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Biofilms are involved in the recalcitrance of infections due to the presence of persister cells. Although the molecular basis of persistence is still largely unknown, toxin-antitoxin modules (TA) are thought to play a role in this process. In this study, we investigated whether TA modules contribute to persistence toward antibiotics in Burkholderia cenocepacia J2315. Sixteen pairs of genes were identified based on their apparent similarity to TA modules. Overexpression of the putative toxins had various effects on growth, persistence, and biofilm formation. Toxins, whose overexpression resulted in growth inhibition, often increased the number of surviving persisters; in contrast, overexpression of putative toxins showing no effects on growth had no positive influence on the number of surviving persisters. Furthermore, the expression of the TA modules was compared between treated and untreated sessile and planktonic wild-type cultures. For 10 toxin-encoding genes, the expression was higher in untreated sessile cells than in untreated planktonic cells. Nine toxin-encoding genes were upregulated after treatment with tobramycin, but none after treatment with ciprofloxacin. These results indicate that most, but not all TA modules contribute to persistence in B. cenocepacia J2315 and that this contribution depends on the mode of growth and the antibiotic used., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Staphylococcus epidermidis strain ET-024 was isolated from a biofilm on an endotracheal tube of a mechanically ventilated patient. This strain is resistant to methicillin, and the draft genome sequence shares some characteristics with other nosocomial S. epidermidis strains (such as S. epidermidis RP62A)., (Copyright © 2014 Vandecandelaere et al.)

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

In this study, we investigated the biotherapeutic potential of previously isolated quorum quenching (QQ) bacteria. Some of them produce and secrete small compounds that inhibit quorum sensing (QS), others quench QS by enzymatic degradation of N-acylhomoserine lactones (AHLs). The supernatant of cultures of these isolates was tested for inhibitory properties against P. aeruginosa PAO1 biofilms. Most isolates had a moderate effect on biofilm formation, as shown by viability staining and/or staining of the biofilm biomass. A substantial part of the isolates reduced P. aeruginosa elastase production in a concentration-dependent manner. Using Caenorhabditis elegans as an in vivo model system for virulence testing, we found that some of the isolates were able to increase survival of P. aeruginosa PAO1 and Burkholderia cenocepacia LGM16656-infected nematodes when co-administered with the pathogen. Altogether, these data indicate that some QQ bacteria, or the active compounds they produce, could be useful to attenuate virulence of P. aeruginosa PAO1 and possibly also other Gram-negative pathogens that use AHLs to regulate the production of virulence factors., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Aim: In this study, we investigated the function of BCAM0257-8-9 located in the BCESM region of the Burkholderia cenocepacia J2315 genome., Materials & Methods: Differential RNA sequencing was used to determine transcription start sites. The phenotype of overexpression mutants was studied and a transcriptome analysis of the BCAM0258 overexpression mutant was performed., Results: BCAM0257 and BCAM0258 were identified as belonging to an operon, positively regulated by BCAM0259. We found that this operon is involved in persistence and that BCAM0258 functions as a regulator influencing quorum sensing and activating pathways related to iron acquisition and biofilm formation. Overexpression of BCAM0257 increased virulence., Conclusion: The BCESM genomic region contains an operon that contributes to quorum sensing and is involved in persistence, biofilm formation and virulence. BCAM0257-8-9 is found in all sequenced B. cenocepacia ET12 genomes and these results may help explain why infections with strains of the B. cenocepacia ET12 lineage are difficult to treat.

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.

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., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Mechanically ventilated patients often develop ventilator-associated pneumonia (VAP). Soon after intubation, a mixed biofilm harboring microbial pathogens is formed on the endotracheal tube (ET). It is believed that this biofilm contributes to the development of VAP. Unfortunately, the causative agent is often not known at the time VAP is suspected, and early therapy often relies on the identification of surveillance cultures (SC). It is thus important to know whether these SC can predict the microbial flora in ET biofilms. In this study, we compare the presence of a number of antibiotic-resistant nosocomial bacteria (Enterobacter aerogenes, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and of Candida albicans in cultures from ET biofilms and SC (i.e. sputum samples, nose swabs, and throat swabs) of 20 mechanically ventilated patients. Our data indicate that there is a good correlation between the presence of (antibiotic-resistant) pathogens in ET biofilms and SC., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Aims: Although several factors contribute to wound healing, bacterial infections and the presence of biofilm can significantly affect healing. Despite that this clearly indicates that therapies should address biofilm in wounds, only few wound care products have been evaluated for their antibiofilm effect. For this reason, we developed a rapid quantification approach to investigate the efficacy of wound care products on wounds infected with Staphylococcus spp., Methods and Results: An in vitro chronic wound infection model was used in which a fluorescent Staph. aureus strain was used to allow the rapid quantification of the bacterial burden after treatment. A good correlation was observed between the fluorescence signal and the bacterial counts. When evaluated in this model, several commonly used wound dressings and wound care products inhibited biofilm formation resulting in a decrease between one and seven log CFU per biofilm compared with biofilm formed in the absence of products. In contrast, most dressings only moderately affected mature biofilms., Conclusion: Our model allowed the rapid quantification of the bacterial burden after treatment. However, the efficacy of treatment varied between the different types of dressings and/or wound care products., Significance and Impact of the Study: Our model can be used to compare the efficacy of wound care products to inhibit biofilm formation and/or eradicate mature biofilms. In addition, the results indicate that treatment of infected wounds should be started as soon as possible and that novel products with more potent antibiofilm activity are needed., (© 2013 The Society for Applied Microbiology.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

