Showing papers on "Biofilm published in 1999"
TL;DR: Improvements in understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
Abstract: Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
11,162 citations
TL;DR: Minimal biofilm eradication concentrations, derived by using the Calgary Biofilm Device, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to beeffective at the MICs.
Abstract: Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.
1,880 citations
TL;DR: Investigating a variety of Staphylococcus aureus strains finds that all strains tested contain the ica locus and that several can form biofilms in vitro, suggesting that cell-cell adhesion and the potential to form biofilmms is conserved within this genus.
Abstract: Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear β-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica) locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Sequence comparison with the S. epidermidis ica genes revealed 59 to 78% amino acid identity. Deletion of the ica locus results in a loss of the ability to form biofilms, produce PIA, or mediate N-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of icaA in several other Staphylococcus species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.
1,113 citations
01 Jan 1999
TL;DR: This paper presents a meta-analysis of the function and structure of the EPS from the Strong Acidophile Thiobacillus ferrooxidans and the role of Enzymes within Microbial Biofilms and the Role of the Extracellular Polymer Matrix Interaction Between Extrace cellular Polysaccharides and EnZymes.
Abstract: Introduction What are Bacterial Extracellular Polymeric Substances?- In Situ Characterization of Extracellular Polymeric Substances (EPS) in Biofilm Systems Extraction of EPS Biofilm Exopolysaccharides Regulation of Matrix Polymer in Biofilm Formation and Dispersion Exopolymers of Sulphate-Reducing Bacteria Analysis and Function of the EPS from the Strong Acidophile Thiobacillus ferrooxidans Physical and Chemical Properties of Extracellular Polysaccharides Associated with Biofilms and Related Systems Chemical Communication Within Microbial Biofilms: Chemotaxis and Quorum Sensing in Bacterial Cells Function of EPS Polysaccharases in Biofilms - Sources - Action - Consequences!- Extracellular Enzymes Within Microbial Biofilms and the Role of the Extracellular Polymer Matrix Interaction Between Extracellular Polysaccharides and Enzymes.
663 citations
TL;DR: The phenotypes of these three groups suggest that the type IV pilus and flageLLum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotics surface, and exopolysaccharide is involved in the formation of three‐dimensional biofilm architecture.
Abstract: We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.
663 citations
01 Jan 1999
TL;DR: Biofilms are accumulations of microorganisms (prokaryotic and eukaryotic unicellular organisms), EPS, multivalent cations, biogenic and inorganic particles as well as colloidal and dissolved compounds that determine the physicochemical and biological properties of biofilms.
Abstract: The vast majority of microorganisms live and grow in aggregated forms such as biofilms and flocs (“planktonic biofilms”). This mode of existence is lumped in the somewhat inexact but generally accepted expression “biofilm”. The common feature of all these phenomena is that the microorganisms are embedded in a matrix of extracellular polymeric substances (EPS). The production of EPS is a general property of microorganisms in natural environments and has been shown to occur both in prokaryotic (Bacteria, Archaea) and in eukaryotic (algae, fungi) microorganisms. Biofilms containing mixed populations of these organisms are ubiquitously distributed in natural soil and aquatic environments, on tissues of plants, animals and man as well as in technical systems such as filters and other porous materials, reservoirs, plumbing systems, pipelines, ship hulls, heat exchangers, separation membranes, etc. (Costerton et al. 1987; 1995; Flemming and Schaule 1996). Biofilms develop adherent to a solid surface (substratum) at solid-water interfaces, but can also be found at water-oil, water-air and solid-air interfaces. Biofilms are accumulations of microorganisms (prokaryotic and eukaryotic unicellular organisms), EPS, multivalent cations, biogenic and inorganic particles as well as colloidal and dissolved compounds. EPS are mainly responsible for the structural and functional integrity of biofilms and are considered as the key components that determine the physicochemical and biological properties of biofilms.
