Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa.

Microbial biofilms are a serious threat to human health. Recent studies have indicated that many clinically relevant biofilms are polymicrobial. In the present study, multispecies biofilms were grown in a reproducible manner in a 96-well microtiter plate. The efficacy of nine commercially available disinfectants against Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa in multispecies biofilms was determined and compared. The results showed that the direction and the magnitude of the effect in a multispecies biofilm depend on the strain and the disinfectant used and challenge the common belief that organisms in multispecies biofilms are always less susceptible than in monospecies biofilms.

Genomewide screening for genes involved in biofilm formation and miconazole susceptibility in Saccharomyces cerevisiae.

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms.

Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi.

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lactone signaling molecules, while dioxazaborocanes structurally resemble previously reported oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all compounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC(50) values in the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein. The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.

Phytosphingosine-1-phosphate is a signaling molecule involved in miconazole resistance in sessile Candida albicans cells.

Previous research has shown that 1% to 10% of sessile Candida albicans cells survive treatment with high doses of miconazole (a fungicidal imidazole). In the present study, we investigated the involvement of sphingolipid biosynthetic intermediates in this survival. We observed that the LCB4 gene, coding for the enzyme that catalyzes the phosphorylation of dihydrosphingosine and phytosphingosine, is important in governing the miconazole resistance of sessile Saccharomyces cerevisiae and C. albicans cells. The addition of 10 nM phytosphingosine-1-phosphate (PHS-1-P) drastically reduced the intracellular miconazole concentration and significantly increased the miconazole resistance of a hypersusceptible C. albicans heterozygous LCB4/lcb4 mutant, indicating a protective effect of PHS-1-P against miconazole-induced cell death in sessile cells. At this concentration of PHS-1-P, we did not observe any effect on the fluidity of the cytoplasmic membrane. The protective effect of PHS-1-P was not observed when the efflux pumps were inhibited or when tested in a mutant without functional efflux systems. Also, the addition of PHS-1-P during miconazole treatment increased the expression levels of genes coding for efflux pumps, leading to the hypothesis that PHS-1-P acts as a signaling molecule and enhances the efflux of miconazole in sessile C. albicans cells.

Synthesis and evaluation of the quorum sensing inhibitory effect of substituted triazolyldihydrofuranones.

Acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a triazole group were synthesized and evaluated for their effect on quorum sensing (QS) and biofilm formation in Burkholderia cenocepacia and Pseudomonas aeruginosa. In addition, the influence of the length of the acyl-mimicking chain was investigated. The compounds showed selectivity between two different AHL QS systems. 3-(1H-1,2,3-Triazol-1-yl)dihydrofuran-2(3H)-ones, in which the 4-substituent best resembled the acyl chain of the native AHL molecule exhibited significant QS agonistic and antagonistic activities. Replacing this aliphatic substituent by a phenyl-containing moiety resulted in active inhibitors of QS. The most active compounds showed biofilm inhibitory as well as biofilm eradicating activities in both test organisms.

In vitro inactivation of endodontic pathogens with Nd:YAG and Er:YAG lasers.

Both Nd:YAG and Er:YAG lasers have been suggested as root canal disinfection aids. The aim of this in vitro study is to compare both wavelengths in terms of irradiation dose required for microbial inactivation, to quantify these irradiation doses and to investigate the influence of certain (laser) parameters on the antimicrobial efficacy. Agar plates containing a uniform layer of Enterococcus faecalis, Candida albicans or Propionibacterium acnes were mounted perpendicularly underneath the laser handpieces (5 mm spot). The Er:YAG laser was operated in single-pulse mode. Pulse energies of 40-400 mJ and pulse lengths of 100, 300, 600, and 1,000 µs were tested. After incubation at 37°C for 48 h, growth on the plates was scored. The pulse energy yielding complete absence of growth over the entire spot area was taken as the total inhibition threshold (TIT). TITs were determined for every species and pulse length. The Nd:YAG laser was operated with pulse trains because single pulses were ineffective. Output power was 15 W and frequency was 100 Hz. Spots were irradiated for 5-120 s. After incubation, the diameters of the inhibition zones were measured. For the Er:YAG laser, TITs varied between 100 and 210 mJ, and differed significantly between species and pulse lengths. Using Nd:YAG irradiation, TITs were around 5,300 J/cm(2) for C. albicans and 7,100 J/cm(2) for P. acnes. No inhibition was observed for E. faecalis. Er:YAG irradiation was superior to Nd:YAG in inactivating microorganisms on agar surfaces.

Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo.

Although the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) has been proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosa and Burkholderia cepacia complex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus (hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepacia complex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitro and in vivo biofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitro the combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although reductions were strain and model dependent. A significantly higher fraction of infected Galleria mellonella larvae and Caenorhabditis elegans survived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepacia more than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.

Superoxide dismutases are involved in Candida albicans biofilm persistence against miconazole.

We investigated the cellular mechanisms responsible for the occurrence of miconazole-tolerant persisters in Candida albicans biofilms. Miconazole induced about 30% killing of sessile C. albicans cells at 75 µM. The fraction of miconazole-tolerant persisters, i.e., cells that can survive high doses of miconazole (0.6 to 2.4 mM), in these biofilms was 1 to 2%. Since miconazole induces reactive oxygen species (ROS) in sessile C. albicans cells, we focused on a role for superoxide dismutases (Sods) in persistence and found the expression of Sod-encoding genes in sessile C. albicans cells induced by miconazole compared to the expression levels in untreated sessile C. albicans cells. Moreover, addition of the superoxide dismutase inhibitor N,N'-diethyldithiocarbamate (DDC) to C. albicans biofilms resulted in an 18-fold reduction of the miconazole-tolerant persister fraction and in increased endogenous ROS levels in these cells. Treatment of biofilms of C. albicans clinical isolates with DDC resulted in an 18-fold to more than 200-fold reduction of their miconazole-tolerant persister fraction. To further confirm the important role for Sods in C. albicans biofilm persistence, we used a Δsod4 Δsod5 mutant lacking Sods 4 and 5. Biofilms of the Δsod4 Δsod5 mutant contained at least 3-fold less of the miconazole-tolerant persisters and had increased ROS levels compared to biofilms of the isogenic wild type (WT). In conclusion, the occurrence of miconazole-tolerant persisters in C. albicans biofilms is linked to the ROS-detoxifying activity of Sods. Moreover, Sod inhibitors can be used to potentiate the activity of miconazole against C. albicans biofilms.

Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite.

BACKGROUND: Burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis (CF). As treatment of infected CF patients is problematic, multiple preventive measures are taken to reduce the infection risk. Besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. However, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of B. cepacia complex bacteria have been reported. In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of reactive oxygen species (ROS), the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2 or NaOCl. RESULTS: The exposure to H2O2 and NaOCl resulted in an upregulation of the transcription of 315 (4.4%) and 386 (5.4%) genes, respectively. Transcription of 185 (2.6%) and 331 (4.6%) genes was decreased in response to the respective treatments. Many of the upregulated genes in the NaOCl- and H2O2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ROS. In addition, multiple upregulated genes encode proteins that are necessary to repair ROS-induced cellular damage. Unexpectedly, a prolonged treatment with H2O2 also resulted in an increased transcription of multiple phage-related genes. A closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6S RNA. CONCLUSION: Our results reveal that the transcription of a large fraction of B. cenocepacia J2315 genes is altered upon exposure of sessile cells to ROS. These observations have highlighted that B. cenocepacia may alter several pathways in response to exposure to ROS and they have led to the identification of many genes not previously implicated in the stress response of this pathogen.

Rapid and direct quantification of viable Candida species in whole blood by use of immunomagnetic separation and solid-phase cytometry.

