Showing papers on "Biofilm published in 2010"

The biofilm matrix

Abstract: The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.

A review of current and emergent biofilm control strategies

Abstract: Microbial adhesion to surfaces and the consequent biofilm formation has been documented in many different environments. Biofilms constitute a protected mode of growth that allows microorganisms to survival in hostile environments, being their physiology and behavior significantly different from their planktonic counterparts. In dairy industry, biofilms may be a source of recalcitrant contaminations, causing food spoilage and are possible sources of public health problems such as outbreaks of foodborne pathogens. Biofilms are difficult to eradicate due to their resistant phenotype. However, conventional cleaning and disinfection regimens may also contribute to inefficient biofilm control and to the dissemination of resistance. Consequently, new control strategies are constantly emerging with main incidence in the use of biosolutions (enzymes, phages, interspecies interactions and antimicrobial molecules from microbial origin). The present review will focus on describing the mechanisms involved in biofilm formation and behavior, deleterious effects associated with their presence, and some of the current and emergent control strategies, providing new insight of concern for food industry.

Oral multispecies biofilm development and the key role of cell–cell distance

Abstract: Growth of oral bacteria in situ requires adhesion to a surface because the constant flow of host secretions thwarts the ability of planktonic cells to grow before they are swallowed. Therefore, oral bacteria evolved to form biofilms on hard tooth surfaces and on soft epithelial tissues, which often contain multiple bacterial species. Because these biofilms are easy to study, they have become the paradigm of multispecies biofilms. In this Review we describe the factors involved in the formation of these biofilms, including the initial adherence to the oral tissues and teeth, cooperation between bacterial species in the biofilm, signalling between the bacteria and its role in pathogenesis, and the transfer of DNA between bacteria. In all these aspects distance between cells of different species is integral for oral biofilm growth.

Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization

Abstract: Commensal bacteria are known to inhibit pathogen colonization; however, complex host-microbe and microbe-microbe interactions have made it difficult to gain a detailed understanding of the mechanisms involved in the inhibition of colonization. Here we show that the serine protease Esp secreted by a subset of Staphylococcus epidermidis, a commensal bacterium, inhibits biofilm formation and nasal colonization by Staphylococcus aureus, a human pathogen. Epidemiological studies have demonstrated that the presence of Esp-secreting S. epidermidis in the nasal cavities of human volunteers correlates with the absence of S. aureus. Purified Esp inhibits biofilm formation and destroys pre-existing S. aureus biofilms. Furthermore, Esp enhances the susceptibility of S. aureus in biofilms to immune system components. In vivo studies have shown that Esp-secreting S. epidermidis eliminates S. aureus nasal colonization. These findings indicate that Esp hinders S. aureus colonization in vivo through a novel mechanism of bacterial interference, which could lead to the development of novel therapeutics to prevent S. aureus colonization and infection.

D-amino acids trigger biofilm disassembly.

Abstract: Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of D-leucine, D-methionine, D-tyrosine, and D-tryptophan that could act at nanomolar concentrations. D-amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of D-amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. D-amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. D-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly.

Biofilm Dispersal: Mechanisms, Clinical Implications, and Potential Therapeutic Uses

Abstract: Like all sessile organisms, surface-attached communities of bacteria known as biofilms must release and disperse cells into the environment to colonize new sites. For many pathogenic bacteria, biofilm dispersal plays an important role in the transmission of bacteria from environmental reservoirs to human hosts, in horizontal and vertical cross-host transmission, and in the exacerbation and spread of infection within a host. The molecular mechanisms of bacterial biofilm dispersal are only beginning to be elucidated. Biofilm dispersal is a promising area of research that may lead to the development of novel agents that inhibit biofilm formation or promote biofilm cell detachment. Such agents may be useful for the prevention and treatment of biofilms in a variety of industrial and clinical settings. This review describes the current status of research on biofilm dispersal, with an emphasis on studies aimed to characterize dispersal mechanisms, and to identify environmental cues and inter- and intracellular signals that regulate the dispersal process. The clinical implications of biofilm dispersal and the potential therapeutic applications of some of the most recent findings will also be discussed.

Amyloid fibers provide structural integrity to Bacillus subtilis biofilms.

Abstract: Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10–15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

Ciprofloxacin Causes Persister Formation by Inducing the TisB toxin in Escherichia coli

Abstract: Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage–induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.

Biofilm formation and the food industry, a focus on the bacterial outer surface

Abstract: The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food-processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.

Pseudomonas aeruginosa biofilms in cystic fibrosis

Abstract: The persistence of chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients is due to biofilm-growing mucoid (alginate-producing) strains. A biofilm is a structured consortium of bacteria, embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. In CF lungs, the polysaccharide alginate is the major part of the P. aeruginosa biofilm matrix. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and resist phagocytosis, as well as other components of the innate and the adaptive immune system. As a consequence, a pronounced antibody response develops, leading to immune complex-mediated chronic inflammation, dominated by polymorphonuclear leukocytes. The chronic inflammation is the major cause of the lung tissue damage in CF. Biofilm growth in CF lungs is associated with an increased frequency of mutations, slow growth and adaptation of the bacteria to the conditions in the lungs, and to antibiotic therapy. Low bacterial metabolic activity and increase of doubling times of the bacterial cells in CF lungs are responsible for some of the tolerance to antibiotics. Conventional resistance mechanisms, such as chromosomal β-lactamase, upregulated efflux pumps, and mutations of antibiotic target molecules in the bacteria, also contribute to the survival of P. aeruginosa biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy, and they can be treated by chronic suppressive therapy.