629 citations
TL;DR: It is shown that the length and/or degree of substitution of the N-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by P. aeruginosa cells are not freely permeable to 3OC12-HSL, and the mexA-mexB-oprM-encoded efflux pump is involved inactive efflux of 3OC 12- HSL.
Abstract: Pseudomonas aeruginosa remains a leading cause of both nosocomial infections in immunocompromised patients and chronic infections in cystic fibrosis patients (reviewed in references 9 and 57). P. aeruginosa virulence depends on cell-associated factors, including alginate and pili (5, 9), and secreted factors, including toxins, exotoxin A, and exoenzyme S (14, 28); proteases, elastase, alkaline protease, and LasA protease (13, 27, 47); and hemolysins, rhamnolipid, and phospholipase (25). Cell-to-cell signaling (quorum sensing) is required for expression of many P. aeruginosa virulence factors (see below) (6, 43).
Intrinsic resistance of P. aeruginosa to many antibiotics and disinfectants also causes clinical problems (49). The intrinsic resistance is due to low outer membrane permeability and to multidrug efflux pumps that reduce the cellular level of antibiotics (reviewed in references 11 and 30). Three known P. aeruginosa multidrug efflux pumps are encoded by the mexAB-oprM, mexCD-oprJ, and mexEF-oprN operons, respectively (18, 45, 46). These pumps consist of a cytoplasmic membrane component of the resistance-nodulation-cell division (RND) family (39) thought to function as a proton antiport exporter (i.e., MexB), an outer membrane component thought to form channels (i.e., OprM), and a membrane fusion protein thought to link MexB and OprM (reviewed in reference 29). Bacterial RND pumps have also been shown to cause the efflux of many other organic compounds, including solvents and inhibitors (24, 29, 50). However, no natural products of P. aeruginosa have yet been shown to be subject to efflux via RND pumps.
Quorum sensing (or autoinduction) is the controlled expression of specific genes in response to extracellular chemical signals produced by the bacteria themselves (6). Typically cells emit an N-acyl homoserine lactone signal called an autoinducer (AI), which is usually synthesized by a LuxI-type AI synthase, into the environment (6). At high cell densities, the AI reaches a threshold concentration and binds to a LuxR-type protein which is then able to activate target genes (6). To date, luxR-luxI-type quorum-sensing systems and their N-acyl homoserine lactone AIs have been found in many different gram-negative bacteria (6). In P. aeruginosa the las (lasR-lasI) (7, 37) and rhl (rhlR-rhlI) (33, 34) quorum-sensing systems direct the synthesis of two distinct AIs, N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) (formerly called PAI-1) (40) and N-butyryl homoserine lactone (C4-HSL) (formerly called PAI-2) (41, 59), respectively. LasR and 3OC12-HSL activate expression of several genes, including lasI itself (54), lasB (encoding elastase) (37), lasA (encoding LasA protease) (8), both xcp operons (xcpPQ and xcpR-Z, encoding the type II secretion apparatus [3]), and rhlR (20, 44). Recently, the las quorum-sensing system was shown to be involved in P. aeruginosa biofilm differentiation (4). C4-HSL and RhlR also activate expression of numerous genes, including rhlI itself (20), the rhamnolipid biosynthesis operon rhlAB (32, 42), lasB (1, 41, 42), and rpoS (encoding the stationary-phase sigma factor ςs) (20).
Although many different N-acyl homoserine lactone AIs have been isolated from various gram-negative bacteria, to date, all differences are in the N-acyl side chain length (from C4 to C14) or degree of substitution (either 3-oxo, 3-hydroxy, saturated, or unsaturated) (6, 26, 48, 55). All AIs have been assumed to be freely diffusible in bacterial cells. This assumption is based on the fact that a radiolabeled Vibrio fischeri AI, N-3-oxo-hexanoyl homoserine lactone ([3H]3OC6-HSL), was shown to freely diffuse into and out of V. fischeri and Escherichia coli cells (15, 42). Here, we studied the uptake and efflux of 3OC12-HSL and C4-HSL by P. aeruginosa cells. Our results indicate that [3H]C4-HSL freely diffuses into and out of P. aeruginosa cells. In contrast, cellular concentrations of [3H]3OC12-HSL are higher than external levels. Our results show that the increased cellular level of [3H]3OC12-HSL is not due to association with LasR or RhlR and suggest that it is not due to active (inward) transport. We propose that the high cellular level of 3OC12-HSL is probably due to partitioning into the cell membranes. By use of P. aeruginosa Δ(mexAB-oprM) mutant cells or poisoned wild-type cells, we also show that 3OC12-HSL is subject to active efflux by the MexAB-OprM pump.
