Effect of Sequence and Structural Properties on 14-Helical β-Peptide Activity against Candida albicans Planktonic Cells and Biofilms

ABSTRACTStatement of problem: Modern polyamide ‘flexible’ denture base materials have increased in popularity for use in removable partial dentures in the last several years. The introduction of these newer products warrants investigation of their relative potential to develop fungal biofilms.PurposeThe purpose of this study was to investigate the potential of three denture base materials to support fungal biofilm formation.Materials and methodsSpecimens of two ‘flexible’ nylon type materials and one traditional heat processed, methyl methacrylate resin material were studied (both polished and unpolished surfaces). The specimens were coated with saliva and evaluated for fungal (Candida albicans) biofilm formation. The fungal biofilm mass formed on denture substrates were evaluated by dry weight analysis and by determining the number of viable fungal cells in the biofilm by MTT viability assay. Alteration in fungal metabolic function following the treatment of the biofilmC. albicanswith nystatin and fluconazole was determined by XTT assay.ResultsIn general, the unpolished surfaces of the denture disks favored the fungal biofilm, the most being on polyamide specimen, Valplast. Significantly, less biofilm was formed on Duraflex and Lucitone surfaces. Biofim on C. albicans was also found to be resistant to antifungal agents. As compared to freshly incubated (grown) planktonic cells, biofilm fungal cells required significantly higher concentrations of nystatin and fluconazole in order to obtain 50% reduction in metabolic activity.ConclusionThis study demonstrated the differences in denture materials to support fungal biofilm formation, and also difference between polished and unpolished denture material surfaces. The results demonstrated that one of the polyamide materials (duraflex) had lesser potential to biofilm formation than the others.Clinical significanceUnfavorable tissue responses can ensue from the presence of fungal biofilms on dental prosthetics. Resistance to biofilm formation is a factor for dental materials in their selection and usage. This study helps to quantify, evaluate and compare biofilm formation on polished and unpolished surfaces of three commonly used denture base materials. The results of this study helped to identify materials, which may, therefore, be better indicated in clinical applications. Evaluations for the newer denture base materials, specific to these testing methods, appear to be novel in the scientific literature.How to cite this articleJain V, Babu J, Ahuja S, Wicks R, Garcia-Godoy F. Comparison of Fungal Biofilm Formation on Three Contemporary Denture Base Materials. Int J Experiment Dent Sci 2015;4(2):104-108.

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.

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

The human pathogen Listeria monocytogenes forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation effectively inactivates planktonic Listeria, but no information is available on the relative efficacy of the process against biofilm-associated Listeria. The irradiation sensitivity of planktonic or biofilm cells was determined for L. monocytogenes ATCC 43256 and ATCC 49594 and a commonly used surrogate Listeria innocua ATCC 51742. Biofilms were formed on sterile glass slides incubated for 48 h at 22°C, 28°C, or 37°C. The cultures were gamma irradiated and the irradiation D 10 value was calculated for each combination of isolate/culture/temperature. The effect of temperature of cultivation on the irradiation sensitivity of both planktonic cells and biofilm cells varied for each of the isolates. Depending on isolate and temperature, biofilm cells were equally sensitive or more sensitive (P < 0.05) to irradiation. D 10 values overall tended to increase with temperature of cultivation for L. monocytogenes 49594 and L. innocua 51742, but tended to decrease with increasing temperature for L. monocytogenes 43256. The D 10 values of the various culture/temperature combinations differed significantly among the isolates examined. Irradiation effectively eliminates both planktonic and biofilm-associated cells. The extent to which the biofilm habitat modifies the antimicrobial efficacy of irradiation is dependent on the specific isolate examined and the temperature at which it forms. This study is the first inquiry to show that biofilm Listeria cells are as sensitive or more sensitive to irradiation compared with planktonic cells and that this response is dependent on biofilm formation conditions.

Enhanced expression of catechol 1,2 dioxygenase gene in biofilm forming Pseudomonas mendocina EGD-AQ5 under increasing benzoate stress

Chlorhexidine, an antimicrobial with a broad inhibitory spectrum, is commonly used to treat oral infections as an active ingredient in mouthwash. While typically used at high concentrations (1–2 mg/ml), oral bacteria can be exposed to sublethal concentrations due to the bioavailability and protective barrier of biofilms (dental plaques). Sublethal concentrations can cause transcriptional remodelling of bacteria such as Streptococcus mutans, a key player in dental caries. Using an RNA-seq approach, this report provides a compendium on the effect of sublethal concentrations of chlorhexidine on the transcriptome of S. mutans as planktonic cells and in biofilm states. Streptococcus mutans showed major transcriptional remodelling between planktonic and biofilm states. The transcriptional response towards chlorhexidine was more pronounced in planktonic cells compared to sessile cells. However, the response observed for biofilm-associated cells was not specific to chlorhexidine, as the transcriptional response in biofilms exposed to the β-lactam amoxicillin was similar to those observed for chlorhexidine. Furthermore, we found that S. mutans modulates the transcription of a multitude of ABC transporters in both planktonic and biofilm-associated cells upon exposure to these antimicrobials.

