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PubMed1994

Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms.

Bacterial infections associated with indwelling medical devices often demonstrate an intrinsic resistance to antimicrobial therapies. In order to explore the possibility of transport limitation to biofilm bacteria as a contributing factor, the penetration of a fluoroquinolone antibiotic, ciprofloxacin, through Pseudomonas aeruginosa biofilms was investigated. Attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry was employed to monitor bacterial colonization of a germanium substratum, transport of ciprofloxacin to the biofilm-substratum interface, and interaction of biofilm components with the antibiotic in a flowing system. Transport of the antibiotic to the biofilm-substratum interface during the 21-min exposure to 100 micrograms/ml was found to be significantly impeded by the biofilm. Significant changes in IR bands of the biofilm in regions of the spectrum associated with RNA and DNA vibrational modes appeared following exposure to the antibiotic, indicating chemical modification of biofilm components. These results suggest that transport limitations may be an important factor in the antimicrobial resistance of biofilm bacteria and that ATR/FT-IR spectrometry may be used to follow the time course of antimicrobial action in biofilms in situ.

Biofilms↗
PubMed1997

Influence of electric fields and pH on biofilm structure as related to the bioelectric effect.

Mixed species biofilms of Klebsiella pneumoniae, Pseudomonas fluorescens, and Pseudomonas aeruginosa were grown in a flow cell fitted with two platinum wire electrodes. The biofilm growing on the wires reached a thickness of approximately 50 microm after 3 days. When a voltage was applied with oscillating polarity, the biofilm attached to the wire expanded and contracted. The biofilm expanded by approximately 4% when the wire was cathodic but was reduced to 74% of the original thickness when the wire was anodic. The phenomenon was reproduced by alternately flushing the flow cell with media adjusted to pH 3 and pH 10 with no electric current. At pH 10 the biofilm was unaltered, but it became compacted to 69% of the original thickness at pH 3. We explained these phenomena in terms of the molecular interactions between charged acidic groups in the biofilm slime and the bacterial cell walls. Contraction of the biofilm under acidic conditions may be caused by (i) the elimination of electrostatic repulsion from neutralization of negatively charged carboxylate groups through protonation and (ii) subsequent hydrogen bonding between the carboxylic acids and oxygen atoms in the sugars. Electrostatic interactions between negatively charged groups in the biofilm and the charged wire may also be expected to cause biofilm expansion when the wire was cathodic and contraction when the wire was anodic. The consequences of the explanation of the increased susceptibility of biofilm cells to antibiotics in an electric field, the "bioelectric effect," are discussed.

Analysis of Variance↗
PubMed2004

Dynamics and spatial distribution of beta-lactamase expression in Pseudomonas aeruginosa biofilms.

The development of resistance to beta-lactam antibiotics is a problem in the treatment of chronic Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. The main resistance mechanism is high-level expression of the chromosomally encoded AmpC beta-lactamase of P. aeruginosa cells growing in biofilms. Several genes have been shown to influence the level of ampC expression, but little is known about the regulation of ampC expression in P. aeruginosa biofilms. To study the expression of ampC in P. aeruginosa biofilms, we constructed a reporter that consisted of the fusion of the ampC promoter to gfp(ASV) encoding an unstable version of the green fluorescent protein. In vitro biofilms of P. aeruginosa were exposed to the beta-lactam antibiotics imipenem and ceftazidime. Sub-MICs of imipenem significantly induced the monitor system of the biofilm bacteria in the peripheries of the microcolonies, but the centers of the microcolonies remained uninduced. However, the centers of the microcolonies were physiologically active, as shown by experiments with another monitor construction consisting of an arabinose-inducible promoter fused to gfp(ASV). The whole biofilm was induced in the presence of increased imipenem concentrations. Ceftazidime induced the monitor system of the biofilm bacteria as well, but only bacteria in the peripheries of the microcolonies were induced in the presence of even very high concentrations. The experiments illustrate for the first time the dynamic and spatial distributions of beta-lactamase induction in P. aeruginosa cells growing in biofilms. Thus, our experiments show that P. aeruginosa cells growing in biofilms constitute a heterogeneous population unit which may create different antibiotic-selective environments for the bacteria in the biofilm.

Anti-Bacterial Agents↗
PubMed2005

Secretion of proteases by Pseudomonas aeruginosa biofilms exposed to ciprofloxacin.

Pseudomonas aeruginosa proteases are considered important virulence factors which damage host tissues and interfere with host antibacterial defense mechanisms. P. aeruginosa biofilm cells are not completely killed by antibacterials, and therefore this study addresses the question whether ciprofloxacin attenuates the virulence of biofilm communities by abolishing their secretion of proteases. The surviving cells of the colony biofilms studied, despite their cyclical exposure to four doses of ciprofloxacin at bactericidal concentrations (one dose a day), still secreted active proteases to the environment surrounding the biofilms. The biofilm cells secreted elastase B (LasB) over the duration of the experiments as confirmed by Western immunoblot analysis. The colony biofilms did not secrete LasA-a protease with staphylolytic activity. The same profiles on zymogram gels with gelatin were observed for the proteases secreted by both ciprofloxacin-exposed and unexposed (control) biofilms. Total proteolytic activities of the colony biofilms studied were significantly reduced after exposure to ciprofloxacin at bactericidal concentrations-after 96 h of exposure they dropped to 38% for the strain intermediate resistant to ciprofloxacin and to 65% for the strain highly resistant to the antibiotic, relative to the control biofilms. The surviving cells of the colony biofilms after their release into a fresh medium displayed transient increased resistance to ciprofloxacin compared to their planktonic counterparts.

