JAC Advance Access originally published online on February 26, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1062-1065; doi:10.1093/jac/dkn072
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Original research |
Resistance of planktonic and biofilm-grown Burkholderia cepacia complex isolates to the transition metal gallium
Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium
* Corresponding author. Tel: +32-9-2648141; Fax: +32-9-2648195; E-mail: tom.coenye{at}ugent.be
Received 6 September 2007; returned 17 December 2007; revised 24 January 2008; accepted 1 February 2008
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Abstract |
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Objectives: The Burkholderia cepacia complex is a group of pathogens that can cause severe pulmonary infections in cystic fibrosis (CF) patients. The aim of the present study was to investigate the in vitro activity of gallium against planktonic and biofilm-grown B. cepacia complex isolates.
Methods: Six B. cepacia complex isolates (belonging to three different species) as well as Pseudomonas aeruginosa PAO1 were included in the present study. MICs of Ga(NO3)3 for planktonic cells were determined using a broth microdilution method. Biofilms were formed in 96-well microtitre plates, and the fraction of surviving cells following Ga(NO3)3 treatment was determined using resazurin as a marker for cell viability. The antimicrobial effect of Ga(NO3)3 was assessed in the presence (50 µM) and absence of Fe3+.
Results: When tested against planktonic cells, the MICs of Ga(NO3)3 in the absence of Fe3+ were 64 mg/L for all B. cepacia complex strains investigated. However, the addition of 50 µM Fe3+ in the presence of 64 mg/L Ga(NO3)3 resulted in increased growth for all B. cepacia complex strains investigated. In sessile cells, resistance to Ga(NO3)3 and the extent of the protective effect of 50 µM Fe3+ against Ga(NO3)3 appear to be strain-dependent: the Burkholderia cenocepacia strains investigated are insensitive to Ga(NO3)3 in the presence of 50 µM Fe3+, whereas the presence of Fe3+ has no protective effect for both Burkholderia multivorans strains investigated.
Conclusions: As maximal tolerable Ga3+ levels in plasma are estimated to be ~200 µM and considering the high levels of Fe3+ in the lungs of people with CF, our data suggest that the added value of a Ga(NO3)3 treatment of B. cepacia complex-infected patients may be limited.
Keywords: iron metabolism , infection , cystic fibrosis , Pseudomonas aeruginosa
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Introduction |
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Patients with cystic fibrosis (CF) are at particular risk for respiratory infections caused by members of the Burkholderia cepacia complex. Antibiotic resistance is widespread within the B. cepacia complex and is considered a key factor in the excessive mortality observed in CF patients, following B. cepacia complex infections.1 Bacterial biofilms can play an important role in the pathogenesis of various human infections, and one of the striking properties of sessile (biofilm-grown) cells is their increased resistance to antimicrobial agents.2 It has been previously shown that many B. cepacia complex bacteria readily form biofilms in vitro and that these sessile bacteria are more resistant to antibiotics than their planktonic counterparts.3 The use of the transition metal gallium was recently proposed to treat infections caused by another important CF pathogen, Pseudomonas aeruginosa.4 In vitro and in vivo studies showed that Ga3+ inhibits P. aeruginosa growth and biofilm formation, and kills planktonic and sessile cells, in part by interfering with iron signalling through the transcriptional regulator pvdS.4 The aim of the present study was to investigate whether Ga3+ could also be used to inhibit growth and biofilm formation of B. cepacia complex organisms.
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Materials and methods |
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Strains and culture conditions
The following B. cepacia complex strains were used: Burkholderia cenocepacia LMG 16656 and LMG 18828, Burkholderia multivorans LMG 18822 and LMG 18825, and Burkholderia dolosa LMG 18941 and LMG 18943. All strains were isolated from CF patients and were obtained from the BCCM/LMG Bacteria Collection (Gent, Belgium). For comparison, we also included P. aeruginosa reference strain PAO1. All isolates were cultured on Nutrient Agar (Oxoid, Hampshire, UK) at 37°C.
Antibacterial susceptibility testing
MICs of Ga(NO3)3 (Sigma, Bornem, Belgium) were determined using a broth microdilution method based on EUCAST Discussion Document E.Dis 5.1.5 Isolates were cultured in flat-bottomed 96-well plates (TPP, Trasadingen, Switzerland) using a chemically defined minimal salt medium (CDM) without iron.6 The microtitre plates were incubated for 20 h at 37°C. Optical densities were measured using a Wallac Victor2 multilabel microtitre plate reader (Perkin Elmer LAS, Waltham, MA, USA) (
= 535 nm). MICs were also determined in CDM supplemented with Fe3+ (final concentration 50 µM) (added as FeCl3·6H2O) (designated as CDM-Fe). The MIC was recorded as the lowest concentration that completely inhibited growth.