Sodium hypochlorite (NaOCl) is widely recognized for its broad-spectrum antimicrobial efficacy in skin wound care. This study investigates the effectiveness of NaOCl against a range of bacterial and fungal isolates from pressure ulcer (PU) patients. We analyzed 20 bacterial isolates from PU patients, comprising carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), along with 5 Candida albicans isolates. Antibiotic resistance profiles were determined using standard susceptibility testing. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARGs) and disinfectant resistance genes (DRGs). Genetic determinants of biofilm formation were also assessed. The antimicrobial activity of NaOCl was evaluated by determining the minimum inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for both planktonic and biofilm-associated cells. CRKP and MDRAB showed resistance to fluoroquinolones and carbapenems, while MRSA exhibited resistance to β-lactams and levofloxacin. MSSA displayed a comparatively lower resistance profile. WGS identified significant numbers of ARGs in CRKP and MDRAB, with fewer DRGs compared to MRSA and MSSA. All isolates possessed genes associated with fimbriae production and adhesion, correlating with pronounced biofilm biomass production. NaOCl demonstrated substantial antimicrobial activity against both planktonic cells and biofilms. The MIC90 for planktonic bacterial cells was 0.125 mg/mL, and the MBEC90 ranged from 0.225 to 0.5 mg/mL. For planktonic C. albicans, the MIC90 was 0.150 mg/mL, and the MBEC90 was 0.250 mg/mL. These results highlight the challenge in treating biofilm-associated infections and underscore the potential of NaOCl as a robust antimicrobial agent against difficult-to-treat biofilm infections at concentrations lower than those typically found in commercial disinfectants.

The virulence of Candida albicans is conditioned by several virulence factors, one of which is the formation of biofilm which reduces the sensitivity of the yeast to conventional antimycotics. This study determines the antifungal and antibiofilm activity of five essential oils (EOs) of the Lamiaceae family: Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, and Hyssopus officinalis. In the preliminary research, the antifungal effect of eachof the EOs was tested in the concentration range of 200-0.4 mg/mL on planktonic Candida albicans (C. albicans) cells. A total of 13 C. albicans clinical isolates and one reference strain were evaluated on biofilm formation. Nine isolates (69.2%) showed weak biofilm production and four strains (30.8%) were detected as moderate biofilm producers. The EOs of Thymus vulgaris and Origanum vulgare were seen as effective antifungal agents on planktonic cells with the MIC 0.4 mg/mL. The highest average MIC values were recorded in Salvia officinalis EO (24.0 and 14.8 mg/mL). All isolates were used to determine EOs efficacy on the inhibition of adherence phase and biofilm formation. The biofilm production of C. albicans after exposition by EOs was quantitatively examined by crystal violet dye. The most effective for adherence phase and biofilm formation were EOs of Origanum vulgare (0.1 mg/mL and 0.3 mg/mL) and Thymus vulgaris (0.1 mg/mL and 0.4 mg/mL). The obtained results show that EOs of Thymus vulgaris and Origanum vulgare are potential agents for antifungal treatment or prophylaxis by reducing the resistance of pathogen.

Fungal biofilm formation on voice prosthesis (VP) is a major health problem that requires repeated replacement of the prosthesis. Candida albicans is one of the pathogens that frequently inhabits the VP. We proposed that coating VPs with sustained-release varnish (SRV) containing clotrimazole (CTZ) might prevent fungal biofilm formation. The long-term antifungal activities of SRV-CTZ- versus SRV-placebo-coated VPs was tested daily by measuring the inhibition zone of C. albicans seeded on agar plates or by measuring the fungal viability of C. albicans in suspension. The extent of biofilm formation on coated VPs was analyzed by confocal microscopy and scanning electron microscopy. We observed that SRV-CTZ-coated VPs formed a significant bacterial inhibition zone around the VPs and prevented the growth of C. albicans in suspension during the entire testing period of 60 days. Fungal biofilms were formed on placebo-coated VPs, while no significant biofilms were observed on SRV-CTZ-coated VPs. HPLC analysis shows that CTZ is continuously released during the whole test period of 60 days at a concentration above the minimal fungistatic concentration. In conclusion, coating VPs with an SRV-CTZ film is a potential effective method for prevention of fungal infections and biofilm formation on VPs.

