Searching for mechanisms of action of antimicrobials.

Although development of antimicrobial resistance by bacteria is a natural phenomenon, the antibiotic resistance crisis is a reality that leads us in a gap of antimicrobial alternatives on therapeutics. Considering this cruel reality and committed to contribute to look for new antibacterial agents, this manuscript presents a review of easy laboratory methods that evaluate the mode of action of compounds, since it is a basic requirement for the discovery and development of new drugs. The literature was screened by searching the keywords "mode of action", "antibiotic", "antimicrobial activity", "inhibition mode", "method" and "bacteria" in Pubmed, Scopus, Science Direct and BVS in a period of time from 2000 to 2019. This review demonstrates the wide variety of methods that can be employed in research on mechanisms of action of antibacterial substances. Therefore, we compiled different protocols (presented in the supplementary material), available in the literature, with the purpose of facilitating the start of experiments.

Combined potential of copaifera officinalis oleoresin and chitosan against oral pathogens.

Copaifera officinalis Oleoresin (COR) and Chitosan (CH) were combined to test the potential to inhibit oral bacteria. First, COR was analyzed by GC-MS to identify its main constituents and then Minimum Inhibitory Concentration (MIC) assays and Minimum Microbiocidal Concentration (MMC) of the compounds alone against 17 pathogens were performed. Sixteen primary compounds were identified in COR, but the major constituent was ß-Caryophyllene (40.5%). COR showed MIC concentrations of 26.04 to 46.87 µg/mL and CH 0.1 mg/mL to 0.8 mg/mL. Second, the combination against oral bacteria strains was tested using a checkerboard test with the determination of Fractional Inhibitory Concentration (FIC) for synergistic effect, followed by the bacterial biofilm aggregation test using monospecies and mixed biofilm. The combination of COR and CH showed a synergistic effect for S. oralis (ATCC 10557) and an additive effect for the other strains tested, promoting bactericidal activity, as well as reducing the concentrations needed to cause bacterial inhibition. In addition, it showed good activity in inhibiting biofilm formation, with inhibition percentages close to Azithromycin. The results of this study highlight the synergistic potential of COR and CH combination as a promising strategy in the search for innovative antimicrobial therapies for infections related to oral bacterial biofilms.

Insights into the bioactive potential of the Amazonian species Acmella oleracea leaves extract: A focus on wound healing applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Acmella oleracea is traditionally used by Amazonian folks to treat skin and mucous wounds, influenza, cough, toothache, bacterial and fungal infections. Its phytoconstituents, such as alkylamides, phenolic compounds, and terpenes, are reported to produce therapeutic effects, which justify the medicinal use of A. oleracea extracts. However, the scientific evidence supporting the application A. oleracea bioactive products for wound treatment of remains unexplored so far. OBJECTIVE: This work aimed to characterize the phytochemical composition of methanolic extract of A. oleracea leaves (AOM) and to investigate their antioxidant, anti-inflammatory, antimicrobial and healing potential focusing on its application for wound healing. MATERIAL AND METHODS: The dried leaves from A. oleracea submitted to static maceration in methanol for 40 days. The phytochemical constitution of AOM was analyzed based on the total phenolic dosage method and by UFLC-QTOF-MS analysis. Antioxidant activity was assessed by DPPH and NO scavenging activities, as well as MDA formation, evaluation of ROS levels, and phosphomolybdenum assays. In vitro anti-inflammatory activities were assessed by reduction of NO, IL-6, and TNF-α production and accumulation of LDs in peritoneal macrophages cells. Antimicrobial activity was evaluated by determining MIC and MBC/MFC values against P. aeruginosa, E. coli, S. epidermidis, S. aureus and C. albicans, bacterial killing assay, and biofilm adhesion assessment. In vitro wound healing activity was determined by means of the scratch assay with L929 fibroblasts. RESULTS: Vanillic acid, quercetin, and seven other alkamides, including spilanthol, were detected in the UFLC-QTOF-MS spectrum of AOM. Regarding the biocompatibility, AOM did not induce cytotoxicity in L929 fibroblasts and murine macrophages. The strong anti-inflammatory activity was evidenced by the fact that AOM reduced the cellular production of inflammatory mediators IL-6, TNF-α, NO, and LDs in macrophages by 100%, 96.66 ± 1.95%, 99.21 ± 3.82%, and 67.51 ± 0.72%, respectively. The antioxidant effects were confirmed, since AOM showed IC50 values of 44.50 ± 4.46 and 127.60 ± 14.42 µg/mL in the DPPH and NO radical inhibition assays, respectively. Additionally, AOM phosphomolybdenium reducing power was 63.56 ± 13.01 (RAA% of quercetin) and 104.01 ± 21.29 (RAA% of rutin). Finally, in the MDA quantification assay, AOM showed 63,69 ± 3.47% of lipid peroxidation inhibition. It was also observed that the production of ROS decreased by 69.03 ± 3.85%. The MIC values of AOM ranged from 1000 to 125 µg/mL. Adhesion of S. aureus, P. Aeruginosa, and mixed biofilms was significantly reduced by 44.71 ± 4.44%, 95.50 ± 6.37 %, and 51.83 ± 1.50%, respectively. AOM also significantly inhibited the growth of S. aureus (77.17 ± 1.50 %) and P. aeruginosa (62.36 ± 1.01%). Furthermore, AOM significantly enhanced the in vitro migration of L929 fibroblasts by 97.86 ± 0.82% compared to the control (P < 0.05). CONCLUSIONS: This study is the first to report total antioxidant capacity and intracellular LD reduction by AOM. The results clearly demonstrated that AOM exerts potent anti-inflammatory, antioxidant, antimicrobial, and wound healing effects, encouraging its further investigation and promising application in wound treatment.

Maintenance of sterility in SMS packaging in dental environments: concern with biosafety

Healthcare services must be guided by biosafety practices and microbial control. This control is highly influenced by humidity, which directly impacts the maintenance of sterility of the materials used in the appointments. High concentration of moisture, in the form of aerosol, splashes and spills, is caused during dental care. During the COVID-19 times the contamination by aerosol and droplets worries greatly. Considering that it could cause harm to the sterility of an autoclaved material, especially in dental environments, the objective was to evaluate the behavior of SMS sterilization packages (Spunbonded / Meltblown / Spunbonded) against microbial penetration in an aqueous vehicle. SMS of three brands were challenged, equally divided into two groups: virgin and processed (subjected to a single autoclaving cycle). Each specimen was aseptically deposited on Macconkey agar. Subsequently, 5 µL of Escherichia coliATCC 25922 saline solution [108CFU mL-1] was deposited in center of the SMS specimen and the dish incubated at36°C/ 48h. Reading was performed by the presence or absence of bacterial growth typical of the species under the SMS, observed on the back of Petri dish. The lowest penetration rate observed was 60% for one of the brands in the virgin condition, and 75% for two brands in the processed condition. Statistical analysis showed an association between bacterial penetration and the evaluated group, this association being valid only in the virgin condition. The different SMS behave similarly in terms of resistance to bacterial penetration after being processed. The data show that moisture can assist in bacterial transport through sterilized SMS. Therefore, SMS packages are not able to prevent bacterial penetration, and possibly other microorganisms, when in aqueous vehicles, offering a potential risk of breaking the aseptic chain. Thus, care must be taken in routines for handling and storage sterile packaging.