Aim: To compare the antimicrobial efficacy of two-high power lasers (Nd:YAG and Er:YAG) and two commercial antimicrobial photodynamic therapy (aPDT) systems with that of sodium hypochlorite (NaOCl) action on Enterococcus faecalis biofilms grown on dentine discs., Methodology: Enterococcus faecalis biofilms were grown on dentine discs in a microtiter plate, incubated for 24 h and subjected to the following treatments: aPDT (Denfotex and Helbo system), Er:YAG laser irradiation (2940 nm, 50 mJ or 100 mJ, 15 Hz, 40 s), Nd:YAG laser irradiation (1064 nm, 2 W, 15 Hz, 40 s) and immersion in 2.5% (w/v) NaOCl for 1, 5, 10 and 30 min. Surviving bacteria were harvested, and the number of CFU per disc was determined by plate counting., Results: Significant reductions (anova, P ≤ 0.05) in viable counts were observed for aPDT (Helbo) (2 log(10) reduction), Er:YAG irradiation using 100 mJ pulses (4.3 log(10) reduction) and all NaOCl treatments (>6 log(10) reduction). NaOCl (2.5%) for 5 min effectively eliminated all bacteria. aPDT (Denfotex), Er:YAG irradiation using 50 mJ pulses and Nd:YAG treatment caused a reduction in the viable counts of <1 log(10) unit; these results were not significantly different from the untreated controls., Conclusion: Within the limitations of this particular laboratory set-up, NaOCl was the most effective in E. faecalis biofilm elimination, while Er:YAG laser treatment (100 mJ pulses) also resulted in high reductions in viable counts. The use of both commercial aPDT systems resulted in a weak reduction in the number of E. faecalis cells. Nd:YAG irradiation was the least effective., (© 2012 International Endodontic Journal.)