Fluconazole and biogenic silver nanoparticles-based nano-fungicidal system for highly efficient elimination of multi-drug resistant Candida biofilms

Salmonella has emerged as a well-recognized food-borne pathogen, with many strains able to form biofilms and thus cause cross-contamination in food processing environments where acid-based disinfectants are widely encountered. In the present study, RNA sequencing was employed to establish complete transcriptome profiles of Salmonella Enteritidis in the forms of planktonic and biofilm-associated cells cultured in Tryptic Soytone Broth (TSB) and acidic TSB (aTSB). The gene expression patterns of S. Enteritidis significantly differed between biofilm-associated and planktonic cells cultivated under the same conditions. The assembled transcriptome of S. Enteritidis in this study contained 5,442 assembled transcripts, including 3,877 differentially expressed genes (DEGs) identified in biofilm and planktonic cells. These DEGs were enriched in terms such as regulation of biological process, metabolic process, macromolecular complex, binding and transferase activity, which may play crucial roles in the biofilm formation of S. Enteritidis cultivated in aTSB. Three significant pathways were observed to be enriched under acidic conditions: bacterial chemotaxis, porphyrin-chlorophyll metabolism and sulfur metabolism. In addition, 15 differentially expressed novel non-coding small RNAs (sRNAs) were identified, and only one was found to be up-regulated in mature biofilms. This preliminary study of the S. Enteritidis transcriptome serves as a basis for future investigations examining the complex network systems that regulate Salmonella biofilm in acidic environments, which provide information on biofilm formation and acid stress interaction that may facilitate the development of novel disinfection procedures in the food processing industry.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. It is also a model organism for bacterial biofilm formation. Acute infections are often associated with planktonic or free-floating cells, high virulence and fast growth. Conversely, chronic infections are often associated with the biofilm mode of growth, low virulence and slow growth that resembles that of planktonic cells in stationary phase. Biofilm formation and type III secretion have been shown to be reciprocally regulated, and it has been suggested that factors related to acute infection may be incompatible with biofilm formation. In a previous proteomic study of the interrelationships between planktonic cells, colonies and continuously grown biofilms, we showed that biofilms under the growth conditions applied are more similar to planktonic cells in exponential phase than to those in stationary phase. In the current study, we investigated how these conditions influence the production of virulence factors using a transcriptomic approach. Our results show that biofilms express the type III secretion system, whereas planktonic cells do not. This was confirmed by the detection of PcrV in the cellular and secreted fractions of biofilms, but not in those of planktonic cells. We also detected the type III effector proteins ExoS and ExoT in the biofilm effluent, but not in the supernatants of planktonic cells. Biofilm formation and type III secretion are therefore not mutually exclusive in P. aeruginosa, and biofilms could play a more active role in virulence than previously thought.

Candida albicans lives as a commensal in most healthy humans but can cause superficial skin infections to life-threatening systemic infections. C. albicans also forms biofilms on biotic and abiotic surfaces. Biofilm cells are difficult to treat and highly resistant to antifungals. A specific set of genes is differentially regulated in biofilm cells as compared to free-floating planktonic cells of C. albicans. In this study, we addressed how a variant histone H3VCTG, a previously identified negative regulator of biofilm formation, modulates gene expression changes. By providing compelling evidence, we show that biased eviction of H3VCTG nucleosomes at the promoters of biofilm-relevant genes facilitates the accessibility of both transcription activators and repressors to modulate gene expression. Our study is a comprehensive investigation of genome-wide nucleosome occupancy in both planktonic and biofilm states, which reveals transition to an open chromatin landscape during biofilm mode of growth in C. albicans, a medically relevant pathogen.

Tyrosol and farnesol are quorum-sensing molecules produced by Candida albicans which accelerate and block, respectively, the morphological transition from yeasts to hyphae. In this study, we have investigated the secretion of tyrosol by C. albicans and explored its likely role in biofilm development. Both planktonic (suspended) cells and biofilms of four C. albicans strains, including three mutants with defined defects in the Efg 1 and Cph 1 morphogenetic signaling pathways, synthesized extracellular tyrosol during growth at 37 degrees C. There was a correlation between tyrosol production and biomass for both cell types. However, biofilm cells secreted at least 50% more tyrosol than did planktonic cells when tyrosol production was related to cell dry weight. The addition of exogenous farnesol to a wild-type strain inhibited biofilm formation by up to 33% after 48 h. Exogenous tyrosol appeared to have no effect, but scanning electron microscopy revealed that tyrosol stimulated hypha production during the early stages (1 to 6 h) of biofilm development. Experiments involving the simultaneous addition of tyrosol and farnesol at different concentrations suggested that the action of farnesol was dominant, and 48-h biofilms formed in the presence of both compounds consisted almost entirely of yeast cells. When biofilm supernatants were tested for their abilities to inhibit or enhance germ tube formation by planktonic cells, the results indicated that tyrosol activity exceeds that of farnesol after 14 h, but not after 24 h, and that farnesol activity increases significantly during the later stages (48 to 72 h) of biofilm development. Overall, our results support the conclusion that tyrosol acts as a quorum-sensing molecule for biofilms as well as for planktonic cells and that its action is most significant during the early and intermediate stages of biofilm formation.

Growth promotion of greenhouse tomatoes with Pseudomonas sp. and Bacillus sp. biofilms and planktonic cells