In vitro activity and synergy of bismuth thiols and tobramycin against Burkholderia cepacia complex

Wilfredo G. Veloira1,3, Philip Domenico2,3,*, John J. LiPuma4, Jonathan M. Davis1,3, Ellen Gurzenda3 and Jeffrey A. Kazzaz2,3

Departments of 1 Pediatrics (Pulmonary Medicine and Neonatology) and 2 Medicine, and the 3 CardioPulmonary Research Institute, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola, NY; 4 Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA

Received 10 June 2003; returned 27 July 2003; revised 11 September 2003; accepted 13 September 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To determine the susceptibility of Burkholderia multivorans and Burkholderia cenocepacia to bismuth-thiols (BTs), and to examine the synergistic effects of tobramycin and subinhibitory concentrations of BTs against these organisms.

Methods: The susceptibilities of 25 clinical isolates each of B. multivorans and B. cenocepacia to six BTs were measured by broth dilution in accordance with NCCLS protocols. Ten strains were selected to evaluate the antimicrobial interaction between BTs and tobramycin. Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC) indices were calculated to assess synergy.

Results: B. multivorans and B. cenocepacia showed a wide range of susceptibilities to BTs. Bismuth ethanedithiol (BisEDT) was one of the more potent BTs against these organisms (MIC50 7.8 µM), and was selected for synergy studies. Selected strains were highly resistant to tobramycin. The addition of subinhibitory concentrations of BisEDT (2 µM) reduced the MIC and MBC of tobramycin against all strains, achieving synergy in many instances. The FIC index was in the range 0.28–0.66 and the FBC in the range 0.12–0.85. Most strains became susceptible to tobramycin at clinically achievable concentrations in the presence of non-toxic BisEDT levels.

Conclusions: Treatment with subinhibitory BisEDT and tobramycin reduces the MICs and MBCs for B. multivorans and B. cenocepacia. BTs may represent an important adjunctive therapy for resistant Burkholderia cepacia complex.

Keywords: cystic fibrosis, antibiotics, biofilms, bacterial polysaccharide, aminoglycosides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Initially recognized as a plant pathogen, Burkholderia cepacia has emerged as an opportunistic human pathogen, particularly in cystic fibrosis (CF) and certain immunodeficiency syndromes.13 Infection with B. cepacia affects a relatively small proportion of patients with CF.4 However, the presence of this organism has had a significant impact on morbidity and mortality, with an accelerated decline in pulmonary function, lower median survival rates, and significantly higher mortality post-lung transplantation compared with CF patients without B. cepacia infection.58

Recently, taxonomists have identified several distinct species (genomovars) among organisms previously identified as B. cepacia.9 There are currently nine species in what is now referred to as the B. cepacia complex. All have received formal binomial designations and have been cultured from sputa of CF patients.911 However, two species, Burkholderia multivorans and Burkholderia cenocepacia—formerly called genomovar II and genomovar III, respectively—account for >90% of isolates recovered from patients in North America and some European countries.1215

The primary treatment of pulmonary infections in CF patients is antibiotic therapy. Treatment often decreases bacterial density and virulence factor production, leading to decreased inflammation and clinical improvement.16,17 Unfortunately, the B. cepacia complex is generally refractory to antibiotics. Furthermore, the development of resistance during therapy can result in cross-resistance to other antimicrobial agents.9,1821 With limited alternative therapies, antibiotics continue to be used in a variety of combinations.1720 However, new treatment strategies are urgently needed.

Bismuth-thiols (BTs) are a new class of antibacterial agents, with antibiofilm activity against Gram-positive and -negative bacteria.2225 The thiol component functions as a lipophilic carrier that promotes bismuth uptake into bacteria, thus enhancing the effects of bismuth up to 1000-fold.22 Once inside the cell, bismuth acts as a metabolic poison, resulting in growth inhibition and cell death.22,25,26 At subinhibitory concentrations, BTs inactivate bacterial respiratory enzymes, suppress exopolysaccharide expression and prevent biofilm formation in Gram-positive and -negative bacteria. BTs also interfere with bacterial adherence and colonization, and increase the susceptibility of bacteria to host defences.2325

The objectives of this study were to determine the susceptibility of B. multivorans and B. cenocepacia to BTs, and to examine the synergistic effects of tobramycin and BTs against resistant strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

Twenty-five clinical strains each of B. multivorans and B. cenocepacia were obtained from independent CF sputum isolates referred to the B. cepacia Research Laboratory and Repository at the University of Michigan. Frozen stocks were prepared in trypticase soy broth containing 20% glycerol. Strains were subcultured weekly on nutrient agar and stored at 4°C.

Bismuth thiols and antibiotics

Six BT formulations were evaluated against B. multivorans and B. cenocepacia. The dithiols used in the BT formulations included: 2,3-dimercaptopropanol (BisBAL), 2,3-dimercaptotoluene (BisTOL) and 1,2-ethanedithiol (BisEDT). Monothiol complexes contained pyrithione (BisPYR), 3-mercapto-2-butanol (BisMBO) and p-chlorothiophenol (Bis{Phi}CL). All thiols were purchased from Sigma/Aldrich (St Louis, MO, USA). Stock BT solutions were prepared in propylene glycol as described previously.2225 BTs were added directly to growth media at various concentrations just prior to testing. Tobramycin (Eli Lilly & Co., Indianapolis, IN, USA) was also employed. BT concentrations are expressed in bismuth units, since the molar ratio of bismuth to thiol varied. Bismuth was mixed with dithiols at a 2:1 or 1:1 molar ratio, and with monothiols at a 1:2 ratio.

