Microbial resistance to sanitizers in the food industry: review.

Hygiene programs which comprise the cleaning and sanitization steps are part of the Good Hygiene Practices (GHP) and are considered essential to ensure food safety and quality. Inadequate hygiene practices may contribute to the occurrence of foodborne diseases, development of microbial resistance to sanitizers, and economic losses. In general, the sanitizer resistance is classified as intrinsic or acquired. The former is an inherent characteristic, naturally present in some microorganisms, whereas the latter is linked to genetic modifications that can occur at random or after continuous exposure to a nonnormal condition. The resistance mechanisms can involve changes in membrane permeability or in the efflux pump, and enzymatic activity. The efflux pump mechanism is the most elucidated in relation to the resistance caused by the use of different types of sanitizers. In addition, microbial resistance to sanitizers can also be favored in the presence of biofilms due to the protection given by the glycocalyx matrix and genetic changes. Therefore, this review aimed to show the main microbial resistance mechanisms to sanitizers, including genetic modifications, biofilm formation, and permeability barrier.

A review of the unsolvable task in dog communication and cognition: comparing different methodologies.

Communication between dogs and humans is a topic of growing interest, and the "unsolvable task" is a common method used to measure human-directed communication. In this task, dogs learn how to solve a problem to obtain a reward. After a fixed number of trials, the reward becomes impossible to access, arguably leading to communicative attempts from the dog. Although useful to observe dogs' communicative behaviors in a fairly naturalistic situation, the methodology varies among studies regarding apparatus, number of trials, and other factors. The proxies used, for instance, gaze duration or frequency of gaze alternation, also vary, and there are discrepancies and a debate regarding what the task actually measures. Therefore, in this study, we reviewed the usage of the unsolvable task in canids of the genus Canis, searching Web of Science and Scopus for the terms "dog*", "Canis", "dingo*", "wolf" or "wolves" in the title and "unsolvable task" or "impossible task" in the topic. We included thirty-five studies in this review and discussed their different methodologies and proxies, such as different apparatuses, number of solvable trials, and different interpretations of "looking back", pointing out how they can affect results and hinder comparisons. Lastly, we used current data to propose strategies to homogenize the use of this important paradigm, with an ethogram of possible behaviors and their interpretation and a predefined set of methodological aspects for future research.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 µm; truncated with protruding hilum and varied in length from 1.0 to 1.5 µm and diameters from 2.0 to 2.2 µm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

Antifungal activity of geraniol and citronellol, two monoterpenes alcohols, against Trichophyton rubrum involves inhibition of ergosterol biosynthesis.

CONTEXT: Trichophyton rubrum is the most common fungus causing chronic dermatophytosis in humans. Antifungal activity of promising agents is of great interest. Geraniol and citronellol are monoterpenes with antimicrobial properties. OBJECTIVE: This study aimed to investigate the inhibitory effects and possible mechanism of antifungal activity of geraniol and citronellol against strains of T. rubrum. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of each drug against 14 strains was determined by broth microdilution. The effects of the drugs on dry mycelial weight, conidial germination, infectivity on human nail fragments, and morphogenesis of T. rubrum were analyzed. The effects on the cell wall (test with sorbitol) and cell membrane (release of intracellular material and ergosterol biosynthesis) were investigated. RESULTS: MIC values of geraniol ranged between 16 and 256 µg/mL while citronellol showed MIC values from 8 to 1024 µg/mL. The drugs (MIC and 2 × MIC) inhibited the mycelial growth, conidia germination, and fungal growth on nail fragments. The drugs (half of MIC) induced the formation of wide, short, and crooked hyphae in T. rubrum morphology. With sorbitol, geraniol MIC was increased by 64-fold and citronellol by 32-fold. The drugs caused leakage of intracellular material and inhibited ergosterol biosynthesis. DISCUSSION: The results suggest that the drugs damage cell wall and cell membrane of T. rubrum through a mechanism that seems to involve the inhibition of the ergosterol biosynthesis. CONCLUSION: This study confirms that geraniol and citronellol can be regarded as potential drugs for controlling T. rubrum growth, with great potential against agents of dermatophytosis.

Metronidazole alone or with amoxicillin as adjuncts to non-surgical treatment of chronic periodontitis: a secondary analysis of microbiological results from a randomized clinical trial.