Candida species are a common source of nosocomial bloodstream infections in critically ill patients. The sensitivity of the traditional diagnostic procedure based on blood culture is variable, and it usually takes 2 to 4 days before growth of Candida species is detected. We developed a 4-h method for the quantification of Candida species in blood, combining immunomagnetic separation (IMS) with solid-phase cytometry (SPC) using viability labeling. Additionally, Candida albicans cells could be identified in real time by using fluorescent in situ hybridization. By analysis of spiked blood samples, our method was shown to be sensitive and specific, with a low detection limit (1 cell/ml of blood). In a proof-of-concept study, we applied the IMS/SPC method to 16 clinical samples and compared it to traditional blood culture. Our method proved more sensitive than culture (seven samples were positive with IMS/SPC but negative with blood culture), and identification results were in agreement. The IMS/SPC data also suggest that mixed infections might occur frequently, as C. albicans and at least one other Candida species were found in five samples. Additionally, in two cases, high numbers of cells (175 to 480 cells/ml of blood) were associated with an endovascular source of infection.

Fungicidal activity of miconazole against Candida spp. biofilms.

OBJECTIVES: Although azole antifungals are considered to be fungistatic, miconazole has fungicidal activity against planktonic Candida albicans cells, presumably associated with the induction of reactive oxygen species (ROS) production. Only few data are available concerning the effect of miconazole against sessile C. albicans cells. In the present study, the fungicidal activity of miconazole against in vitro-grown mature Candida biofilms, and its relationship with the induction of ROS and ROS-dependent apoptosis were examined. METHODS: The effect of miconazole on mature biofilms formed by 10 C. albicans strains and 5 strains from other Candida species was evaluated by plate counting and measuring the level of ROS induction. MIC tests were performed in the absence and presence of ascorbic acid, a quencher of ROS. The apoptotic population in C. albicans cells was determined using annexin-Cy3. RESULTS: Miconazole showed a significant fungicidal effect against all mature Candida biofilms tested and caused elevated ROS levels, both in planktonic and sessile cells. Addition of ascorbic acid drastically reduced these levels. While ROS quenching decreased the susceptibility to miconazole of planktonic cells of most Candida strains, no reduced fungicidal activity of miconazole against biofilms was observed. Miconazole did not cause a significant increase in apoptosis. CONCLUSIONS: ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment.

Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression.

BACKGROUND: Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model. RESULTS: HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model. CONCLUSIONS: Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.

What can be learned from genotyping of fungi?

Multiple genotyping studies have been carried out in order to clarify the epidemiology of fungal infections, more specifically to determine the sources, transmission routes, and colonization patterns of fungal isolates. In this review, the results obtained in genotyping investigations of Aspergillus isolates are summarized and discussed. Furthermore, we examine the epidemiologic studies of Candida albicans, Exophiala dermatitidis and Scedosporium apiospermum infections in patients with cystic fibrosis. Relative to Aspergillus fumigatus, colonization of the respiratory tract by multiple strains, and of deep organs by only a single strain were observed. On the other hand, the few studies which focused on other fungi isolated from patients with cystic fibrosis have suggested that colonization occurs primarily by a dominant genotype.

Candida albicans biofilm formation on peptide functionalized polydimethylsiloxane.

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.

Prevention of Candida albicans biofilm formation by covalently bound dimethylaminoethylmethacrylate and polyethylenimine.

Candida albicans biofilms are a major cause of voice prosthesis deterioration in laryngectomized patients. The aim of this study was to produce a surface capable of inhibiting C. albicans biofilm formation. Dimethylaminoethylmethacrylate (DMAEMA) and polyethylenimine (PEI) moieties were covalently bound to the surface of polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA) and subsequently quaternized. Physicochemical characterization of the grafted surfaces was carried out and their effect on C. albicans cell numbers was assessed using a modified Robbins device to grow the biofilms. Covalently bound quaternized polyDMAEMA (polyDMAEMAq) and PEI (PEIq) inhibited biofilm growth, with reductions up to 92%. Our approach may show promise for future application in medical devices such as catheters and prostheses.

Inhibition of Candida albicans biofilm formation by antimycotics released from modified polydimethyl siloxane.

Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.