615 citations
TL;DR: The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity.
Abstract: The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.
569 citations
TL;DR: It is shown that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase, which seems to result from changes in multiple environmental physicochemical conditions.
Abstract: To get further information on bacterial surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless lacZ gene, was performed with an ompR234 adherent strain, and a simple screen was developed to assess changes in gene expression in biofilm cells versus planktonic cells. This screen revealed that major changes in the pattern of gene expression occur during biofilm development: the transcription of 38% of the genes was affected within biofilms. Different cell functions were more expressed in sessile bacteria: the OmpC porin, the high-affinity transport system of glycine betaine (encoded by the proU operon), the colanic acid exopolysaccharide (wca locus, formerly called cps), tripeptidase T (pepT), and the nickel high-affinity transport system (nikA). On the other hand, the syntheses of flagellin (fliC) and of a putative protein of 92 amino acids (f92) were both reduced in biofilms. Such a genetic reprogramming of gene expression in biofilms seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase.
524 citations
TL;DR: Evidence of QS control of genes essential for relieving oxidative stress is provided, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.
Abstract: Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-L-homoserine lactone) and PAI-2 (N-butyryl-L-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. beta-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.
TL;DR: Electrostatic interactions and hydrogen bonds are found to be the dominating forces among macromolecules within the biofilm.
TL;DR: Susceptibility data for these pairs show that the ET acts as a reservoir for infecting microorganisms which exhibit significantly greater antibiotic resistance than their tracheal counterparts.
Abstract: Objective: To determine the relationship between, and antibiotic resistance of, endotracheal tube (ET) biofilm and pulmonary pathogens in ventilator-associated pneumonia (VAP).¶Setting: General intensive care units in two university teaching hospitals.¶Design: The microbiology of ET biofilm and tracheal samples from patients with and without VAP were compared. For individual patients, matching pairs of pathogens were confirmed as identical and characterised for antibiotic susceptibility.¶Patients: 40 intensive care unit patients – 20 with VAP, 20 without VAP as control. The duration of intubation (median and range) was 6.5 days (3–17) and 5 days (2–10), respectively.¶Measurements and results: Samples of tracheal secretions were taken during ventilation for bacteriological culture. Following extubation, ETs were examined for the presence of biofilm. Isolates of high pathogenic potential included Staphylococcus aureus, enterococci, Enterobacteriaceae, pseudomonads and Candida spp. Where the same microorganism was found on tracheal and ET samples by phenotyping, these were confirmed as identical by genotyping and characterised for antibiotic susceptibility in both the free floating and biofilm forms. Seventy per cent of patients with VAP had identical pathogens isolated from both ET biofilm and tracheal secretions. No pairing of pathogens was observed in control patients (p < 0.005). Susceptibility data for these pairs show that the ET acts as a reservoir for infecting microorganisms which exhibit significantly greater antibiotic resistance than their tracheal counterparts.¶Conclusion: This investigation provides further evidence for the role of ET biofilm in VAP. The difficulty in eradicating an established microbial biofilm using antibiotics implies that increased attention must be directed towards modification of the ET to prevent or substantially reduce biofilm formation.
TL;DR: The results showed that analyzing wash waters is essential in quantifying biofilm EPS; the contribution of this step varied from 8-50% of the total carbohydrate yield, depending on the extraction method.
TL;DR: It is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older.
