Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci

Background: The ability of biofilm formation seems to play an essential role in the virulence of coagulase-negative staphylococci (CNS). The most clearly characterized component of staphylococcal biofilms is the polysaccharide intercellular adhesin (PIA) encoded by the icaADBC operon. Biofilm production was studied in 80 coagulase-negative staphylococci (CNS) strains isolated from clinical specimens of newborns with infection hospitalized at the Neonatal Unit of the University Hospital, Faculty of Medicine of Botucatu, and in 20 isolates obtained from the nares of healthy individuals without signs of infection. The objective was to compare three phenotypic methods with the detection of the icaA, icaD and icaC genes by PCR. Findings: Among the 100 CNS isolates studied, 82% tested positive by PCR, 82% by the tube test, 81% by the TCP assay, and 73% by the CRA method. Using PCR as a reference, the tube test showed the best correlation with detection of the ica genes, presenting high sensitivity and specificity. Conclusions: The tube adherence test can be indicated for the routine detection of biofilm production in CNS because of its easy application and low cost and because it guarantees reliable results with excellent sensitivity and specificity. © 2010 Cunha et al; licensee BioMed Central Ltd.

Effect of brushing with conventional versus whitening dentifrices on surface roughness and biofilm formation of dental ceramics

The aim of this study was to evaluate the effect of conventional and whitening dentifrices on the weight loss, surface roughness, and early in situ biofilm formation on the surface of dental ceramics. Standardized feldspar ceramic specimens (Vita VM7 and Vita VM13) were submitted to the following experimental conditions: no brushing; brushing without a dentifrice; brushing with a conventional dentifrice; and brushing with a whitening dentifrice. A brushing machine was used to simulate brushing. The mass and surface roughness of all specimens from the test groups were evaluated prior to and after brushing. Ten participants used an oral device for eight hours to evaluate the biofilm formed in situ on the specimens. Scanning electron microscopy was used for qualitative and quantitative analysis of the biofilm. ANOVA and Tukey tests were used to analyze the results of weight loss, surface roughness, and presence of bacteria. A one-way Kruskal-Wallis test was used for bacterial colonization results. For both ceramics, brushing with a whitening dentifrice resulted in weight loss that was significantly greater when compared to brushing without a dentifrice or with a conventional dentifrice. Increased surface roughness was noticed on VM13 ceramic samples with both dentifrices, whereas only conventional dentifrice had a significant effect on the surface roughness of VM7 samples. For both VM7 and VM13, no difference was found between the experimental conditions with regard to the presence or number of bacteria. Cocci and short rods were the predominant microbial morphotypes. Granular or fibrillar acellular material partially covered the specimens. Brushing with a whitening dentifrice resulted in significant weight loss of ceramic restorations, while brushing with both conventional and whitening dentifrices can roughen ceramic surfaces. The increase in roughness was not clinically significant to contribute to increased biofilm formation.

Effectiveness of mechanical brushing with different denture cleansing agents in reducing in vitro Candida albicans biofilm viability

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis, a common disease diagnosed among denture wearers. A routine of denture cleansing is essential to prevent biofilm formation and the onset of this infection. The aim of this study was to investigate the effectiveness of combining brushing and cleansing agents in killing C. albicans biofilm. Disks of acrylic resin were made, sterilized, and inoculated with C. albicans (107 cfu/mL). After incubation (37°C/48 h), specimens were randomly assigned to 10 experimental groups (n=9): 5 subjected to brushing with distilled water or cleansing agents - dentifrice slurry, 2% chlorhexidine gluconate (CHX), 1% sodium hypochlorite (NaOCl), and Polident fresh cleanse® (combined method) - and 4 exposed to the cleansing agents without brushing (immersion). Non-cleansed specimens were used as positive controls. The viability of cells was evaluated by XTT reduction method. Results were analyzed by Mann-Whitney and Kruskal-Wallis tests (α=0.05). The combined method was significantly more effective (p<0.0001) in reducing biofilm viability than the immersion. Brushing with CHX and NaOCl resulted in 100% removal of the biofilm. Immersion in the agents reduced significantly (p<0.0001) the biofilm viability, with CHX being the most effective (p<0.0001). The use of the combined method of brushing with cleansing agents is an effective method to reduce C. albicans biofilm, being CHX and NaOCl the most effective solutions.

