Candida albicans as an Essential “Keystone” Component within Polymicrobial Oral Biofilm Models?

Background: Existing standardized biofilm assays focus on simple mono-species or bacterial-only models. Incorporating Candida albicans into complex biofilm models can offer a more appropriate and relevant polymicrobial biofilm for the development of oral health products. Aims: This study aimed to assess the importance of interkingdom interactions in polymicrobial oral biofilm systems with or without C. albicans, and test how these models respond to oral therapeutic challenges in vitro. Materials and Methods: Polymicrobial biofilms (two models containing 5 and 10 bacterial species, respectively) were created in parallel in the presence and absence of C. albicans and challenged using clinically relevant antimicrobials. The metabolic profiles and biomasses of these complex biofilms were estimated using resazurin dye and crystal violet stain, respectively. Quantitative PCR was utilized to assess compositional changes in microbial load. Additional assays, for measurements of pH and lactate, were included to monitor fluctuations in virulence “biomarkers.” Results: An increased level of metabolic activity and biomass in the presence of C. albicans was observed. Bacterial load was increased by more than a factor of 10 in the presence of C. albicans. Assays showed inclusion of C. albicans impacted the biofilm virulence profiles. C. albicans did not affect the biofilms’ responses to the short-term incubations with different treatments. Conclusions: The interkingdom biofilms described herein are structurally robust and exhibit all the hallmarks of a reproducible model. To our knowledge, these data are the first to test the hypothesis that yeasts may act as potential “keystone” components of oral biofilms.

Effects of resveratrol on the growth and enzyme production of Stenotrophomonas maltophilia: a burn wound pathogen

Objective: The purpose of this study was to identify the potential of resveratrol in inhibiting the growth and production of two enzymes, hyaluronidase and protease, in Stenotrophomonas maltophilia, which has become a burn wound pathogen of great significance. Method: Stenotrophomonas maltophilia (ATCC 17666) was cultured in nutrient broth and the microbial load was standardised to 0.5 McFarland standard at 600nm. The study included antimicrobial assays (well diffusion and resazurin dye binding method), hyaluronidase expression regulation assay (hyaluronic acid hydrolysis assay and turbidity assay) and protease expression regulation assay (casein hydrolysis assay and determination of specific activity of protease using tyrosine standard). Results: The minimum inhibitory concentration (MIC) of resveratrol against Stenotrophomonas maltophilia was found to be 125µg/ml. Hyaluronidase production in the organism treated with resveratrol was found to be half that in the untreated organism. The specific activity of protease produced by the organism treated with resveratrol was found to be one-quarter that in the untreated organism, as analysed by the tyrosine standard estimation protocol. Conclusion: Resveratrol was found to be a potent compound to treat Stenotrophomonas maltophilia infections. In addition to the antimicrobial and enzyme-regulatory properties of resveratrol, it also shows anti-oxidant and anti-inflammatory properties. This finding has great scope clinically as resveratrol may prove to be an ideal drug to treat burn wound infections.

In Vitro Assessment of the Antioxidant and Antitumor Potentials of Biogenic Silver Nanoparticle

Silver nanoparticles (AgNPs) were biosynthesized using the cell free supernatant of putative probiotic Lactobacillus paracasei A26. Several biological activities of biogenic AgNPs were investigated in respect to in vitro anti-oxidant and anti-tumor potentials. Anti-oxidant potentials were screened in terms of free radical scavenging activity against two free radicals, 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and resazurin dye. AgNPs exhibited a potent scavenging activity against resazurin dye (91±0.046%) with an EC50 concentration of 146.823 µg/ml, while scavenging of DPPH was significantly (P≤0.05) reduced to 72.330±0.114% using a higher EC50 concentration of 176.12 µg/ml. The anti-tumor potentials of biogenic AgNPs were studied in relation to the cytotoxicity against two human breast cancer cell lines (CAL-51 and MCF7), using crystal violet dye assay. The viability of AgNPs-treated cancerous cells was significantly decreased in a time- and concentration manner, as compared to insignificant cytotoxic effects against the normal cell line. However, the anti-proliferative activity of AgNPs did not exceed the value of 63.85±0.019% in both cancer cell lines. CAL-51 cells were the most sensitive to the introduced AgNPs, with a maximum decrease in viability of 49.889±0.021% being reached using an IC50 value of 98.65µg/ml for 48h exposure time. The inhibition percentage was increased to 60.13±0.005% when the used IC50 value was significantly declined to 40.73µg/ml with an exposure time expanded to 72h. MCF7 cells showed lower sensitivity than CAL-51 cells, but with a similar inhibition trend of 59.523±0.01% with an IC50 concentration of 66.54 µg/ml for 48h which was increased to 63.857±0.019% when the IC50 was reduced to 62.63 µg/ml and the exposure time expanded to 72h. The morphological changes of AgNPs-treated cells were apparent at 72h exposure time, with cells showing apoptotic-like features such as shrinkage and losing of regular fusiform shape. Moreover, cells became detached to surfaces and from each other.

Antifungal activity of paracloacal gland secretion of Caiman yacare (DAUDIN, 1802)

The objective of this study was to evaluate the in vitro antifungal activity of ethanolic and aqueous extracts obtained from Caiman yacare paracloacal gland (PG) against Candida albicans (ATCC 10231), as well as a bibliographical survey on the chemical composition of exudates of PG. The PG were collected from the disposal generated during the slaughter of C. yacare by regularized industry. Two extracts were made from these glands, one ethanolic and the other aqueous. The Minimal Inhibitory Concentration (MIC) of the substances were determined by dilution of the extract in series using the microdilution technique in the culture medium Sabouraud broth, carried out in a 96-well microplate visually read after 48 hours of incubation, confirmed by the method using 0.01% aqueous resazurin dye. The ethanolic extract had MIC at the concentration of 25 µg / L. The Minimum Fungicidal Concentration (MFC) was determined by subcultures of MIC in Sabouraud agar medium. The ethanolic extract presented MFC at a concentration of 50 µg / mL. The aqueous extract showed no antifungal activity at the concentrations tested. This work is the first work to assess an activity of the PG secretion and reveals pharmacological potential in a local product previously discarded.

The effect of anthracene-based chalcone derivatives in the resazurin dye reduction assay mechanisms for the investigation of Gram-positive and Gram-negative bacterial and fungal infection

A novel series of anthracene-based chalcone derivatives were synthesized and evaluated in terms of their antibacterial and antifungal activities.

Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

ABSTRACTThe search for drugs that can kill replicating and nonreplicatingMycobacterium tuberculosisfaces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds againstM. tuberculosisunder conditions that prevent the replication ofM. tuberculosisoften involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicatingM. tuberculosismay arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whetherM. tuberculosisbacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.