Abstract: In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.
TL;DR: It is shown that Vibrio cholerae El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity.
Abstract: While much has been learned regarding the genetic basis of host-pathogen interactions, less is known about the molecular basis of a pathogen's survival in the environment. Biofilm formation on abiotic surfaces represents a survival strategy utilized by many microbes. Here it is shown that Vibrio cholerae El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity. In contrast, attachment of V. cholerae to chitin is shown to be independent of the MSHA pilus, suggesting divergent pathways for biofilm formation on nutritive and nonnutritive abiotic surfaces.
TL;DR: Results indicate that the structure of a C. albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems.
Abstract: Two model biofilm systems, involving growth on disks of catheter material or on cylindrical cellulose filters, were used to investigate the structure of Candida albicans biofilms To assess the importance of dimorphism in biofilm development, biofilms produced by two wild-type strains were compared with those formed by two morphological mutants, incapable of yeast and hyphal growth, respectively Scanning electron microscopy and thin sections of biofilms examined by light microscopy revealed that biofilms of the wild-type strains formed on catheter disks consisted of two distinct layers: a thin, basal yeast layer and a thicker, but more open, hyphal layer The hypha- mutant produced only the basal layer, whereas the yeast- mutant formed a thicker, hyphal biofilm equivalent to the outer zone of the wild-type structures Biofilms of the yeast- mutant were more easily detached from the catheter surface than the others, suggesting that the basal yeast layer has an important role in anchoring the biofilm to the surface Biofilms formed on cylindrical cellulose filters were quite different in appearance The hypha- mutant and both wild types produced exclusively yeast-form biofilms whereas the yeast- mutant generated a dense hyphal mat on the top of the filter All these biofilms, irrespective of morphological form, were resistant to the antifungal agent, amphotericin B Overall, these results indicate that the structure of a C albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems
TL;DR: The ribosome number is a reliable indicator of growth rate in bacteria growing in balanced growth and has been used as a standard for growth rates in biofilm-embedded bacteria as well.
Abstract: Publisher Summary This chapter describes methods for the handling and analysis of microbial behavior of organisms in biofilm communities at both microscopic and macroscopic levels. Only methods and reporter systems that can be applied without disturbing the spatial organization of the organisms in the biofilm are presented. The in situ methods described in this chapter can be used for more than just identifying or tracing cells or genes in biofilms. By combining promoters that respond to specific environmental signals with appropriate marker genes, it may be possible to tag specific organisms and use these as monitor systems to estimate local chemical composition directly in the biofilms. Changes in environmental conditions will also have significant effects on the physiological state of the organisms. Such shifting conditions may result in several responses, such as altered growth rates, stress response, starvation, or even cell death. Most of these responses can be visualized directly using specific promoter–reporter fusions. The ribosome number is a reliable indicator of growth rate in bacteria growing in balanced growth and has been used as a standard for growth rates in biofilm-embedded bacteria as well.
TL;DR: RM4440, a Pseudomonas aeruginosa FRD1 derivative that carries a plasmid-basedrecA-luxCDABE fusion, is a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems and has proved to be a useful tool to study microbial communities in a noninvasive manner.
Abstract: We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a Pseudomonas aeruginosa FRD1 derivative that carries a plasmid-based recA-luxCDABE fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner.
TL;DR: Transmission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofilms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grownBiofilm aged revealed a reduced matrix distribution and enhanced cell density were observed as the biofilm aged.