Daily biofilm control and oral health: Consensus on the epidemiological challenge-Latin American Advisory Panel

Our understanding of dental plaque biofilm has evolved since the nonspecific plaque hypothesis that considered plaque as a nonspecific mass of native microorganisms that, because of lack of oral hygiene, builds up in proportions great enough to overcome the host resistance threshold and affect the tooth structure and tooth supporting tissues. A great diversity of microorganisms-over 700 species-was detected in the oral cavity, and evidence shows that the investigation of specific microorganisms or associations of microorganisms as etiological agents for periodontal diseases and caries is not a simplistic approach. Although clinical evidence shows that oral mechanical hygiene is fundamental to prevent and control caries and periodontal disease, it is important to highlight that optimal control is not achieved by most individuals. Thus the complementary use of chemotherapeutic agents has been investigated as a way to overcome the deficiencies of mechanical oral hygiene habits, insofar as they reduce both plaque formation and gingival inflammation, and represent a valid strategy to change the biofilm and maintain dental and periodontal health. The role of the dental professional is to monitor patients and offer them the best recommendations to preserve oral health throughout their life. With this in mind, chemical control should be indicated as part of daily oral hygiene, together with mechanical procedures, for all individuals who present supragingival and/or subgingival biofilm, taking into account age, physical and/or psychological limitations, allergies, and other factors.

Candida species: Current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options

The incidence of fungal infections has increased significantly, so contributing to morbidity and mortality. This is caused by an increase in antimicrobial resistance and the restricted number of antifungal drugs, which retain many side effects. Candida species are major human fungal pathogens that cause both mucosal and deep tissue infections. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth. Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm production is also associated with a high level of antimicrobial resistance of the associated organisms. The ability of Candida species to form drugresistant biofilms is an important factor in their contribution to human disease. The study of plants as an alternative to other forms of drug discovery has attracted great attention because, according to the World Health Organization, these would be the best sources for obtaining a wide variety of drugs and could benefit a large population. Furthermore, silver nanoparticles, antibodies and photodynamic inactivation have also been used with good results. This article presents a brief review of the literature regarding the epidemiology of Candida species, as well as their pathogenicity and ability to form biofilms, the antifungal activity of natural products and other therapeutic options. © 2013 SGM.

Comparative analysis of Enterococcus faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37°C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (μm3), live bacteria biovolume (μm3), and substrate coverage (%) were quantified by using the BioImage-L software. Results obtained were analyzed by nonparametric tests (P =.05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P <.05) and hydroxyapatite the highest at 21 days (P <.05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P <.05). The percentages of coverage were similar among all substrates at 21 days (P >.05), but at 14 days, bovine bone presented the highest coverage (P <.05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated. © 2013 American Association of Endodontists.

Photodynamic inactivation of biofilm: Taking a lightly colored approach to stubborn infection

Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents. © 2013 Informa UK Ltd.

N-acetylcysteine and vancomycin alone and in combination against staphylococci biofilm

Introduction: The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere and to live on artificial surfaces and to resist to the host immune factors and antibiotics. Staphylococcal infections have become increasingly difficult to treat due their antibiotic resistance. Therefore, there is a continuous need for new and effective treatment alternatives against staphylococcal infections. The main goal of this study was to test N-acetylcysteine (NAC) and vancomycin alone and in combination against S. epidermidis and S. aureus biofilms. Methods: Biofilms were treated with NAC at minimum inhibitory concentration (MIC) and 10 × MIC concentrations and vancomycin at MIC and peak serum concentrations. Results: The use of NAC 10 × MIC alone showed a significant antibactericidal effect, promoting a 4-5 log10 CFU/ mL reduction in biofilm cells. The combination of NAC 10 × MIC with vancomycin (independently of the concentration used) reduced significantly the number of biofilm cells for all strains evaluated (5-6 log10). Conclusion: N-acetylcysteine associated to vancomycin can be a potential therapeutic strategy in the treatment of infections associated to biofilms of S. epidermidis or S. aureus.