AIM: To evaluate the changes occurring in the subgingival microbial profile of subjects with generalized chronic periodontitis (ChP) treated by scaling and root planing (SRP) alone or with metronidazole (MTZ) or MTZ + amoxicillin(AMX). A secondary aim was to examine a possible added effect of chlorhexidine(CHX) to these therapies. METHODS: One hundred and eighteen subjects were randomly assigned to receive SRP-only or with MTZ [400 mg/thrice a day (TID)] or MTZ + AMX (500 mg/TID) for 14 days. Half of the subjects in each group rinsed with 0.12% CHX twice a day (BID) for 2 months. Nine subgingival plaque samples/subject were analysed by checkerboard DNA­DNA hybridization for 40 bacterial species at baseline, 3, 6 and 12 months post-therapy. RESULTS: At 12 months, the antibiotic-treated groups harboured lower mean counts and proportions of key periodontal pathogens than the SRP group (p < 0.05). These benefits were observed at initially deep and shallow sites. Initial reductions in periodontal pathogens obtained with SRP partially rebound after 12 months. CHX rinsing enhanced the microbiological effects of the MTZ + AMX treatment in shallow sites. CONCLUSION: The adjunctive use of MTZ and MTZ + AMX results in a greater reduction in the levels of periodontal pathogens in generalized ChP subjects compared to SRP alone.

Membranes composed of poly(lactic acid)/poly(ethylene glycol) and Ora-pro-nóbis (Pereskia aculeata Miller) extract for dressing applications.

Wounds are considered one of the most critical medical conditions that must be managed appropriately due to the psychological and physical stress they cause for patients, as well as creating a substantial financial burden on patients and global healthcare systems. Nowadays, there is a growing interest in developing nanofiber mats loaded with varying plant extracts to meet the urgent need for advanced wound ressings. This study investigated the development and characterization of poly(lactic acid) (PLA)/ poly(ethylene glycol) (PEG) nanofiber membranes incorporated with Ora-pro-nóbis (OPN; 12.5, 25, and 50 % w/w) by the solution-blow-spinning (SBS) technique. The PLA/PEG and PLA/PEG/OPN nanofiber membranes were characterized by scanning electron microscopy (SEM), thermal properties (TGA and DSC), Fourier transform infrared spectroscopy (FTIR), contact angle measurements and water vapor permeability (WVTR). In addition, the mats were analyzed for swelling properties in vitro cell viability, and fibroblast adhesion (L-929) tests. SEM images showed that smooth and continuous PLA/PEG and PLA/PEG/OPN nanofibers were obtained with a diameter distribution ranging from 171 to 1533 nm. The PLA/PEG and PLA/PEG/OPN nanofiber membranes showed moderate hydrophobicity (~109-120°), possibly preventing secondary injuries during dressing removal. Besides that, PLA/PEG/OPN nanofibers exhibited adequate WVTR, meeting wound healing requirements. Notably, the presence of OPN gave the PLA/PEG membranes better mechanical properties, increasing their tensile strength (TS) from 3.4 MPa (PLA/PEG) to 5.3 MPa (PLA/PEG/OPN), as well as excellent antioxidant properties (Antioxidant activity with approximately 45 % oxidation inhibition). Therefore, the nanofiber mats based on PLA/PEG, especially those incorporated with OPN, are promising options for use as antioxidant dressings to aid skin healing.

Dogs understand the role of a human partner in a cooperative task.

Humans are exceptionally flexible in cooperation, partly due to our ability to recognize the roles of cooperative partners. While some non-human animals understand the need for a partner in such interactions, it is unclear whether they grasp the consequences of their partner's actions and adjust accordingly. Previous studies utilizing economic games with non-human animals yielded mixed results. We investigated dogs, known for their close cooperation with humans, in a stag hunt game. Dogs could cooperate for better rewards or defect for lower ones, while their human partners would either cooperate, never cooperate, or act randomly. We control for attraction to food, side bias, and local enhancement. Dogs were more likely to coordinate with their partners when it led to better rewards, suggesting that they understood their partner's actions. By highlighting this cognitive skill in dogs, we advance our knowledge of the intricate mechanisms driving cooperative behavior in non-human animals.

Investigation on mechanism of antifungal activity of eugenol against Trichophyton rubrum.