Biofilms were formed in 96-well microtitre plates and quantified using resazurin as a marker for cell viability. In brief, wells of a round-bottomed polystyrene 96-well microtitre plate (TPP) were inoculated with 100 µL of medium containing 107 cfu. Following 4 h of adhesion, the supernatant with non-adherent cells was removed from each well and plates were rinsed using 100 µL of 0.9% (w/v) NaCl. Subsequently, 100 µL of fresh medium was added to each well and the plates were further incubated for 24 h, after which the supernatant was again removed and the wells were rinsed with 100 µL of 0.9% (w/v) NaCl. In the resazurin assay, a commercially available resazurin solution (CellTiter-Blue, CTB, Promega, Madison, WI, USA) was used. To each well, 100 µL of 0.9% (w/v) NaCl was added, followed by the addition of 20 µL CTB. Fluorescence was measured (ex: 560 nm and em: 590 nm) using the Wallac Victor2 multilabel microtitre plate reader. Biofilms were treated with two concentrations of Ga(NO3)3 (32 and 64 mg/L) in CDM or CDM-Fe for 24 h. Treatment was initiated after 4 or 28 h of biofilm formation.
Analysis of variance with the Scheffe post hoc test was performed using SPSS 15.0 software (SPSS, Chicago, IL, USA).
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Results and discussion |
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The MICs of Ga(NO3)3 in CDM were 64 mg/L for all B. cepacia complex strains tested (Figure 1). This corresponds to ~250 µM Ga3+. The addition of 50 µM Fe3+ (a concentration similar to the total Fe3+ concentration found in lungs of CF patients7) in the presence of 64 mg/L Ga(NO3)3 resulted in increased growth for all B. cepacia complex strains investigated, and no clear MIC of Ga(NO3)3 in CDM-Fe could be determined (Figure 1). The MICs observed for the B. cepacia complex strains are higher than the concentrations required to obtain a 100-fold reduction in Rhodococcus equi cell numbers (50 µM)8 and similar to the concentrations required to inhibit the growth of Mycobacterium tuberculosis and Mycobacterium avium complex in broth culture.9 For control purposes, we also included P. aeruginosa PAO1 in our experiments. It has previously been shown that the use of low Ga3+ concentrations (2–10 µM) results in growth inhibition and significant killing of planktonic P. aeruginosa PAO1 cells.4 We observed that in the absence of Fe3+, complete inhibition of planktonic growth required at least 16 mg/L (62.5 µM) of Ga(NO3)3. In CDM-Fe, partial growth inhibition (i.e. the optical density measured in the treated wells is less than half of the optical density in the control wells) was observed starting from a Ga(NO3)3 concentration of 2 mg/L (7.8 µM), confirming previous results4 describing that a molar ratio Fe3+:Ga3+ of at least 5:1 is necessary to overcome the inhibiting effects of Ga3+ in P. aeruginosa PAO1.