AI-2 quorum-sensing inhibitors affect the starvation response and reduce virulence in several Vibrio species, most likely by interfering with LuxPQ.

The increase of disease outbreaks caused by Vibrio species in aquatic organisms as well as in humans, together with the emergence of antibiotic resistance in Vibrio species, has led to a growing interest in alternative disease control measures. Quorum sensing (QS) is a mechanism for regulating microbial gene expression in a cell density-dependent way. While there is good evidence for the involvement of auto-inducer 2 (AI-2)-based interspecies QS in the control of virulence in multiple Vibrio species, only few inhibitors of this system are known. From the screening of a small panel of nucleoside analogues for their ability to disturb AI-2-based QS, an adenosine derivative with a p-methoxyphenylpropionamide moiety at C-3' emerged as a promising hit. Its mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of Vibrio harveyi AI-2 QS mutants. Our results indicate that this compound, as well as a truncated analogue lacking the adenine base, block AI-2-based QS without interfering with bacterial growth. The active compounds affected neither the bioluminescence system as such nor the production of AI-2, but most likely interfered with the signal transduction pathway at the level of LuxPQ in V. harveyi. The most active nucleoside analogue (designated LMC-21) was found to reduce the Vibrio species starvation response, to affect biofilm formation in Vibrio anguillarum, Vibrio vulnificus and Vibrio cholerae, to reduce pigment and protease production in V. anguillarum, and to protect gnotobiotic Artemia from V. harveyi-induced mortality.

In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria.

OBJECTIVES: The goal of the present study was to obtain a comprehensive overview of the bacteriostatic and bactericidal effects of six commonly used antibiotics on planktonic as well as on sessile Burkholderia cepacia complex cells. METHODS: The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods. RESULTS: Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms. CONCLUSIONS: Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria.

Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia.

Burkholderia cepacia complex strains are opportunistic pathogens causing life-threatening infections in cystic fibrosis patients. B. cepacia complex strains are resistant to many antimicrobial agents and commonly produce biofilms in vitro and in vivo. This contributes to their virulence and makes Burkholderia infections difficult to treat. Recently, the quorum sensing (QS) system of Burkholderia spp. has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. However, detailed information about the anti-biofilm effect of these compounds is still lacking. In the present study, we evaluated the anti-biofilm effect of several known QS inhibitors. The effect on Burkholderia spp. biofilm formation was examined using crystal violet, resazurin and SYTO9 staining, confocal laser scanning microscopy as well as plating. When used at subinhibitory concentrations, several compounds interfered with biofilm formation by Burkholderia spp. Our results suggest that the QS inhibitors affect later stages of biofilm formation and detachment.

Resistance of planktonic and biofilm-grown Burkholderia cepacia complex isolates to the transition metal gallium.

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(NO(3))(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(NO(3))(3) treatment was determined using resazurin as a marker for cell viability. The antimicrobial effect of Ga(NO(3))(3) was assessed in the presence (50 microM) and absence of Fe(3+). RESULTS: When tested against planktonic cells, the MICs of Ga(NO(3))(3) in the absence of Fe(3+) were 64 mg/L for all B. cepacia complex strains investigated. However, the addition of 50 microM Fe(3+) in the presence of 64 mg/L Ga(NO(3))(3) resulted in increased growth for all B. cepacia complex strains investigated. In sessile cells, resistance to Ga(NO(3))(3) and the extent of the protective effect of 50 microM Fe(3+) against Ga(NO(3))(3) appear to be strain-dependent: the Burkholderia cenocepacia strains investigated are insensitive to Ga(NO(3))(3) in the presence of 50 microM Fe(3+), whereas the presence of Fe(3+) has no protective effect for both Burkholderia multivorans strains investigated. CONCLUSIONS: As maximal tolerable Ga(3+) levels in plasma are estimated to be approximately 200 microM and considering the high levels of Fe(3+) in the lungs of people with CF, our data suggest that the added value of a Ga(NO(3))(3) treatment of B. cepacia complex-infected patients may be limited.