Effect of diamond-like carbon thin film coated acrylic resin on candida albicans biofilm formation

Purpose: The purpose of this study was to evaluate the effect of diamond-like carbon thin films doped and undoped with silver nanoparticles coating poly(methyl methacrylate) (PMMA) on Candida albicans biofilm formation. The control of biofilm formation is important to prevent oral diseases in denture users. Materials and Methods: Forty-five PMMA disks were obtained, finished, cleaned in an ultrasonic bath, and divided into three groups: Gc, no surface coating (control group); Gdlc, coated with diamond-like carbon film; and Gag, coated with diamond-like carbon film doped with silver nanoparticles. The films were deposited using a reactive magnetron sputtering system (physical vapor deposition process). The specimens were characterized by optical profilometry, atomic force microscopy, and Rutherford backscattering spectroscopy analyses that determined differences in chemical composition and morphological structure. Following sterilization of the specimens by γ-ray irradiation, C. albicans (ATCC 18804) biofilms were formed by immersion in 2 ml of Sabouraud dextrose broth inoculated with a standardized fungal suspension. After 24 hours, the number of colony forming units (cfu) per specimen was counted. Data concerning biofilm formation were analyzed using ANOVA and the Tukey test (p < 0.05). Results: C. albicans biofilm formation was significantly influenced by the films (p < 0.00001), reducing the number of cfu, while not affecting the roughness parameters (p > 0.05). The Tukey test showed no significant difference between Gdlc and Gag. Films deposited were extremely thin (∼50 nm). The silver particles presented a diameter between 60 and 120 nm and regular distribution throughout the film surface (to Gag). Conclusion: Diamond-like carbon films, doped or undoped with silver nanoparticles, coating the base of PMMA-based dentures could be an alternative procedure for preventing candidosis in denture users. © 2013 by the American College of Prosthodontists.

Susceptibility of multispecies biofilm to photodynamic therapy using Photodithazine®

This in vitro study evaluated the effect of photodynamic therapy (PDT) on the multispecies biofilm of Candida albicans, Candida glabrata, and Streptococcus mutans. Standardized fungal and bacterial suspensions were cultivated appropriately for each species and inoculated in 96-well microtiter plates for mix-biofilm formation. After 48 h of incubation, the biofilms were submitted to PDT (P + L+) using Photodithazine® (PDZ) at 100, 150, 175, 200, or 250 mg/mL for 20 min and 37.5 J/cm2 of light-emitting diode (LED) (660 nm). Additional samples were treated only with PDZ (P + L-) or LED (P-L+), or neither (control, P-L-). Afterwards, the biofilms were evaluated by quantification of colonies (CFU/mL), metabolic activity (XTT reduction assay), total biomass (crystal violet staining), and confocal scanning laser microscopy (CSLM). Data were analyzed by one-way ANOVA and Tukey tests (p < 0.05). Compared with the control, PDT promoted a significant reduction in colonies viability of the three species evaluated with 175 and 200 mg/mL of PDZ. PDT also significantly reduced the metabolic activity of the biofilms compared with the control, despite the PDZ concentration. However, no significant difference was found in the total biomass of samples submitted or not to PDT. For all analysis, no significant difference was verified among P-L-, P + L-, and P-L+. CSLM showed a visual increase of dead cells after PDT. PDT-mediated PDZ was effective in reducing the cell viability of multispecies biofilm. © 2013 Springer-Verlag London.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic escherichia coli strain

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains. ©2013, American Society for Microbiology.

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L.monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L.monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30°C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20°C for 24h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35°C for 48h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (. p=0.3847). We conclude that L.monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm. © 2013 Elsevier Ltd.

NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biochemical and microbiological characteristics of in situ biofilm formed on materials containing fluoride or amorphous calcium phosphate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)