Trichophyton rubrum is a worldwide agent responsible for chronic cases of dermatophytosis which have high rates of resistance to antifungal drugs. Attention has been drawn to the antimicrobial activity of aromatic compounds because of their promising biological properties. Therefore, we investigated the antifungal activity of eugenol against 14 strains of T. rubrum which involved determining its minimum inhibitory concentration (MIC) and effects on mycelial growth (dry weight), conidial germination and morphogenesis. The effects of eugenol on the cell wall (sorbitol protect effect) and the cell membrane (release of intracellular material, complex with ergosterol, ergosterol synthesis) were investigated. Eugenol inhibited the growth of 50% of T. rubrum strains employed in this study at an MIC = 256 µg/ml, as well as mycelial growth and conidia germination. It also caused abnormalities in the morphology of the dermatophyte in that we found wide, short, twisted hyphae and decreased conidiogenesis. The results of these studies on the mechanisms of action suggested that eugenol exerts antifungal effects on the cell wall and cell membrane of T. rubrum. Eugenol act on cell membrane by a mechanism that seems to involve the inhibition of ergosterol biosynthesis. The lower ergosterol content interferes with the integrity and functionality of the cell membrane. Finally, our studies support the potential use of the eugenol as an antifungal agent against T. rubrum.

Correction: Freitas et al. Bacterial Cellulose/Tomato Puree Edible Films as Moisture Barrier Structures in Multicomponent Foods. Foods 2022, 11, 2336.

In the original publication [...].

Obtaining poly (lactic acid) nanofibers encapsulated with peppermint essential oil as potential packaging via solution-blow-spinning.

The development of active packaging based on biodegradable material and incorporating active compounds, such as essential oil, is a new technique to ensure food safety without harming the environment. In this study, nanofiber mats of poly (lactic acid)/ polyethylene glycol (PLA/PEG) blend incorporated with peppermint essential oil (PO) at different ratios (5-20 % v/w) were produced by solution-blow-spinning (SBS) for potential packaging application. Electron microscopy showed a cylindrical and interlaced morphology for PLA/PEG/PO and a significant increase in the diameter (139-192 nm) of the nanofibers by increasing PO content. All nanofibers showed high thermal stability (278-345 °C) suitable for use in the food industry. Nuclear magnetic resonance (13C NMR) spectrum confirmed PO in the nanofibers after SBS. ATR-FTIR spectral analysis supported the chemical composition of the nanofiber mats. PO addition led to obtaining hydrophobic nanofibers, enhancing the contact angle to 122° and decreasing water vapor permeability (60 % reduction compared to the PLA/PEG (3.0 g.mm.kPa-1.h-1.m-2). Although the PLA/PEG/20%PO nanofibers did not show halo formation in 24 h, they effectively extended the strawberries' shelf-life at 25 °C, evidencing PO release over time. It also reduced weight loss (2.5 % and 0.3 % weight loss after 5 days for PLA/PEG and PLA/PEG/20%PO, respectively) and increased firmness (8-12 N) for strawberries packed with the nanofiber mats. It is suggested that PLA/PEG films incorporating PO may be used as an active, environmentally friendly packaging material.

Development of iron ore tailings based wood-cement composite panels.

This article evaluated the effect of using mining waste on the mechanical, physical, thermal, and microstructural properties of cement-wood panels. The percentages of 10, 20, 30, and 40% were evaluated as replacing cement with mining waste. The wood particles of Pinus oocarpa were evaluated for their chemical, anatomical, and physical characteristics, and the mining waste was evaluated for its granulometry, chemical composition, and inhibition index. The composites were evaluated for their physical-mechanical properties, X-ray diffraction and infrared spectroscopy analysis, microstructural, thermal, and durability. All evaluated treatments met the requirements of the Bison standard for MOR and MOE for static bending and internal bonding, even after accelerated aging. It was possible to conclude that it is feasible to replace 40% of cement with mining waste for the production of cement-wood panels in industrial scale.

Actigraphic characterization of sleep and circadian phenotypes of PER3 gene VNTR genotypes.

