harbouring plasmids showed reduced susceptibility to ertapenem and ceftazidime, but meropenem, imipenem and cefepime were unaffected. blaOXA-2 was carried on a 2910 bp partial class 1 integron containing aacA4-blaOXA-2-qacEΔ1-sul1 on an IncA/C2plasmid, which was not present in the earlier ST258 isolates possessing blaKPC-2 with intact promoters. Hydrolysis of ertapenem by OXA-2 was confirmed using purified enzyme.

Conclusions

Production of OXA-2 was associated with reduced ceftazidime and ertapenem susceptibility in a K. pneumoniae ST258 isolate.

Issue Section: ORIGINAL RESEARCH

© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Antibiotics; Drugs Resistance; Beta-lactams; Carbapenem; Ceftazidime; Ertapenem; Meropenem; Imipenem; Cefepime.

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#Ceftazidime – #Avibactam in Combination With #Fosfomycin: A Novel #Therapeutic Strategy Against #MDR #Pseudomonas aeruginosa (J Infect Dis., abstract)

[Source: Journal of Infectious Diseases, full page: (LINK). Abstract, edited.]

Ceftazidime-Avibactam in Combination With Fosfomycin: A Novel Therapeutic Strategy Against Multidrug-Resistant Pseudomonas aeruginosa

Krisztina M Papp-Wallace, Elise T Zeiser, Scott A Becka, Steven Park, Brigid M Wilson, Marisa L Winkler, Roshan D’Souza, Indresh Singh, Granger Sutton, Derrick E Fouts, Liang Chen, Barry N Kreiswirth, Evelyn J Ellis-Grosse, George L Drusano, David S Perlin, Robert A Bonomo

The Journal of Infectious Diseases, jiz149, https://doi.org/10.1093/infdis/jiz149

Published: 17 May 2019

 

Abstract

Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to β-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-avibactam-fosfomycin was superior to either drug alone. By employing a “mechanism-based approach” to combination chemotherapy, we show that ceftazidime-avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-β-lactamases.

Pseudomonas aeruginosa, β-lactams, fosfomycin, combination therapy

Topic:  pseudomonas aeruginosa – ceftazidime – fosfomycin – lactams – infection – mice – avibactam – avibactam/ceftazidime

Issue Section: Major Article

Keywords: Antibiotics; Drugs Resistance; Pseudomonas aeruginosa; Avibactam; Ceftazidime; Fosfomycin.

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Emergence and dominance of #E coli ST131 CTX-M-27 in a community #paediatric cohort study: independent host factors and #bacterial genetic determinants (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Emergence and dominance of E. coli ST131 CTX-M-27 in a community paediatric cohort study: independent host factors and bacterial genetic determinants

André Birgy, Corinne Levy, Marie-Hélène Nicolas-Chanoine, Aurélie Cointe, Claire A. Hobson, Mélanie Magnan, Stéphane Bechet, Philippe Bidet, Robert Cohen, Stéphane Bonacorsi

DOI: 10.1128/AAC.00382-19

 

ABSTRACT

The recent emergence and diffusion in the community of Escherichia coli isolates belonging to the multidrug-resistant and CTX-M-27-producing ST131 C1-M27 cluster, makes this cluster potentially as epidemic as the worldwide E coli ST131 subclade C2 composed of multidrug resistant isolates producing CTX-M-15. Thirty-five extended-spectrum beta-lactamase (ESBL) producing ST131 isolates were identified in a cohort of 1,885 French children over a 5 year-period. They were sequenced to characterize the ST131 E. coli isolates producing CTX-M-27 recently emerging in France. ST131 isolates producing CTX-M-27 (n=17), and particularly those belonging to the C1-M27 cluster (n=14), carried many resistance-encoding genes and predominantly a F1:A2:B20 plasmid type. In multivariate analysis, having been hospitalized since birth (OR=10.9; 95%CI=2.4;48.8; p=0.002) and being cared for in a day-care center (OR=9.4;95%; CI=1.5;59.0; p=0.017) were independent risk factors for ST131 CTX-M-27 fecal carriage compared with ESBL-producing non-ST131 isolates. No independent risk factor was found when comparing CTX-M-15 (n=11) and CTX-M-1/14 (n=7)-producing ST131 isolates with ESBL-producing non-ST131 isolates or with non-ESBL-producing isolates. Several factors may contribute to the increase in fecal carriage of CTX-M-27-producing E. coli isolates: resistance to multiple antibiotics, capacity of the CTX-M-27 enzyme to hydrolyze both cefotaxime and ceftazidime, carriage of a peculiar F-type plasmid, and/or capacity to colonize children who have been hospitalized since birth or who attend day-care centers.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; E. Coli; Pediatrics; Cefotaxime; Ceftazidime.

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