Anti-Bacterial Agents↗
PubMed1998

Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation.

The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 mumol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.

Alkaline Phosphatase↗
PubMed2000

Evaluation of fluorescently labeled lectins for noninvasive localization of extracellular polymeric substances in Sphingomonas biofilms.

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.

Biofilms↗
PubMed2001

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Bacterial Adhesion↗
PubMed2002

Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule.

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 micro M) and incubated at 37 degrees C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 micro M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.

Animals↗
PubMed2002

Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation.

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.

Biofilms↗
PubMed2002

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans.

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.

Amino Acid Sequence↗
PubMed2003

Natural genetic transformation in monoculture Acinetobacter sp. strain BD413 biofilms.

Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp. strain BD413.

Acinetobacter↗
PubMed2003

Effects of current velocity on the nascent architecture of stream microbial biofilms.

Current velocity affected the architecture and dynamics of natural, multiphyla, and cross-trophic level biofilms from a forested piedmont stream. We monitored the development and activity of biofilms in streamside flumes operated under two flow regimes (slow [0.065 m s(-1)] and fast [0.23 m s(-1)]) by combined confocal laser scanning microscopy with cryosectioning to observe biofilm structure and composition. Biofilm growth started as bacterial microcolonies embedded in extracellular polymeric substances and transformed into ripple-like structures and ultimately conspicuous quasihexagonal networks. These structures were particularly pronounced in biofilms grown under slow current velocities and were characterized by the prominence of pennate diatoms oriented along their long axes to form the hexagons. Microstructural heterogeneity was dynamic, and biofilms that developed under slower velocities were thicker and had larger surface sinuosity and higher areal densities than their counterparts exposed to higher velocities. Surface sinuosity and biofilm fragmentation increased with thickness, and these changes likely reduced resistance to the mass transfer of solutes from the water column into the biofilms. Nevertheless, estimates of dissolved organic carbon uptake and microbial growth suggested that internal cycling of carbon was more important in thick biofilms grown in slow flow conditions. High-pressure liquid chromatography-pulsed amperometric detection analyses of exopolysaccharides documented a temporal shift in monosaccharide composition as the glucose levels decreased and the levels of rhamnose, galactose, mannose, xylose, and arabinose increased. We attribute this change in chemical composition to the accumulation of diatoms and increased incorporation of detrital particles in mature biofilms.

Bacteria↗
PubMed2003

Growth of Escherichia coli in model distribution system biofilms exposed to hypochlorous acid or monochloramine.

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH(2)Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH(2)Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila, E. coli, and beta and delta proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.

Bacteria↗
PubMed2004

Metamorphosis of a scleractinian coral in response to microbial biofilms.

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.

Animals↗
PubMed2004

Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata.

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A Delta alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.

Bacterial Proteins↗
PubMed2005

Three stages of a biofilm community developing at the liquid-liquid interface between polychlorinated biphenyls and water.

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.

Bacteria↗
PubMed2005

Sensitivity of planktonic and biofilm-associated Salmonella spp. to ionizing radiation.

Salmonella enterica forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation has been used to inactivate Salmonella on a variety of foods and contact surfaces, but the relative efficacy of the process against biofilm-associated cells versus free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm-associated cells was determined for three food-borne-illness-associated isolates of Salmonella. Biofilms were formed on sterile glass slides in a coincubation apparatus, using inoculated tryptic soy broth, incubated at 37 degrees C for 48 h. Resulting biofilms were 18 to 24 microm in height as determined by confocal scanning laser microscopy. The planktonic and biofilm cultures were gamma irradiated to doses of 0.0 (control), 0.5, 1.0, 1.5, 2.0 and 2.5 kGy. The D(10) value (the dose of radiation required to reduce a population by 1 log(10), or 90%) was calculated for each isolate-culture based on surviving populations at each radiation dose. The D(10) values of S. enterica serovar Anatum were not significantly (P < 0.05) different for biofilm-associated (0.645 kGy) and planktonic (0.677 kGy) cells. In contrast, the biofilm-associated cells of S. enterica serovar Stanley were significantly more sensitive to ionizing radiation than the respective planktonic cells, with D(10) values of 0.531 and 0.591 kGy, respectively. D(10) values of S. enterica serovar Enteritidis were similarly reduced for biofilm-associated (0.436 kGy) versus planktonic (0.535 kGy) cells. The antimicrobial efficacy of ionizing radiation is therefore preserved or enhanced in treatment of biofilm-associated bacteria.

Biofilms↗
PubMed2005

Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms.

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.

Anti-Bacterial Agents↗