The molecular circadian timing system involves genes known as "clock genes," such as the PER3 gene. Studies have demonstrated associations among a repeat polymorphism (VNTR) of the PER3 gene with chronotypes, with the occurrence of circadian rhythm disorders and with sleep homeostasis phenotypes. The aim of this study was to investigate, by actigraphy, sleep and circadian rhythm profiles of people with different genotypes for the VNTR polymorphism of the PER3 gene. We genotyped 467 individuals (46,39% male) for the PER3 VNTR polymorphism. The mean age of the participants was 21.84 ± 2.64, ranging from 18 to 30 y old. Actigraphy data were collected from a subsample of 81 subjects with PER3 4-repeats homozygous (PER34/4) or 5-repeats homozygous (PER35/5) genotypes from April to June of 2021. From this sample, 48 PER34/4 and 33 PER35/5 subjects wore a wrist actigraph between 12 and 19 d. The sleep onset (weekdays, p = 0.015; weekend, p = 0.022) and offset (weekdays, p = 0.004; weekend, p = 0.041) of the PER35/5 group occurred later than the PER34/4 group. Similar results were observed for the mid-sleep phase of weekdays (MSW) (p = 0.008) and free days (MSF) (p = 0.019), and for the mid-sleep phase corrected for sleep debt accumulated over the week (MSFsc) (p = 0.024). Despite the phase differences found between the PER34/4 and PER35/5 groups, no differences were found in sleep duration and social jet lag. However, the PER34/4 group presented, on average, a longer sleep rebound on the days off when compared to the PER35/5 (p = 0.002). The PER35/5 group showed lower interdaily stability (IS) (p = 0.032) and higher daily activity rhythm variability (IV) (p = 0.035). The findings of the present study revealed associations between the PER3 gene, sleep, and circadian rhythms. In general, we found that the gene is associated with the expression of sleep timing and duration and to the phase of the activity rhythm. The experiments carried out here occurred in the COVID-19 pandemic scenario, which should be considered as an environmental element with potential effects on the results obtained.

Patients with type 2 diabetes and severe periodontitis harbor a less pathogenic subgingival biofilm than normoglycemic individuals with severe periodontitis.

BACKGROUND: Whether, and to what extent, diabetes mellitus (DM) can affect the subgingival biofilm composition remains controversial. Thus, the aim of this study was to compare the composition of the subgingival microbiota of non-diabetic and type 2 diabetic patients with periodontitis using 40 "biomarker bacterial species." METHODS: Biofilm samples of shallow (probing depth [PD] and clinical attachment level [CAL] ≤3 mm without bleeding) and deep sites (PD and CAL ≥5 mm with bleeding) of patients with or without type 2 DM were evaluated for levels/proportions of 40 bacterial species by checkerboard DNA-DNA hybridization. RESULTS: A total of 828 subgingival biofilm samples from 207 patients with periodontitis (118 normoglycemic and 89 with type 2 DM) were analyzed. The levels of most of the bacterial species evaluated were reduced in the diabetic compared with the normoglycemic group, both in shallow and in deep sites. The shallow and deep sites of patients with type 2 DM presented higher proportions of Actinomyces species, purple and green complexes, and lower proportions of red complex pathogens than those of normoglycemic patients (P < 0.05). CONCLUSIONS: Patients with type 2 DM have a less dysbiotic subgingival microbial profile than normoglycemic patients, including lower levels/proportions of pathogens and higher levels/proportions of host-compatible species. Thus, type 2 diabetic patients seem to require less remarkable changes in biofilm composition than non-diabetic patients to develop the same pattern of periodontitis.

Molecular detection of feline hemoplasmas and retroviruses in free-roaming and shelter cats within a university campus.

Objectives: The aim of the present study was to assess the frequency of hemoplasma, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections in cats living in an on-campus shelter and free-roaming cats within a university campus in Brazil. Methods: Blood samples were tested using quantitative PCR for hemoplasma, FIV and FeLV. Positive hemoplasma samples were sequenced. Associations between hemoplasma detection and living situation, sex, flea and/or tick parasitism, and coinfection with FIV and FeLV, were assessed using Fisher's exact test and the respective odds ratios were calculated. Results: Overall, 6/45 (13.3%) cats tested positive: four (8.9%) were infected with 'Candidatus Mycoplasma haemominutum' and two (4.4%) with Mycoplasma haemofelis. All positive samples were from free-roaming cats (6/15; 40.0%) and had statistically significantly lower packed cell volumes (P = 0.037). Although 5/23 (21.7%) males and 1/22 (4.6%) females were positive, no statistically significant association between sex and hemoplasma infection was found (P = 0.19). Viral quantitative PCR (qPCR) was performed on 43/45 samples, among which 2/43 (4.7%) were positive for FIV and none for FeLV. Only one cat (2.3%) was coinfected with hemoplasma and FIV (P = 0.26). In addition, 4/6 (66.7%) cats that tested positive for hemoplasmas were infested by fleas (P = 0.0014) and/or ticks (P = 0.25). Conclusions and relevance: These results show that even if the free-roaming cat population is clinically healthy and has adequate access to food, it may present flea infestation and hemoplasma infection with lower packed cell volume values.