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In order to determine whether Ga(NO3)3 had any activity against B. cepacia complex biofilms, we treated these biofilms with high doses of Ga(NO3)3 (32 and 64 mg/L) for 24 h and determined the fraction of surviving (i.e. metabolically active) cells in the biofilm (Table 1). Treating young (4 h old) B. cepacia complex biofilms with Ga(NO3)3 in the absence of Fe3+ resulted in statistically significant reductions of the number of viable cells in most cases, although there were considerable differences between strains. Except for B. multivorans LMG 18825, reductions were at least 45.2% and 45.4% when Ga(NO3)3 concentrations of 32 or 64 mg/L were used, respectively. Although reductions were higher when using the higher Ga(NO3)3 concentration for five of the six strains tested, the differences in reductions between both concentrations were only statistically significant for both B. multivorans isolates and for B. dolosa LMG 18941 (P < 0.001). In preliminary experiments, we obtained similar results using the plate count method (data not shown). The use of Ga(NO3)3 against mature (28 h old) biofilms in the absence of iron also resulted in strain-dependent reductions in the number of viable cells, with the highest reductions observed for B. multivorans LMG 18822 (78.3% and 80.9% for 32 and 64 mg/L, respectively) and the lowest reductions for B. multivorans LMG 18825 (31.1% and 38.1%). Similarly, as observed for the young biofilms, reductions were higher when using the higher Ga(NO3)3 concentration (except for B. cenocepacia LMG 18828), but this difference was only significant (P < 0.001) for both B. dolosa strains. The presence of 50 µM Fe3+ resulted in a statistically significant (P < 0.01) loss of susceptibility to Ga(NO3)3 (i.e. lower reductions) for both B. cenocepacia strains investigated. This protective effect was not observed in the B. multivorans and B. dolosa isolates investigated (except for B. dolosa LMG 18943, 64 mg/L). It remains to be seen whether the lack of protection against the action of Ga(NO3)3 by Fe3+ is common to all B. multivorans isolates. Similarly, it is at present unclear whether the loss of susceptibility to Ga(NO3)3 in the presence of Fe3+ is common to all B. cenocepacia isolates or whether this is a strain-dependent characteristic. Kaneko et al.4 reported that low (0.5 µM) Ga(NO3)3 concentrations were sufficient to completely inhibit P. aeruginosa biofilm formation, whereas mature biofilms could be killed by treating them with higher Ga(NO3)3 concentrations (100 and 1000 µM). Although there was no quantification of the anti-biofilm effect (biofilm killing was assessed using propidium iodide to visualize dead cells), a close inspection of the pictures presented by Kaneko et al. suggests that at least half of the cells were dead after 24 h of treatment with 1000 µM Ga(NO3)3. This is roughly in agreement with the reductions observed in the present study (Table 1). The addition of iron (50 µM) to the medium resulted in a partial but significant (P < 0.01) reduction of the anti-biofilm effect of Ga(NO3)3 in P. aeruginosa PAO1; higher concentrations are probably required to completely restore biofilm biomass to control levels. Overall, our data confirm the results obtained by Kaneko et al.,4 and the minor differences observed between both studies probably relate to differences in methodology (growth medium, incubation temperature and biofilm model system).
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Our data clearly indicate that biofilm-grown B. cepacia complex organisms are resistant to high levels of Ga(NO3)3. The exact level of resistance and the extent of the protective effect of 50 µM Fe3+ against Ga(NO3)3 appear to be strain- or species-dependent. As maximal tolerable Ga3+ levels in plasma are estimated to be ~200 µM10 and considering the high levels of Fe3+ in the lungs of patients with CF7, our data suggest that the added value of a Ga(NO3)3 treatment of B. cepacia complex-infected patients may be limited, although further research is required.
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Funding |
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This research was financially supported by the BOF of Ghent University and FWO-Vlaanderen.
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Transparency declarations |
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None to declare.
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References |
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1 Burns J. Antibiotic resistance of Burkholderia spp. In: Burkholderia: Molecular Biology and Genomics—Coenye T, Vandamme P, eds. (2007) Wymondham: Horizon Press. 81–91.
2 Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet (2001) 358:135–8.[CrossRef][Web of Science][Medline]
3 Caraher E, Reynolds G, Murphy P, et al. Comparison of antibiotic susceptibility of Burkholderia cepacia complex organisms when grown planktonically or as biofilm in vitro. Eur J Clin Microbiol Infect Dis (2007) 26:213–6.[CrossRef][Web of Science][Medline]
4 Kaneko Y, Thoendel M, Olakanmi O, et al. The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity. J Clin Invest (2007) 117:877–88.[CrossRef][Medline]
5 European Committee for Antimicrobial Susceptibility Testing (EUCAST). Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. Clin Microbiol Infect (2003) 9:ix–xv.[CrossRef]
6
McKenney D, Allison DG. Effects of growth rate and nutrient limitation on virulence factor production in Burkholderia cepacia. J Bacteriol (1995) 177:4140–3.
7 Reid DW, Withers NJ, Francis L, et al. Iron deficiency in cystic fibrosis: relationship to lung disease severity and chronic Pseudomonas aeruginosa infection. Chest (2002) 121:48–54.[CrossRef][Web of Science][Medline]
8 Harrington JR, Martens RJ, Cohen ND, et al. Antimicrobial activity of gallium against virulent Rhodococcus equi in vitro and in vivo. J Vet Pharmacol Ther (2006) 29:121–7.[Medline]
9
Olakanmi O, Britigan BE, Schlesinger LS. Gallium disrupts iron metabolism of mycobacteria residing within human macrophages. Infect Immun (2000) 68:5619–27.
10
Bernstein LR. Mechanisms of therapeutic activity for gallium. Pharmacol Rev (1998) 50:665–82.
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