Propionibacterium acnes is a Gram-positive bacterium that plays an important role in the pathogenesis of acne vulgaris. This organism is capable of biofilm formation and the decreased antimicrobial susceptibility of biofilm-associated cells may hamper efficient treatment. In addition, the prolonged use of systemic antibiotic therapy is likely to lead to the development and spread of antimicrobial resistance. In the present study we investigated whether P. acnes biofilms could be eradicated by plant extracts or their active compounds, and whether other mechanisms besides killing of biofilm cells could be involved. Out of 119 plant extracts investigated, we identified five with potent antibiofilm activity against P. acnes (extracts from Epimedium brevicornum, Malus pumila, Polygonum cuspidatum, Rhodiola crenulata and Dolichos lablab). We subsequently identified icariin, resveratrol and salidroside as active compounds in three of these extracts. Extracts from E. brevicornum and P. cuspidatum, as well as their active compounds (icariin and resveratrol, respectively) showed marked antibiofilm activity when used in subinhibitory concentrations, indicating that killing of microbial cells is not their only mode of action., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1 mM N-(3-oxo-dodecanoyl)-L-homoserine lactone and 2 mM N-(3-oxo-hexanoyl)-L-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Background: Many bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs., Methodology/principal Findings: By evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an α,β unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus., Conclusions/significance: Several new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic respiratory pathogens which are particularly difficult to eradicate from the lungs of cystic fibrosis (CF) patients because of their innate resistance to antimicrobials and their capacity to form biofilms. The goal of the present study was to evaluate the bacteriostatic and bactericidal activities of temocillin against planktonic and sessile Bcc bacteria. 37 strains belonging to 17 Bcc species were tested. 75.7% of the strains were susceptible when grown planktonically (minimal inhibitory concentration <16 μg/ml). The minimal bactericidal concentrations were higher than 128 μg/ml for most strains. No remarkable differences in resistance between exponentially growing planktonic cells and 4 h old biofilms were observed: when grown in a biofilm, 59.5% of the strains were susceptible. After treating 24 h biofilms with a concentration of 10×MIC, only a minor reduction was seen in the number of sessile cells, indicating a limited bactericidal activity against biofilms. Our data indicate that temocillin has a good bacteriostatic in vitro activity against planktonic and 4 h old biofilms, but seems of limited use to eradicate 24 h old biofilms., (Copyright © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Objective: Ocular bioadhesive minitablets containing gentamicin and vancomycin were developed using different powder mixtures of pregelatinized starch and Carbopol (physical or cospray-dried mixtures)., Methods: Drug content, antimicrobial activity, and radical formation of the powders used for tablet preparation were evaluated immediately and 30 days after gamma sterilization. Tablet properties and in vitro drug release from the sterilized minitablets were determined. Storage stability of vancomycin and gentamicin in sterilized bioadhesive mixtures was examined by LC-UV/MS and a microbiological assay, respectively. A bioadhesive powder mixture containing only vancomycin was irradiated by X electron-magnetic radiation to evaluate vancomycin stability following sterilization through irradiation., Results: The antimicrobial activity of gentamicin against Staphylococcus epidermidis was not altered in comparison to nonsterilized formulations. Only after an overkill dose of 50 kGy, the concentration of vancomycin decreases to an extent that was pharmaceutically significant. No significant difference in radiation stability between drug substance and product (i.e., powder mixture) was observed. A shift in stability profile was not observed at 6 weeks after irradiation. All other degradation products were present only in small quantities not exceeding 1.0%. The in vitro drug release from the minitablets prepared with physical powder mixtures of pregelatinized starch and Carbopol® 974P NF (96 : 4) was faster compared to the cospray-dried mixtures of starch with Carbopol® 974P NF (ratio: 95:5 and 85:15). The electron paramagnetic resonance signals of the radicals formed during sterilization were still visible after storage for 30 days. The slug mucosal irritation test indicated mild irritation properties of the bioadhesive powder mixtures although no tissue damage was observed.

Aims: The yeast Saccharomyces boulardii is used as a probiotic for the prevention and treatment of diarrhoea. In this study, the quality of 15 probiotic products containing S. boulardii was verified., Methods and Results: Using microsatellite typing, the identity of all Saccharomyces strains in the products was confirmed as S. boulardii. Additionally, solid-phase cytometry (SPC) and a plate method were used to enumerate S. boulardii cells. SPC was not only able to produce results more rapidly than plating (4h compared to 48h) but the cell counts obtained with SPC were significantly higher than the plate counts. Finally, we found that <1% of the S. boulardii cells survived 120min in gastric conditions and storage for 3months at 40°C with 75% relative humidity., Conclusions: We developed a SPC method for the quantification of viable S. boulardii cells in probiotics. Additionally, we demonstrated that gastric conditions and storage have a marked effect on the viability of the yeast cells., Significance and Impact of the Study:   To our knowledge, this is the first time SPC is used for the quality control of probiotics with S. boulardii. Additionally, we demonstrated the need for gastric protection and accurate storage., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65-80% of all human infections is thought to be biofilm-related, this presents a serious challenge. Biofilm model systems are essential to gain a better understanding of the mechanisms involved in biofilm formation and resistance. In this review a comprehensive overview of various in vitro and in vivo systems is presented, and their advantages and disadvantages are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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 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.

Biofilm formation is often associated with persistent Candida albicans infections. Treatment of these infections is difficult, since sessile C. albicans cells show increased resistance towards antifungal agents. The molecular mechanisms behind biofilm resistance in C. albicans are not yet understood. In the present study, we investigated the transcriptional response in young and mature in vitro-grown biofilms after a short and longer exposure time to high doses of fluconazole or amphotericin B. Treatment of biofilms with high doses of antifungal agents resulted in a drug-specific transcriptional response. Exposure of biofilms to fluconazole induced upregulation of genes encoding enzymes involved in ergosterol biosynthesis (ERG1, ERG3, ERG11 and ERG25). Treatment of biofilms with amphotericin B resulted in an overexpression of KRE1 and SKN1, two genes encoding proteins involved in beta-1,6-glucan biosynthesis. Our data indicate that sessile C. albicans cells show controlled regulation of gene expression, as they quickly mount a drug-specific transcriptional response in the presence of high doses of antifungal agents. These transcriptional changes suggest upregulation of ergosterol biosynthesis (fluconazole) and upregulation of beta-1,6-glucan biosynthesis (amphotericin B) in sessile C. albicans cells that might contribute to a resistant biofilm phenotype., ((c) 2010 Elsevier Masson SAS. All rights reserved.)