Antibacterial activity

Susceptibility studies were performed by broth dilution in 96-well tissue culture plates (Nalge Nunc International, Denmark) in accordance with NCCLS protocols.27 Briefly, overnight bacterial cultures were used to prepare 0.5 McFarland standard suspensions, which were further diluted 1:50 (~2 x 106 cfu/mL) in cation-adjusted Mueller–Hinton broth medium (BBL, Cockeysville, MD, USA). BTs were added at incremental concentrations, keeping the final volume constant at 0.2 mL. Cultures were incubated for 24 h at 37°C and turbidity was assessed by absorption at 630 nm using an ELISA plate reader (Biotek Instruments, Winooski, VT, USA). The MIC was expressed as the lowest drug concentration inhibiting growth for 24 h. Viable bacterial counts (cfu/mL) were determined by standard plating on nutrient agar. The MBC was expressed as the concentration of drug that reduced initial viability by 99.9% at 24 h of incubation.

Synergy studies

Five strains each of B. multivorans and B. cenocepacia were selected for further characterization. A BisEDT concentration of 2 µM was used for synergy, excluding strains that were growth-inhibited at 2 µM BisEDT. The chequerboard method was used to assess the activity of antimicrobial combinations. The fractional inhibitory concentration index (FICI) and the fractional bactericidal concentration index (FBCI) were calculated according to Eliopoulos & Moellering.28 Synergy was defined as an FICI or FBCI index of <=0.5, no interaction at >0.5–4 and antagonism at >4.29 Established breakpoints for tobramycin against aerobic, Gram-negative bacteria were <=4 mg/L (susceptible), >4 to <16 mg/L (intermediate), and >=16 mg/L (resistant).30 All assays were performed in triplicate and repeated three times.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility studies

Several BTs were active against most Burkholderia strains tested. The inhibitory and bactericidal concentrations of 50 clinical isolates to six BTs are summarized in Table 1. An MIC50 of 7.8 µM was obtained with BisEDT, BisPYR and BisMBO. BisMBO inhibited 90% of strains at 15.6 µM, and the MBC50 for BisMBO was 15.6 µM. Although effective against B. multivorans and B. cenocepacia, excessive BT concentrations were necessary to inhibit or kill most isolates. To keep the BT concentration below toxic levels, synergy studies were performed with tobramycin, an aminoglycoside antibiotic commonly used in the treatment of CF infections.


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Table 1. Antibacterial BT concentrations against 50 B. cepacia complex isolates
 
Synergy studies

Tobramycin synergy was assessed with our lead BT compound, BisEDT, against 10 B. cepacia complex strains. The MIC of tobramycin against resistant B. multivorans and B. cenocepacia (Table 2) was in the range 64–1024 mg/L. These tobramycin-resistant strains were susceptible to BisEDT, with MICs in the range 2–8 µM. The three strains inhibited by BisEDT at 2 µM were excluded from further study.


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Table 2. MIC of tobramycin and BisEDT versus B. cepacia complex
 
Growth inhibition of B. multivorans and B. cenocepacia strains by tobramycin was markedly affected by the presence of subinhibitory BisEDT. The MIC of tobramycin against these organisms was reduced 4–64 fold in the presence of 2 µM BisEDT. Five of the seven highly resistant strains became susceptible or intermediately resistant to tobramycin when combined with BisEDT. The FIC index indicated synergy or no interaction against all strains (mean 0.52 ± 0.11). No antagonism between tobramycin and BisEDT was noted.

BTs proved bactericidal, but at relatively high concentrations (Table 1). Both B. multivorans and B. cenocepacia were resistant to BT killing. For this reason, we assessed the potential bactericidal synergy of BTs and tobramycin. Among the isolates selected, the MBC of tobramycin was in the range 128–1024 mg/L, whereas that of BisEDT was in the range 4–64 µM (Table 3). The addition of 2 µM BisEDT reduced the MBC of tobramycin against these bacteria by about 12-fold. Two of the 10 resistant strains became susceptible to tobramycin killing (<16 mg/L). The FBC index showed synergy in seven of 10 strains, whereas near synergistic effects were noted in two of the three remaining isolates. One strain of B. multivorans remained unaffected by combined therapy.