Systemic Antibiotics and Chlorhexidine Associated with Periodontal Therapy: Microbiological Effect on Intraoral Surfaces and Saliva.

The effect of systemic antibiotics on the microbial profile of extracrevicular sites after periodontal treatment is currently the subject of research. This study evaluated the microbiological effects on different oral cavity sites of scaling and root planing (SRP) combined with antimicrobial chemical control in the treatment of periodontitis. Sixty subjects were randomly assigned to receive SRP alone or combined with metronidazole (MTZ) + amoxicillin (AMX) for 14 days, with or without chlorhexidine mouth rinse (CHX) for 60 days. Microbiological samples were evaluated by checkerboard DNA-DNA hybridization until 180 days post therapy. The adjunctive use of antibiotics plus CHX significantly reduced the mean proportions of red complex species from subgingival biofilm and saliva (p < 0.05). Furthermore, the analysis of all intraoral niches showed a significantly lower mean proportion of the red complex species in the same group. In conclusion, the concomitant use of antimicrobial chemical control (systemic and local) demonstrated a beneficial effect on the composition of the oral microbiota.

Species Identification and Mycotoxigenic Potential of Aspergillus Section Flavi Isolated from Maize Marketed in the Metropolitan Region of Asunción, Paraguay.

Zea mays var. amylacea and Zea mays var. indurata are maize ecotypes from Paraguay. Aspergillus section Flavi is the main spoilage fungus of maize under storage conditions. Due to its large intraspecific genetic variability, the accurate identification of this fungal taxonomic group is difficult. In the present study, potential mycotoxigenic strains of Aspergillus section Flavi isolated from Z. mays var. indurata and Z. mays var. amylacea that are marketed in the metropolitan region of Asunción were identified by a polyphasic approach. Based on morphological characters, 211 isolates were confirmed to belong to Aspergillus section Flavi. A subset of 92 strains was identified as Aspergillus flavus by mass spectrometry MALDI-TOF and the strains were classified by MALDI-TOF MS into chemotypes based on their aflatoxins and cyclopiazonic acid production. According to the partial sequencing of ITS and CaM genes, a representative subset of 38 A. flavus strains was confirmed. Overall, 75 A. flavus strains (86%) were characterized as producers of aflatoxins. The co-occurrence of at least two mycotoxins (AF/ZEA, FUM/ZEA, and AF/ZEA/FUM) was detected for five of the Z. mays samples (63%). Considering the high mycological bioburden and mycotoxin contamination, maize marketed in the metropolitan region of Asunción constitutes a potential risk to food safety and public health and requires control measures.

In vitro and ex vivo antibiofilm activity of riparin 1, and its nor and dinor homologs, against dermatophytes.

Dermatophytosis is one of the most frequent superficial mycoses in the world. They are mainly caused by the dermatophytes Trichophyton rubrum and Microsporum canis. Biofilm production is an essential factor in the pathogenesis of dermatophytes; it confers drug resistance and significantly impairs antifungal effectiveness. Therefore, we evaluated the antibiofilm activity of an alkamide-type alkaloid called riparin 1 (RIP1) against clinically relevant dermatophytes. We also produced synthetic nor (NOR1) and dinor (DINOR1) homologs for pharmacological evaluation, with a 61-70% yield. We used in vitro (96-well polystyrene plates) and ex vivo (hair fragments) models to verify the effects of these compounds on the formation and viability of biofilms. RIP1 and NOR1 showed antifungal activity against strains of T. rubrum and M. canis, but DINOR1 showed no significant antifungal activity against the dermatophytes. Furthermore, RIP1 and NOR1 significantly reduced the viability of biofilms in vitro and ex vivo (P < 0.05). RIP1 was more potent than NOR1, possibly due to the distance between the p-methoxyphenyl and the phenylamide moieties in these compounds. Due to the significant antifungal and antibiofilm activities observed for RIP1 and NOR1, we suggest that they could be useful in the treatment of dermatophytosis.