Abstract: Four slime-producing isolates of Staphylococcus aureus were used in an antibiotic susceptibility assay for biofilms developed on 96-well polystyrene tissue culture plates. The study involved 11 antibiotics, two biofilm ages (6 and 48 h), two biofilm growth media (tryptone soy broth (TSB) and delipidated milk) and three antibiotic concentrations (4 x MBC, 100 mg/L and 500 mg/L). ATP-bioluminescence was used for automated bacterial viability determination after a 24 h exposure to antibiotics, to avoid biofilm handling. Under the conditions applied, viability in untreated biofilms (controls) was lower when biofilm growth was attempted in milk rather than in TSB. Various antibiotics had a greater effect on viability when used at higher (> or =100 mg/L) antibiotic concentrations and on younger (6 h) biofilms. Increased antibiotic effect was observed in milk-grown rather than TSB-grown biofilms. Phosphomycin and cefuroxime, followed by rifampicin, cefazolin, novobiocin, vancomycin, penicillin, ciprofloxacin and tobramycin significantly affected biofilm cell viability at least under some of the conditions tested. Gentamicin and erythromycin had a non-significant effect on cell viability. Transmission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofilms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grown biofilms. A reduced matrix distribution and enhanced cell density were observed as the biofilm aged. The S. aureus biofilm test discriminated antibiotics requiring shorter (3 h or 6 h) from those requiring longer (24 h) exposure and yielded results which may be complementary to those obtained by conventional tests.
TL;DR: The results of this study demonstrated that the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H2O2, particularly at high H2 O2 concentrations; KatB is induced in both planktoni and biofilms cells in response to H 2O2 insult, but plays a relatively small role in biofilm resistance.
Abstract: The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H(2)O(2)) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H(2)O(2). Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H(2)O(2), whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H(2)O(2). The katB mutant, lacking the H(2)O(2)-inducible catalase KatB, was similar to the wild-type strain with respect to H(2)O(2) resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H(2)O(2), while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H(2)O(2). Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H(2)O(2), suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H(2)O(2), while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of beta-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H(2)O(2), particularly at high H(2)O(2) concentrations; KatB is induced in both planktonic and biofilm cells in response to H(2)O(2) insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H(2)O(2) are sublethal.
TL;DR: Some of the recent work on biofilm biology is reviewed and its role in urinary tract infections, particularly those associated with urinary catheters, is highlighted.
Abstract: The biofilm mode of growth has been implicated in the majority of human bacterial infections. In the urinary tract, notable biofilm-associated infections include prostatitis, chronic cystitis, struvite urolithiasis, and catheter-associated infections. Biofilms protect the causative organisms from host defences and antimicrobial therapy. Biofilm formation has traditionally been considered to result from adhesion and capsule formation by adherent microorganisms. Recent work has shown that a large number of genes are activated during this process, some of which have been associated with twitching motility, quorum sensing, and slow growth. In this paper, we review some of the recent work on biofilm biology and highlight its role in urinary tract infections, particularly those associated with urinary catheters.
TL;DR: The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM).
Abstract: The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga–Flavobacterium group as major parts of the attached community The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe During initial biofilm development, beta-Proteobacteria dominated the bacterial population This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga–Flavobacterium group In mature biofilms, alpha-Proteobacteria and Cytophaga–Flavobacteria continued to be the prevalent bacterial groups Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms
TL;DR: Gram-negative bacteria regulate various functions including bioluminescence, plasmid conjugal transfer, biofilm formation, motility, antibiotic biosynthesis, and the production of virulence factors in plant and animal pathogens.
TL;DR: Testing two strains of Escherichia coli and its isogenic ΔrpoS derivative suggest that rpoS is important for biofilm physiology.
Abstract: Slow growth has been hypothesized to be an essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (DeltalacZ rpoS(+)) and its isogenic DeltarpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h(-1)) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P < 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h(-1). Overall, these studies suggest that rpoS is important for biofilm physiology.
TL;DR: It is shown that the negative effect of the cpxA mutation on biofilm formation results from a decreased transcription of the curlin encoding csgA gene, and the existence of a new signal transduction pathway involved in the adherence process in addition to the EnvZ-OmpR two-component system is proposed.
