scholarly journals “Switching Partners”: Piperacillin-Avibactam Is a Highly Potent Combination against Multidrug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Elise T. Zeiser ◽  
Scott A. Becka ◽  
Brigid M. Wilson ◽  
Melissa D. Barnes ◽  
John J. LiPuma ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Burkholderia Cepacia ◽  
Multidrug Resistant ◽  
Burkholderia Cepacia Complex ◽  
Content Type ◽  
Burkholderia Gladioli ◽  
Alternative Antibiotic ◽  
And Control

ABSTRACT In persons with cystic fibrosis (CF), airway infection with Burkholderia cepacia complex (Bcc) species or Burkholderia gladioli presents a significant challenge due to inherent resistance to multiple antibiotics. Two chromosomally encoded inducible β-lactamases, a Pen-like class A and AmpC are produced in Bcc and B. gladioli. Previously, ceftazidime-avibactam demonstrated significant potency against Bcc and B. gladioli isolated from the sputum of individuals with CF; however, 10% of the isolates tested resistant to ceftazidime-avibactam. Here, we describe an alternative antibiotic combination to overcome ceftazidime-avibactam resistance. Antimicrobial susceptibility testing was performed on Bcc and B. gladioli clinical and control isolates. Biochemical analysis was conducted on purified PenA1 and AmpC1 β-lactamases from Burkholderia multivorans ATCC 17616. Analytic isoelectric focusing and immunoblotting were conducted on cellular extracts of B. multivorans induced by various β-lactams or β-lactam-β-lactamase inhibitor combinations. Combinations of piperacillin-avibactam, as well as piperacillin-tazobactam plus ceftazidime-avibactam (the clinically available counterpart), were tested against a panel of ceftazidime-avibactam nonsusceptible Bcc and B. gladioli. The piperacillin-avibactam and piperacillin-tazobactam-ceftazidime-avibactam combinations restored susceptibility to 99% of the isolates tested. Avibactam is a potent inhibitor of PenA1 (apparent inhibitory constant [Ki app] = 0.5 μM), while piperacillin was found to inhibit AmpC1 (Ki app = 2.6 μM). Moreover, piperacillin, tazobactam, ceftazidime, and avibactam, as well as combinations thereof, did not induce expression of blapenA1 and blaampC1 in the B. multivorans ATCC 17616 background. When ceftazidime-avibactam is combined with piperacillin-tazobactam, the susceptibility of Bcc and B. gladioli to ceftazidime and piperacillin is restored in vitro. Both the lack of blapenA1 induction and potent inactivation of PenA1 by avibactam likely provide the major contributions toward susceptibility. With in vivo validation, piperacillin-tazobactam-ceftazidime-avibactam may represent salvage therapy for individuals with CF and highly drug-resistant Bcc and B. gladioli infections.

scholarly journals In Vitro Activity of Ceftolozane-Tazobactam and Other Antimicrobial Agents against Burkholderia cepacia Complex and Burkholderia gladioli

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Dale M. Mazer ◽  
Carol Young ◽  
Linda M. Kalikin ◽  
Theodore Spilker ◽  
John J. LiPuma
Keyword(s):  
Cystic Fibrosis ◽  
Burkholderia Cepacia ◽  
Antimicrobial Agents ◽  
Vitro Activity ◽  
Burkholderia Cepacia Complex ◽  
In Vitro Activity ◽  
Content Type ◽  
Burkholderia Gladioli

ABSTRACT We tested the activities of ceftolozane-tazobactam and 13 other antimicrobial agents against 221 strains of Burkholderia cepacia complex and Burkholderia gladioli. Most strains (82%) were cultured from persons with cystic fibrosis, and most (85%) were recovered since 2011. The ceftolozane-tazobactam MIC was ≤8 μg/ml for 77% of the strains. However, the MIC range was broad (≤0.5 to >64 μg/ml; MIC50/90, 2/32 μg/ml). Significant differences in susceptibility to some antimicrobial agents were observed between species.