Solid-phase cytometry (SPC) was used to determine the total number and the number of itraconazole-resistant Aspergillus fumigatus cells in 60 air samples. Of the 570 A. fumigatus cells that were recovered, 10 (1.8%) were resistant. SPC proved more specific and rapid than culture and allowed high-troughput susceptibility testing., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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.

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus. In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus, which requires a high discriminatory power.

Aspergillus fumigatus is an ubiquitous fungus capable of causing severe infections such as aspergilloma, allergic bronchopulmonary aspergillosis, and invasive aspergillosis, especially in immunocompromised patients. Monitoring the number of Aspergillus fumigatus spores in the air is crucial for infection control. In the present study, a novel approach for the quantification of Aspergillus fumigatus, based on solid-phase cytometry (SPC) and immunofluorescent labeling, was developed. The sensitivity and specificity of the assay were confirmed by testing pure cultures. Paecilomyces variotii and Rhizopus stolonifer were codetected but could be excluded on the basis of morphology of the microcolonies. The SPC method has considerable advantages compared to the culture-based method, including its low detection limit (4 cells/m3), its speed (results are obtained within 24 h), and the straightforward microscopic identification of Aspergillus fumigatus. Additionally, comparison of results obtained with both methods demonstrated that they are equally accurate for the quantification of Aspergillus fumigatus in environmental air samples.

Aim: To assess the antibacterial action of laser irradiation (Nd:YAG, KTP), photo activated disinfection (PAD) and 2.5% sodium hypochlorite (NaOCl) on Enterococcus faecalis, in an aqueous suspension and in an infected tooth model., Methodology: Root canals of 60 human teeth with single straight canals were prepared to apical size 50, autoclaved, inoculated with an E. faecalis suspension and incubated for 48 h. They were randomly allocated to four treatment and one control groups. After treatment, the root canals were sampled by flushing with physiological saline, and the number of surviving bacteria in each canal was determined by plate count and solid phase cytometry. The same experimental or control treatments were completed on aqueous suspensions of E. faecalis, and the number of surviving bacteria was determined in the same way., Results: In aqueous suspension, PAD and NaOCl resulted in a significant reduction in the number of E. faecalis cells (P < 0.001), whilst Nd:YAG or KTP had no effect. In the infected tooth model, only the PAD and NaOCl treated teeth yielded significantly different results relative to the untreated controls (P < 0.001)., Conclusions: The laser systems as well as PAD were less effective than NaOCl in reducing E. faecalis, both in aqueous suspension and in the infected tooth model.

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.

This protocol describes the use of solid-phase cytometry for the enumeration of airborne bacteria and fungi. In contrast with conventional methods, accurate results can be obtained in real time, especially for air samples with low numbers of microorganisms. Air samples are collected by impaction on a water-soluble polymer that is subsequently dissolved. Part of the sample can be filtered over two membrane filters with different pore sizes. One filter is used to obtain a total count of all viable microorganisms, and a second filter is used to determine the number of airborne fungi. Microorganisms present on the filter are labeled with a viability substrate and subsequently detected and quantified using a solid-phase cytometer. The detected spots are microscopically validated using an epifluorescence microscope to discriminate between bacteria, fungi and fluorescent particles. The whole procedure takes 5 h to complete and results in the accurate quantification of airborne bacteria and fungi for samples with a low or high microbial load.

Aim: To compare the survival of four bacterial strains (Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa) in pharmaceutical oils, including jojoba oil/tea tree oil, carbol oil, jojoba oil and sesame oil., Methods and Results: Oils were spiked with the test bacteria in a concentration of 10(4) CFU ml(-1). Bacteria were extracted from oils with phosphate-buffered saline containing 0.5% Tween 20. Aliquots of the pooled water layers were analysed by solid-phase cytometry and plate counting. Plate counts dropped to zero for all test strains exposed for 24 h to three of the four oils. In contrast, significant numbers of viable cells were still detected by SPC, except in the jojoba oil/tea tree oil mixture and partly in sesame oil., Conclusions: Exposure of bacteria for 24 h to the two oils containing an antimicrobial led to a loss of their culturability but not necessarily of their viability. The antibacterial activity of the jojoba oil/tea tree oil mixture supersedes that of carbol oil., Significance and Impact of the Study: These in vitro data suggest that the jojoba oil/tea tree oil mixture more than carbol oil inhibits bacterial proliferation when used for intermittent self-catherization.

