Activity of Cysteamine against the Cystic Fibrosis Pathogen Burkholderia cepacia Complex

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00498-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 1

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.

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Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00975-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6003-6012 ◽

Cited By ~ 14

Author(s):

Saira Ahmad ◽

Jean Tyrrell ◽

William G. Walton ◽

Ashutosh Tripathy ◽

Matthew R. Redinbo ◽

...

Keyword(s):

Antimicrobial Activity ◽

Lung Disease ◽

Biofilm Formation ◽

Burkholderia Cepacia ◽

Burkholderia Cepacia Complex ◽

Upper Airways ◽

Content Type ◽

Innate Defense ◽

Opportunistic Bacteria ◽

The Impact

ABSTRACTThe opportunistic bacteria of theBurkholderia cepaciacomplex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, includingB. cenocepaciastrain J2315 (50% inhibitory concentration [IC50] = 0.28 μM), and reduced biofilm formation and attachment (IC50= 0.11 μM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00766-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 14

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.

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

Infection and Immunity ◽

10.1128/iai.01876-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4729-4745 ◽

Cited By ~ 57

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.

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Pseudomonas aeruginosa-Derived Rhamnolipids and Other Detergents Modulate Colony Morphotype and Motility in the Burkholderia cepacia Complex

Journal of Bacteriology ◽

10.1128/jb.00171-17 ◽

2017 ◽

Vol 199 (13) ◽

Cited By ~ 6

Author(s):

Steve P. Bernier ◽

Courtney Hum ◽

Xiang Li ◽

George A. O'Toole ◽

Nathan A. Magarvey ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Burkholderia Cepacia Complex ◽

Interspecies Interactions ◽

Content Type ◽

Low Concentrations ◽

Subinhibitory Concentrations ◽

The Impact ◽

Polymicrobial Infections

ABSTRACT Competitive interactions mediated by released chemicals (e.g., toxins) are prominent in multispecies communities, but the effects of these chemicals at subinhibitory concentrations on susceptible bacteria are poorly understood. Although Pseudomonas aeruginosa and species of the Burkholderia cepacia complex (Bcc) can exist together as a coinfection in cystic fibrosis airways, P. aeruginosa toxins can kill Bcc species in vitro. Consequently, these bacteria become an ideal in vitro model system to study the impact of sublethal levels of toxins on the biology of typical susceptible bacteria, such as the Bcc, when exposed to P. aeruginosa toxins. Using P. aeruginosa spent medium as a source of toxins, we showed that a small window of subinhibitory concentrations modulated the colony morphotype and swarming motility of some but not all tested Bcc strains, for which rhamnolipids were identified as the active molecule. Using a random transposon mutagenesis approach, we identified several genes required by the Bcc to respond to low concentrations of rhamnolipids and consequently affect the ability of this microbe to change its morphotype and swarm over surfaces. Among those genes identified were those coding for type IVb-Tad pili, which are often required for virulence in various bacterial pathogens. Our study demonstrates that manipulating chemical gradients in vitro can lead to the identification of bacterial behaviors relevant to polymicrobial infections. IMPORTANCE Interspecies interactions can have profound effects on the development and outcomes of polymicrobial infections. Consequently, improving the molecular understanding of these interactions could provide us with new insights on the possible long-term consequences of these chronic infections. In this study, we show that P. aeruginosa-derived rhamnolipids, which participate in Bcc killing at high concentrations, can also trigger biological responses in Burkholderia spp. at low concentrations. The modulation of potential virulence phenotypes in the Bcc by P. aeruginosa suggests that these interactions contribute to pathogenesis and disease severity in the context of polymicrobial infections.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00181-19 ◽

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.

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Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

Journal of Medical Microbiology ◽

10.1099/jmm.0.001467 ◽

2022 ◽

Vol 71 (1) ◽

Author(s):

Bailey F. Keefe ◽

Luiz E. Bermudez

Keyword(s):

Cystic Fibrosis ◽

Host Cell ◽

Biofilm Formation ◽

Type Species ◽

Divalent Cations ◽

Mycobacterium Abscessus ◽

Content Type ◽

Link Type ◽

Populations At Risk

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

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