scholarly journals Evaluation of the electron transfer flavoprotein as an antibacterial target in Burkholderia cenocepacia

2017 ◽  
Vol 63 (10) ◽  
pp. 857-863 ◽  
Author(s):  
Maria S. Stietz ◽  
Christina Lopez ◽  
Osasumwen Osifo ◽  
Marcelo E. Tolmasky ◽  
Silvia T. Cardona
Keyword(s):  
Electron Transfer ◽  
Burkholderia Cepacia ◽  
Multidrug Resistant ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Infection Model ◽  
Electron Transfer Flavoprotein ◽  
C Elegans

There are hundreds of essential genes in multidrug-resistant bacterial genomes, but only a few of their products are exploited as antibacterial targets. An example is the electron transfer flavoprotein (ETF), which is required for growth and viability in Burkholderia cenocepacia. Here, we evaluated ETF as an antibiotic target for Burkholderia cepacia complex (Bcc). Depletion of the bacterial ETF during infection of Caenorhabditis elegans significantly extended survival of the nematodes, proving that ETF is essential for survival of B. cenocepacia in this host model. In spite of the arrest in respiration in ETF mutants, the inhibition of etf expression did not increase the formation of persister cells, when treated with high doses of ciprofloxacin or meropenem. To test if etf translation could be inhibited by RNA interference, antisense oligonucleotides that target the etfBA operon were synthesized. One antisense oligonucleotide was effective in inhibiting etfB translation in vitro but not in vivo, highlighting the challenge of reduced membrane permeability for the design of drugs against B. cenocepacia. This work contributes to the validation of ETF of B. cenocepacia as a target for antibacterial therapy and demonstrates the utility of a C. elegans liquid killing assay to validate gene essentiality in an in vivo infection model.

scholarly journals In Vitro Activity of a Novel Glycopolymer against Biofilms of Burkholderia cepacia Complex Cystic Fibrosis Clinical Isolates

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Vidya P. Narayanaswamy ◽  
Andrew P. Duncan ◽  
John J. LiPuma ◽  
William P. Wiesmann ◽  
Shenda M. Baker ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Burkholderia Cepacia ◽  
Laser Scanning Microscopy ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Confocal Laser ◽  
Content Type ◽  
Biofilm Removal ◽  
Increased Risk

ABSTRACT Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia gladioli, Burkholderia dolosa, Burkholderia vietnamiensis, and B. cepacia. These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro. Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 μg/ml PAAG following a 1-h treatment (P < 0.01). A significant reduction in biofilm thickness was observed following a 10-min treatment of Bcc biofilms with PAAG compared to that with vehicle control (P < 0.001) in TCP, MBEC, and CLSM analyses. PAAG also rapidly permeabilizes bacteria within the first 10 min of treatment. Glycopolymers, such as PAAG, are a new class of large-molecule therapeutics that support the treatment of recalcitrant Bcc biofilm.

scholarly journals Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

2014 ◽  
Vol 82 (11) ◽  
pp. 4729-4745 ◽  
Author(s):  
Ute Schwab ◽  
Lubna H. Abdullah ◽  
Olivia S. Perlmutt ◽  
Daniel Albert ◽  
C. William Davis ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Burkholderia Cepacia ◽  
Single Cells ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Test Medium ◽  
Fermentation Products ◽  
Content Type

ABSTRACTThe localization ofBurkholderia cepaciacomplex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection withPseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc andP. aeruginosabacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallelin vitroexperiments examined the growth of two Bcc species,Burkholderia cenocepaciaandBurkholderia multivorans, in environments similar to those occupied byP. aeruginosain the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast,P. aeruginosawas identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions withP. aeruginosain an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions,B. cenocepaciaandB. multivoransused fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence ofP. aeruginosain vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade aP. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibitP. aeruginosabiofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.

scholarly journals Activity of Cysteamine against the Cystic Fibrosis Pathogen Burkholderia cepacia Complex