Antifungal and Antibiofilm Activity of Riparin III against Dermatophytes.

The ability of dermatophytes to develop biofilms is possibly involved in therapeutic failure because biofilms impair drug effectiveness in the infected tissues. Research to find new drugs with antibiofilm activity against dermatophytes is crucial. In this way, riparins, a class of alkaloids that contain an amide group, are promising antifungal compounds. In this study, we evaluated the antifungal and antibiofilm activity of riparin III (RIP3) against Trichophyton rubrum, Microsporum canis, and Nannizzia gypsea strains. We used ciclopirox (CPX) as a positive control. The effects of RIP3 on fungal growth were evaluated by the microdilution technique. The quantification of the biofilm biomass in vitro was assessed by crystal violet, and the biofilm viability was assessed by quantifying the CFU number. The ex vivo model was performed on human nail fragments, which were evaluated by visualization under light microscopy and by quantifying the CFU number (viability). Finally, we evaluated whether RIP3 inhibits sulfite production in T. rubrum. RIP3 inhibited the growth of T. rubrum and M. canis from 128 mg/L and N. gypsea from 256 mg/L. The results showed that RIP3 is a fungicide. Regarding antibiofilm activity, RIP3 inhibited biofilm formation and viability in vitro and ex vivo. Moreover, RIP3 inhibited the secretion of sulfite significantly and was more potent than CPX. In conclusion, the results indicate that RIP3 is a promising antifungal agent against biofilms of dermatophytes and might inhibit sulfite secretion, one relevant virulence factor.

α-Pinene: Docking Study, Cytotoxicity, Mechanism of Action, and Anti-Biofilm Effect against Candida albicans.

Candida albicans is associated with serious infections in immunocompromised patients. Terpenes are natural-product derivatives, widely studied as antifungal alternatives. In a previous study reported by our group, the antifungal activity of α-pinene against C. albicans was verified; α-pinene presented an MIC between 128-512 µg/mL. In this study, we evaluate time-kill, a mechanism of action using in silico and in vitro tests, anti-biofilm activity against the Candida albicans, and toxicity against human cells (HaCaT). Results from the molecular-docking simulation demonstrated that thymidylate synthase (-52 kcal mol-1), and δ-14-sterol reductase (-44 kcal mol-1) presented the best interactions. Our in vitro results suggest that α-pinene's antifungal activity involves binding to ergosterol in the cellular membrane. In the time-kill assay, the antifungal activity was not time-dependent, and also inhibited biofilm formation, while rupturing up to 88% of existing biofilm. It was non-cytotoxic to human keratinocytes. Our study supports α-pinene as a candidate to treat fungal infections caused by C. albicans.

Metronidazole alone or with amoxicillin as adjuncts to non-surgical treatment of chronic periodontitis: a 1-year double-blinded, placebo-controlled, randomized clinical trial.

AIM: To evaluate the effects of the adjunctive use of metronidazole (MTZ) or MTZ + amoxicillin (AMX) in the treatment of generalized chronic periodontitis (ChP). A secondary aim was to examine a possible added effect of chlorhexidine to these therapies. METHODS: One hundred and eighteen subjects received scaling and root planing (SRP) only or with MTZ [400 mg/thrice a day (TID)] or MTZ+AMX (500 mg/TID) for 14 days. Half of the subjects in each group rinsed with 0.12% chlorhexidine twice a day (BID) for 2 months. Subjects were clinically monitored at baseline, 3, 6 and 12-months post-therapy. RESULTS: The two antibiotic groups showed lower mean number of sites with probing depth (PD) ≥5 mm and fewer subjects exhibiting ≥9 of these sites at 1-year post-treatment. Logistic regression analysis showed that antibiotics were the only significant predictors of subjects presenting ≤4 sites with PD ≥5 mm at 1 year (MTZ+AMX: OR, 13.33; 95%CI, 3.75-47.39/p = 0.0000; MTZ: OR, 7.26; 95%CI, 2.26-23.30/p = 0.0004). The frequency of adverse events did not differ between the two antibiotic treatments (p > 0.05). The chlorhexidine subgroups showed a trend (p > 0.05) to present fewer residual sites ≥5 mm compared with the placebo subgroups at 1 year. CONCLUSION: Treatment of generalized ChP is significantly improved by the adjunctive use of MTZ+AMX and MTZ.