Oral Biofilm Architecture on Natural Teeth

Abstract: Periodontitis and caries are infectious diseases of the oral cavity in which oral biofilms play a causative role. Moreover, oral biofilms are widely studied as model systems for bacterial adhesion, biofilm development, and biofilm resistance to antibiotics, due to their widespread presence and accessibility. Despite descriptions of initial plaque formation on the tooth surface, studies on mature plaque and plaque structure below the gum are limited to landmark studies from the 1970s, without appreciating the breadth of microbial diversity in the plaque. We used fluorescent in situ hybridization to localize in vivo the most abundant species from different phyla and species associated with periodontitis on seven embedded teeth obtained from four different subjects. The data showed convincingly the dominance of Actinomyces sp., Tannerella forsythia, Fusobacterium nucleatum, Spirochaetes, and Synergistetes in subgingival plaque. The latter proved to be new with a possibly important role in host-pathogen interaction due to its localization in close proximity to immune cells. The present study identified for the first time in vivo that Lactobacillus sp. are the central cells of bacterial aggregates in subgingival plaque, and that Streptococcus sp. and the yeast Candida albicans form corncob structures in supragingival plaque. Finally, periodontal pathogens colonize already formed biofilms and form microcolonies therein. These in vivo observations on oral biofilms provide a clear vision on biofilm architecture and the spatial distribution of predominant species.

Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

Abstract: Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.

Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms

Abstract: Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.

Biofilm Formation on Dental Restorative and Implant Materials

Abstract: Biomaterials for the restoration of oral function are prone to biofilm formation, affecting oral health. Oral bacteria adhere to hydrophobic and hydrophilic surfaces, but due to fluctuating shear, little biofilm accumulates on hydrophobic surfaces in vivo. More biofilm accumulates on rough than on smooth surfaces. Oral biofilms mostly consist of multiple bacterial strains, but Candida species are found on acrylic dentures. Biofilms on gold and amalgam in vivo are thick and fully covering, but barely viable. Biofilms on ceramics are thin and highly viable. Biofilms on composites and glass-ionomer cements cause surface deterioration, which enhances biofilm formation again. Residual monomer release from composites influences biofilm growth in vitro, but effects in vivo are less pronounced, probably due to the large volume of saliva into which compounds are released and its continuous refreshment. Similarly, conflicting results have been reported on effects of fluoride release from glass-ionomer cements. Finally, biomaterial-associated infection of implants and devices elsewhere in the body is compared with oral biofilm formation. Biomaterial modifications to discourage biofilm formation on implants and devices are critically discussed for possible applications in dentistry. It is concluded that, for dental applications, antimicrobial coatings killing bacteria upon contact are more promising than antimicrobial-releasing coatings.

Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP

Abstract: Microorganisms can switch from a planktonic, free-swimming life-style to a sessile, colonial state, called a biofilm, which confers resistance to environmental stress. Conversion between the motile and biofilm life-styles has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, which identifies VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization on c-di-GMP binding.

Current concepts in biofilm formation of Staphylococcus epidermidis.

Abstract: Staphylococcus epidermidis is a highly significant nosocomial pathogen mediating infections primarily associated with indwelling biomaterials (e.g., catheters and prostheses). In contrast to Staphylococcus aureus, virulence properties associated with S. epidermidis are few and biofilm formation is the defining virulence factor associated with disease, as demonstrated by animal models of biomaterial-related infections. However, other virulence factors, such as phenol-soluble modulins and poly-γ-DL-glutamic acid, have been recently recognized that thwart innate immune system mechanisms. Formation of S. epidermidis biofilm is typically considered a four-step process consisting of adherence, accumulation, maturation and dispersal. This article will discuss recent advances in the study of these four steps, including accumulation, which can be either polysaccharide or protein mediated. It is hypothesized that studies focused on understanding the biological function of each step in staphylococcal biofilm formation will yield new treatment modalities to treat these recalcitrant infections.

Dispersion as an Important Step in the Candida albicans Biofilm Developmental Cycle

Abstract: Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of "virulence factors" important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window.

Abstract: Objective To investigate the hypothesis that newly formed wound biofilms (or bioburdens) are more susceptible to antimicrobial treatment. Method Four separate and distinct models were performed by four separate biofilm research laboratories to evaluate the resistance of biofilms to antimicrobial treatments over time. These included a drip-flow biofilm model along with a hydrodebridement study, a porcine skin punch biopsy ex vivo model, a mouse chronic wound model and clinical longitudinal debridement study. Results All four models showed that, within the first 24 hours, the biofilm community was more susceptible to the selected antibiotics, and after maturing for up to 48 hours became increasingly tolerant. In each model, there was at least a 24-hour period in which the biofilms were more resistant to antibiotics. Each of the models utilised showed a significant decrease in the resistance of the biofilm/burden to gentamicin for up to 24 hours with a confidence interval of at least 95%. The resistance incr...

Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation.

Abstract: Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α2-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.

An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal

Abstract: We review the recent advances in the understanding of the Pseudomonas aeruginosa biofilm lifestyle from studies using in vitro laboratory setups such as flow chambers and microtiter trays. Recent work sheds light on the role of nutrients, motility, and quorum sensing in structure formation in P. aeruginosa biofilms. The second messenger, c-di-GMP, is established as an important regulator of the synthesis of polysaccharide and protein components of the biofilm matrix. Extracellular DNA is shown to be an essential component of the biofilm matrix. It has become apparent that biofilm formation involves interactions between different subpopulations. The molecular mechanisms underlying the tolerance of biofilm bacteria to antimicrobial agents are beginning to be unraveled, and new knowledge has been obtained regarding the environmental cues and regulatory mechanisms involved in biofilm dispersal.

Burkholderia Type VI Secretion Systems Have Distinct Roles in Eukaryotic and Bacterial Cell Interactions

Abstract: Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

Biofilms in chronic infections – a matter of opportunity – monospecies biofilms in multispecies infections

Abstract: It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.

Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system.

Abstract: Microorganisms develop biofilms on indwelling medical devices and are associated with device-related infections, resulting in substantial morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated catheters with Pseudomonas aeruginosa bacteriophages on biofilm formation by P. aeruginosa in an in vitro model. Hydrogel-coated catheters were exposed to a 10 log(10) PFU ml(-1) lysate of P. aeruginosa phage M4 for 2 h at 37 degrees C prior to bacterial inoculation. The mean viable biofilm count on untreated catheters was 6.87 log(10) CFU cm(-2) after 24 h. The pretreatment of catheters with phage reduced this value to 4.03 log(10) CFU cm(-2) (P < 0.001). Phage treatment immediately following bacterial inoculation also reduced biofilm viable counts (4.37 log(10) CFU cm(-2) reduction; P < 0.001). The regrowth of biofilms on phage-treated catheters occurred between 24 and 48 h, but supplemental treatment with phage at 24 h significantly reduced biofilm regrowth (P < 0.001). Biofilm isolates resistant to phage M4 were recovered from catheters pretreated with phage. The phage susceptibility profiles of these isolates were used to guide the development of a five-phage cocktail from a larger library of P. aeruginosa phages. The pretreatment of catheters with this cocktail reduced the 48-h mean biofilm cell density by 99.9% (from 7.13 to 4.13 log(10) CFU cm(-2); P < 0.001), but fewer biofilm isolates were resistant to these phages. These results suggest the potential of applying phages, especially phage cocktails, to the surfaces of indwelling medical devices for mitigating biofilm formation by clinically relevant bacteria.

Antibacterial activity of dental composites containing zinc oxide nanoparticles.

Abstract: The resin-based dental composites commonly used in restorations result in more plaque accumulation than other materials. Bacterial biofilm growth contributes to secondary caries and failure of resin-based dental composites. Methods to inhibit biofilm growth on dental composites have been sought for several decades. It is demonstrated here that zinc oxide nanoparticles (ZnO-NPs) blended at 10% (w/w) fraction into dental composites display antimicrobial activity and reduce growth of bacterial biofilms by roughly 80% for a single-species model dental biofilm. Antibacterial effectiveness of ZnO-NPs was assessed against Streptococcus sobrinus ATCC 27352 grown both planktonically and as biofilms on composites. Direct contact inhibition was observed by scanning electron microscopy and confocal laser scanning microscopy while biofilm formation was quantified by viable counts. An 80% reduction in bacterial counts was observed with 10% ZnO-NP-containing composites compared with their unmodified counterpart, indicating a statistically significant suppression of biofilm growth. Although, 20% of the bacterial population survived and could form a biofilm layer again, 10% ZnO-NP-containing composites maintained at least some inhibitory activity even after the third generation of biofilm growth. Microscopy demonstrated continuous biofilm formation for unmodified composites after 1-day growth, but only sparsely distributed biofilms formed on 10% ZnO-NP-containing composites. The minimum inhibitory concentration of ZnO-NPs suspended in S. sobrinus planktonic culture was 50 microg mL(-1). ZnO-NP-containing composites (10%) qualitatively showed less biofilm after 1-day-anaerobic growth of a three-species initial colonizer biofilm after being compared with unmodified composites, but did not significantly reduce growth after 3 days.

Suppression of bacterial cell–cell signalling, biofilm formation and type III secretion system by citrus flavonoids

Abstract: Aim: This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Methods and Results: Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96-well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi-quantitative PCR. Differential responses for different flavonoids were observed for different cell–cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell–cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. Conclusion: The results of the study indicate a potential modulation of bacterial cell–cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer-mediated cell–cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Significance and Impact of the Study: Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell–cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.