2016 ◽  
Vol 60 (10) ◽  
pp. 6200-6206 ◽  
Author(s):  
Douglas Fraser-Pitt ◽  
Derry Mercer ◽  
Emma Lovie ◽  
Jennifer Robertson ◽  
Deborah O'Neil
Keyword(s):  
Cystic Fibrosis ◽  
Biofilm Formation ◽  
Burkholderia Cepacia ◽  
Standard Of Care ◽  
Burkholderia Cepacia Complex ◽  
Content Type ◽  
Folate Pathway ◽  
The Impact ◽  
Slow Growing

ABSTRACTThere are no wholly successful chemotherapeutic strategies againstBurkholderia cepaciacomplex (BCC) colonization in cystic fibrosis (CF). We assessed the impact of cysteamine (Lynovex) in combination with standard-of-care CF antibioticsin vitroagainst BCC CF isolates by the concentration at which 100% of bacteria were killed (MIC100) and checkerboard assays under CLSI standard conditions. Cysteamine facilitated the aminoglycoside-, fluoroquinolone- and folate pathway inhibitor-mediated killing of BCC organisms that were otherwise resistant or intermediately sensitive to these antibiotic classes. Slow-growing BCC strains are often recalcitrant to treatment and form biofilms. In assessing the impact of cysteamine on biofilms, we demonstrated inhibition of BCC biofilm formation at sub-MIC100s of cysteamine.

scholarly journals In Vitro Activities of Designed Antimicrobial Peptides against Multidrug-Resistant Cystic Fibrosis Pathogens

1999 ◽  
Vol 43 (6) ◽  
pp. 1435-1440 ◽  
Author(s):  
Ute Schwab ◽  
Peter Gilligan ◽  
Jesse Jaynes ◽  
David Henke
Keyword(s):  
Cystic Fibrosis ◽  
Antimicrobial Peptides ◽  
Burkholderia Cepacia ◽  
Wide Spectrum ◽  
Multidrug Resistant ◽  
Medium Composition ◽  
Peptide Antibiotics ◽  
Rpmi Medium ◽  
Multidrug Resistant Pathogens

ABSTRACT The emergence of multidrug-resistant pathogens renders antibiotics ineffective in the treatment of lung infections in patients with cystic fibrosis (CF). Designed antimicrobial peptides (DAPs) are laboratory-synthesized peptide antibiotics that demonstrate a wide spectrum of antibacterial activity. Optimal conditions for susceptibility testing of these peptides have not yet been established. Medium composition is clearly a major factor influencing the results and reproducibilities of susceptibility tests. Using time-kill assays, we tested the effects of different media and buffers on the bactericidal activities of the peptides D2A21 and D4E1 onStaphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853. Each peptide at 1 and 5 μM was incubated with bacteria in the different media and buffers. Both peptides were most active in Tris-HCl buffer against S. aureus andP. aeruginosa. Among the more complex media tested, modified RPMI medium was the medium in which the peptides demonstrated the highest activity, while it supported the growth of the bacteria. The broth microdilution technique was used to test the activities of D2A21 and D4E1 in modified RPMI medium against multidrug-resistant pathogens from patients with CF. The MICs of DAPs for methicillin-resistant S. aureus ranged from 0.25 to 4 μg/ml, those for multidrug-resistant P. aeruginosa ranged from 0.125 to 4 μg/ml, those for Stenotrophomonas maltophilia ranged from 0.5 to 32 μg/ml, and those forBurkholderia cepacia ranged from 32 to ≥64 μg/ml. When the activity of peptide D2A21 was compared with that of the tracheal antimicrobial peptide (TAP), D2A21 had greater potency than TAP againstP. aeruginosa. In addition, no difference in the MICs of D2A21 was seen when it was tested in nutrient broth supplemented with NaCl at different concentrations. Thus, DAPs are a class of salt-insensitive antibiotics potentially useful in the treatment of CF patients harboring multidrug-resistant P. aeruginosa.

scholarly journals Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Katharina Schaufler ◽  
Torsten Semmler ◽  
Lothar H. Wieler ◽  
Darren J. Trott ◽  
Johann Pitout ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Risk ◽  
Multidrug Resistant ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Extended Spectrum Beta Lactamase ◽  
Niche Adaptation ◽  
High Level