Abstract: In a genetic screening directed to identify genes involved in biofilm formation, mutations in the cpxA gene were found to reduce biofilm formation by affecting microbial adherence to solid surfaces. This effect was detected in Escherichia coli K12 as well as in E. coli strains isolated from patients with catheter-related bacteremia. We show that the negative effect of the cpxA mutation on biofilm formation results from a decreased transcription of the curlin encoding csgA gene. The effect of the cpxA mutation could not be observed in cpxR− mutants, suggesting that they affect the same regulatory pathway. The cpxA101 mutation abolishes cpxA phosphatase activity and results in the accumulation of phosphorylated CpxR. Features of the strain carrying the cpxA101 mutation are a reduced ability to form biofilm and low levels of csgA transcription. Our results indicate that the cpxA gene increases the levels of csgA transcription by dephosphorylation of CpxR, which acts as a negative regulator at csgA. Thus, we propose the existence of a new signal transduction pathway involved in the adherence process in addition to the EnvZ-OmpR two-component system.
Journal Article•
TL;DR: The authors discusses antimicrobial strategies to control dental caries, including reducing plaque levels, in general or specific cariogenic bacteria in particular, by antiplaque or antimicrobial agents; reducing bacterial acid production by replacing fermentable carbohydrates in the diet with sugar substitutes, or by interfering with bacterial metabolism with fluoride or antimacterial agents.
TL;DR: The formation of adherent multi-layered biofilms embedded into a glycocalyx composed of exopolysaccharides on implanted devices is believed to be essential for the pathogenesis of S. epidermidis infections.
TL;DR: This chapter discusses the formation of Candida biofilm on catheter disks, on cylindrical cellulose filters, and on perfused biofilm fermenter, which represents a model system in which the growth rate of biofilms can be controlled accurately.
Abstract: Publisher Summary This chapter discusses the formation of Candida biofilm on catheter disks, on cylindrical cellulose filters, and on perfused biofilm fermenter. Growth of the biofilms is monitored quantitatively using dry weight, colorimetric, or radioisotope assays and can be visualized readily by scanning electron microscopy. Different types of catheters, made from various plastics, can be used for biofilm formation. These include central venous catheters (composed of polyvinyl chloride (PVC) or polyurethane) and urinary Foley catheters. A much simpler model system is the formation of biofilm within small, cylindrical, cellulose filters that are perfused with culture medium. The perfused biofilm fermenter represents a model system in which the growth rate of biofilms can be controlled accurately. The chapter discusses the susceptibility of Candida biofilms to antifungal agents. Like bacterial biofilms, Candida biofilms are resistant to a range of antimicrobial agents. Drug susceptibility of biofilms can be assayed using any of these three model systems.
TL;DR: It was found that transfer occurs at a much lower rate in intestinal extracts than in laboratory media, and results suggest that the majority of intestinal plasmid transfer takes place in the viscous mucus layer covering the epithelial cells.
Abstract: The transfer of the R1drd19 plasmid between isogenic strains of Escherichia coli BJ4 in batch cultures of laboratory media and intestinal extracts was compared. Using an estimate of plasmid transfer rate that is independent of cell density, of donor:recipient ratios and of mating time, it was found that transfer occurs at a much lower rate in intestinal extracts than in laboratory media. Furthermore, the results suggest that the majority of intestinal plasmid transfer takes place in the viscous mucus layer covering the epithelial cells. Investigation of plasmid transfer in different flow systems harbouring a dynamic, continuously growing population of constant size showed that transfer kinetics were strongly influenced by bacterial biofilm formation. When donor and recipient populations were subjected to continuous mixing, as in a chemostat, transfer continued to occur at a constant rate. When donor and recipient populations retained fixed spatial locations, as in a biofilm, transfer occurred very rapidly in the initial phase, after which no further transfer was detected. From in vivo studies of plasmid transfer in the intestine of streptomycin-treated mice, results were obtained which were similar to those obtained in the biofilm, but differed markedly from those obtained in the chemostat. In spite of peristaltic movements in the gut, and of apparently even distribution of E. coli as single cells in the intestinal mucus, the intestinal environment displays transfer kinetics different from those expected of a mixed, liquid culture, but quite similar to those of a biofilm.