Summary: In the present study we evaluated the efficacy of various procedures recommended for the disinfection of respiratory equipment and other materials in cystic fibrosis, using both planktonic and sessile Burkholderia cenocepacia cells. A modified European Suspension Test was performed to determine the effects of the disinfection procedures on planktonic cells. The ability of the treatments to kill sessile cells and to remove biofilm biomass was evaluated using two resazurin-based viability assays and a crystal violet staining on biofilms grown and treated in 96-well microtitre plates. The effect of chlorhexidine and hydrogen peroxide treatments on the viability of sessile B. cenocepacia cells was clearly reduced compared to the effects on planktonic cells. Treatments with low concentrations of sodium hypochlorite (0.05%, 5 min) and acetic acid (1.25%, 15 min) also resulted in insufficient reductions in the number of viable sessile cells. There was no relation between the ability of the disinfectants to remove biofilm biomass and their potential to kill biofilm cells. In conclusion, our study indicates that testing of the efficacy of disinfectants should be performed on both planktonic and sessile cells, with particular attention to their effects on cellular viability.

Aims: To evaluate the susceptibility to microbial contamination that occurs during simulated handling of protective devices for the preparation of cytotoxic drug solutions., Methods and Results: Four devices, i.e. Chemoprotect spike, Clave connector, PhaSeal and Securmix were challenged with low and high inocula of micro-organisms. The cells, transferred to the connected vials during repeated manipulations of the devices were counted by means of solid-phase cytometry. Of the four devices, PhaSeal afforded the lowest transfer of micro-organisms. Secondly, the efficiency of procedures for the disinfection of an artificially contaminated rubber stopper was compared prior to connection of the vial to the PhaSeal device. Spraying or swabbing alone was inadequate, as opposed to a combination of spraying [0.5% or 2.0% (w/v) chlorhexidine in isopropanol] and swabbing [70% (v/v) isopropanol]., Conclusions: Although Phaseal afforded the lowest transfer of micro-organisms, adequate disinfection of the vial prior to connection remains required., Significance and Impact of the Study: Unlike aspects of operator protection, which are well documented, the microbiological safety of protective devices for the preparation of cytotoxic drugs has not been addressed in the literature. This study estimates the susceptibility to microbial contamination during handling of four commonly used devices.

New smart surface-modified polypropylene (PP) was prepared for improving the loading and the sustained delivery of vancomycin and, thus, reducing the risk of biofilm formation when used as component of biomedical devices. Isothermal titration calorimetry (ITC) served for screening the most suitable monomers for grafting; the drug preferentially bonding to ionized acrylic acid (AAc). A net-PP-g-PNIPAAm-inter-net-PAAc was synthesized by first grafting and cross-linking of N-isopropylacrylamide onto PP films and then interpenetrating a second network by redox polymerization and cross-linking of AAc. PP-g-PAAc slabs were prepared by grafting AAc and, optionally, cross-linking. The amount and composition of grafted polymer (FTIR-ATR), morphology (SEM), temperature- and pH-responsiveness (swelling measurements), thermal behavior (DSC), friction coefficient (rheometry), drug loading and release rate, and effect against methicillin-resistant Staphylococcus aureus (MRSA) biofilms (modified robbins device) were evaluated. Grafting of AAc notably decreased the friction coefficient from 0.28+/-0.03 to 0.05+/-0.02 and enhanced the vancomycin loading (up to 2.5mg/cm(2)). Drug-loaded films showed a pH-dependent release rate, sustaining the release in pH 7.4 aqueous media at 37 degrees C for several hours. All drug-loaded films reduced biofilm formation by MRSA; the anti-biofilm effect being statistically significant (91.7% reduction, alpha<0.05) for PP-g-PAAc with the thinnest grafting layer.

A series of 256 Aspergillus fumigatus isolates, recovered from eight patients with cystic fibrosis (CF), were genotyped using microsatellite-based typing. Only a limited number of genotypes were shared between patients and co-colonisation with multiple strains was indicated for all patients. Additionally, some genotypes were isolated recurrently, indicating that they are capable of prolonged colonisation.

Aims: To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine, a disinfectant for the maintenance of oral medical devices., Methods and Results: Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus) and allowed the determination of the optimal conditions for its use., Conclusion: The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine exhibits high activity against biofilms formed by the micro-organisms tested., Significance and Impact of the Study: Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.