ABSTRACT The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n = 107) and ST10 (n = 96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.

scholarly journals Disruption of Cross-Feeding Inhibits Pathogen Growth in the Sputa of Patients with Cystic Fibrosis

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Jeffrey M. Flynn ◽  
Lydia C. Cameron ◽  
Talia D. Wiggen ◽  
Jordan M. Dunitz ◽  
William R. Harcombe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Anaerobic Bacteria ◽  
Content Type ◽  
Growth Environment ◽  
Polymicrobial Infections

ABSTRACT A critical limitation in the management of chronic polymicrobial infections is the lack of correlation between antibiotic susceptibility testing (AST) and patient responses to therapy. Underlying this disconnect is our inability to accurately recapitulate the in vivo environment and complex polymicrobial communities in vitro. However, emerging evidence suggests that, if modeled and tested accurately, interspecies relationships can be exploited by conventional antibiotics predicted to be ineffective by standard AST. As an example, under conditions where Pseudomonas aeruginosa relies on cocolonizing organisms for nutrients (i.e., cross-feeding), multidrug-resistant P. aeruginosa may be indirectly targeted by inhibiting the growth of its metabolic partners. While this has been shown in vitro using synthetic bacterial communities, the efficacy of a “weakest-link” approach to controlling host-associated polymicrobial infections has not yet been demonstrated. To test whether cross-feeding inhibition can be leveraged in clinically relevant contexts, we collected sputa from cystic fibrosis (CF) subjects and used enrichment culturing to isolate both P. aeruginosa and anaerobic bacteria from each sample. Predictably, both subpopulations showed various antibiotic susceptibilities when grown independently. However, when P. aeruginosa was cultured and treated under cooperative conditions in which it was dependent on anaerobic bacteria for nutrients, the growth of both the pathogen and the anaerobe was constrained despite their intrinsic antibiotic resistance profiles. These data demonstrate that the control of complex polymicrobial infections may be achieved by exploiting obligate or facultative interspecies relationships. Toward this end, in vitro susceptibility testing should evolve to more accurately reflect in vivo growth environments and microbial interactions found within them. IMPORTANCE Antibiotic efficacy achieved in vitro correlates poorly with clinical outcomes after treatment of chronic polymicrobial diseases; if a pathogen demonstrates susceptibility to a given antibiotic in the lab, that compound is often ineffective when administered clinically. Conversely, if a pathogen is resistant in vitro, patient treatment with that same compound can elicit a positive response. This discordance suggests that the in vivo growth environment impacts pathogen antibiotic susceptibility. Indeed, here we demonstrate that interspecies relationships among microbiotas in the sputa of cystic fibrosis patients can be targeted to indirectly inhibit the growth of Pseudomonas aeruginosa. The therapeutic implication is that control of chronic lung infections may be achieved by exploiting obligate or facultative relationships among airway bacterial community members. This strategy is particularly relevant for pathogens harboring intrinsic multidrug resistance and is broadly applicable to chronic polymicrobial airway, wound, and intra-abdominal infections.

scholarly journals Genomic Analysis Identifies NovelPseudomonas aeruginosaResistance Genes under Selection during Inhaled Aztreonam TherapyIn Vivo

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Kathryn McLean ◽  
Duankun Lee ◽  
Elizabeth A. Holmes ◽  
Kelsi Penewit ◽  
Adam Waalkes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Positive Selection ◽  
Single Gene ◽  
Genomic Analysis ◽  
Maximal Activity ◽  
Content Type ◽  
Cystic Fibrosis Airway ◽  
Bacterial Fitness

ABSTRACTInhaled aztreonam is increasingly used for chronicPseudomonas aeruginosasuppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapyin vivo. We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent beingftsIandampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutantampCalleles and performed artificial evolution ofampCfor maximal activity against aztreonam. We found that naturally occurringampCmutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selectionin vivoandin vitro, show thatampChas a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitnessin vivo.

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