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Table 3. MBC of tobramycin and BisEDT versus B. cepacia complex 
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The B. cepacia complex is generally resistant to a wide range of commonly used antibiotics, including aminoglycosides.9,1721,30 Strains tested in this study were resistant to tobramycin, an otherwise useful antibiotic for CF patients.21 Resistance to aminoglycosides results, in part, from an inability of antibacterial agents to permeate the outer membrane.31 The lipopolysaccharide (LPS) of this organism lacks a critical binding site required for polycation-mediated permeation of the outer membrane.32 Thus, by masking negative charges, LPS inhibits binding of polycationic antibiotics such as tobramycin.31 Cations that stabilize the outer membrane by cross-bridging adjacent LPS molecules are thought to be replaced by proteins.33

BTs are bacteriostatic and bactericidal to a wide variety of Gram-positive and -negative bacteria.2225 In this study, we examined the activity of several BTs against B. multivorans and B. cenocepacia, which account for the majority of B. cepacia complex infections in patients with CF. In general, the strains examined were more resistant than other bacteria to BTs, yet still showed a wide range of susceptibility. Some strains were susceptible to 2 µM BisEDT, whereas others were resistant at 62.5 µM. A previous study showed that the susceptibility of B. cepacia complex does not depend on their genomic species.34

The broad range of susceptibilities implies multiple mechanisms of resistance. Indeed, there may be many targets for BT action in bacteria. Bismuth is known to exchange with thiols and to inactivate enzymes involved in respiration, such as F1-ATPase in Helicobacter pylori.26,35 In Escherichia coli, bismuth reduces intracellular ATP levels and collapses the membrane potential.36 BTs may also work externally, forming electron dense layers on bacterial outer surfaces, and inducing blebbing in organisms such as Pseudomonas aeruginosa.25

Of several BTs currently under investigation, BisEDT was selected for further study because of its overall favourable characteristics, including safety, cost and spectrum of activity. Although effectively inhibiting the growth of most B. cepacia complex isolates, there is concern about the toxicity of BisEDT at higher levels. BisEDT is toxic to cultured 16HBE14o or A549 epithelial cells at relatively low concentrations (LD50; 5 µM or 13 µM, respectively). Eukaryotic cell cultures were rendered completely non-viable between 10 and 20 µM BisEDT, but no cytotoxicity was noted at 2 µM BisEDT.25 Given the somewhat narrow therapeutic ratio suggested by the cell culture data, BTs may not be useful alone against most strains of B. cepacia complex. However, in combination with other frontline antibiotics (e.g. tobramycin), BTs may be beneficial at non-toxic levels.

Although not generally growth inhibitory at 2 µM, BisEDT produces antibacterial effects at these sub-MIC levels. Sub-MIC BTs inhibited exopolysaccharide (EPS) production in Klebsiella pneumoniae or Pseudomonas syringae by 80–90% and by >50% in P. aeruginosa.23,25 Furthermore, P. aeruginosa adherence to lung epithelial cells and collagen matrix was significantly reduced.25 BTs also suppress staphylococcal EPS and biofilm formation.24,37 Thus, low BT levels exhibit a variety of virulence-reducing effects against Gram-positive and -negative bacteria.

B. cepacia complex isolates appear similarly affected by sub-MIC BisEDT. Non-toxic BisEDT levels reduced the MIC and MBC of tobramycin against all strains of B. cenocepacia and B. multivorans tested. The exact mechanism of synergy is currently unknown, but several explanations are under investigation. First, by disrupting LPS and the outer membrane of Burkholderia, sub-MIC BisEDT may facilitate tobramycin uptake. Aminoglycosides and other cationic antibacterial agents appear to work in concert with bismuth.38,39 Second, the thiols in BTs may chelate positively charged antibiotics and facilitate their entry into cells. Third, depolarization of the bacterial cytoplasmic membrane, and reduced energy levels, could affect antibiotic efflux pumps. Finally, biofilm-associated antibiotic resistance may be abrogated by BT action. However, Burkholderia biofilms were not examined in this study.

Interestingly, the highly tobramycin-resistant B. cepacia complex strains are susceptible to low levels of BisEDT. Such high-level resistance determinants can interfere with normal physiological processes in bacteria, causing a reduction in biological activity.40 Thus, a consequence of high antibiotic resistance may be susceptibility to certain biocides. Even sub-MIC levels of BisEDT exhibited a considerable reduction in the MIC and MBC of tobramycin against B. cepacia complex. With the increased efficacy and lower toxicity of nebulized tobramycin in the treatment of CF, the addition of BisEDT to this regimen has great potential.41 Thus, sub-MIC BisEDT, as an adjunct to conventional antibiotic therapy, may be an important alternative in the treatment of resistant B. cepacia complex and multiply resistant bacterial infections. It may also prevent bacterial biofilm formation in the lungs of patients with CF. We are currently assessing the effects of BTs on biofilm formation among the B. cepacia complex. Given the broad spectrum and multiple activities of BTs against bacteria, they may prove to be an important adjunctive therapy in treating acute and chronic pulmonary infections in CF patients.


    Acknowledgements
 
J. J. L. was funded by a grant from the Cystic Fibrosis Foundation. Portions of this work were presented at the 2002 Society of Pediatric Research Meeting, Baltimore, MD, USA.


    Footnotes
 
* Correspondence address. CardioPulmonary Research Institute, Winthrop University Hospital, 222 Station Plaza North, Suite 505, Mineola, NY 1150, USA. Tel: +1-516-663-2654; E-mail: pdomenico{at}winthrop.org Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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