Effect of Carvacrol and Thymol on NorA efflux pump inhibition in multidrug-resistant (MDR) Staphylococcus aureus strains.

Undue exposure to antimicrobials has led to the acquisition and development of sophisticated bacterial resistance mechanisms, such as efflux pumps, which are able to expel or reduce the intracellular concentration of various antibiotics, making them ineffective. Therefore, inhibiting this mechanism is a promising way to minimize the phenomenon of resistance in bacteria. In this sense, the present study sought to evaluate the activity of the Carvacrol (CAR) and Thymol (THY) terpenes as possible Efflux Pump Inhibitors (EPIs), by determining the Minimum Inhibitory Concentration (MIC) and the association of these compounds in subinhibitory concentrations with the antibiotic Norfloxacin and with Ethidium Bromide (EtBr) against strains SA-1199 (wild-type) and SA-1199B (overexpresses NorA) of Staphylococcus aureus. In order to verify the interaction of the terpenes with the NorA efflux protein, an in silico molecular modeling study was carried out. The assays used to obtain the MIC of CAR and THY were performed by broth microdilution, while the Efflux Pump inhibitory test was performed by the MIC modification method of the antibiotic Norfloxacin and EtBr. docking was performed using the Molegro Virtual Docker (MVD) program. The results of the study revealed that CAR and THY have moderate bacterial activity and are capable of reducing the MIC of Norfloxacin antibiotic and EtBr in strains of S. aureus carrying the NorA efflux pump. The docking results showed that these terpenes act as possible competitive NorA inhibitors and can be investigated as adjuvants in combined therapies aimed at reducing antibiotic resistance.

Evaluation of antibacterial and modifying action of catechin antibiotics in resistant strains.

Diseases caused by bacteria are today one of the great problems of present time. Access to over-the-counter medication, the indiscriminate use of antibacterials and high rates of hospitalization have contributed to the increase in cases. This picture has led to the search for new alternative drugs. Thus, secondary metabolites have been reported as a possible treatment option, being evidenced in many researches to ascertain their combination with existing drugs. This research aimed to evaluate the antibacterial effect and the antibiotic activity modifying action of the catechin compound against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus multiresistant strains. The determination of the Minimum Inhibitory Concentration (MIC) and the evaluation of the antimicrobial and potentiating effect were performed by broth microdilution. The MIC obtained forcatechin against all the used strains was indicated as not clinically relevant. The combination of catechin and antibacterial drugs, both Gram-negative and Gram-positive, was synergistic and antagonistic in some drugs. Therefore, phenolic compounds can assist in the fight against infections caused by bacteria.

HPLC-DAD analysis and antimicrobial activities of Spondias mombin L. (Anacardiaceae).

Spondias mombin is used in the folk medicine for the treatment of diarrhea and dysentery, indicating that extracts obtained from this species may present pharmacological activities against pathogenic microorganisms. The purpose of this work was to investigate the chemical composition and evaluate the antimicrobial activity of extracts obtained from the leaves (aqueous) and bark (hydroethanolic) of S. mombin both as single treatments and in combination with conventional drugs. Following a qualitative chemical prospection, the extracts were analyzed by HPLC-DAD. The antimicrobial activities were evaluated by microdilution. The combined activity of drugs and extracts was verified by adding a subinhibitory concentration of the extract in the presence of variable drug concentrations. The Minimum Fungicidal Concentration (MFC) was determined by a subculture of the microdilution test, while the effect of the in vitro treatments on morphological transition was analyzed by subculture in moist chambers. While the qualitative analysis detected the presence of phenols and flavonoids, the HPLC analysis identified quercetin, caffeic acid, and catechin as major components in the leaf extract, whereas kaempferol and quercetin were found as major compounds in the bark extract. The extracts showed effective antibacterial activities only against the Gram-negative strains. With regard to the combined activity, the leaf extract potentiated the action of gentamicin and imipenem (against Staphylococcus aureus), while the bark extract potentiated the effect of norfloxacin (against S. aureus), imipenem (against Escherichia coli), and norfloxacin (against Pseudomonas aeruginosa). A more significant antifungal (fungistatic) effect was achieved with the bark extract (even though at high concentrations), which further enhanced the activity of fluconazole. The extracts also inhibited the emission of filaments by Candida albicans and Candida tropicalis. Together, these findings suggest that that the extract constituents may act by favoring the permeability of microbial cells to conventional drugs, as well as by affecting virulence mechanisms in Candida strains.

Evaluation of isoeugenol in inhibition of Staphylococcus aureus efflux pumps and their toxicity using Drosophila melanogaster model.

The Staphylococcus aureus bacteria is a pathogen considered opportunistic and that has been acquiring resistance to several classes of antibiotics, mainly due to the synthesis of efflux pumps, which are proteins that expel these drugs intracellularly, reducing their effectiveness. The objective of this study was to evaluate the ability of isoeugenol to inhibit S. aureus efflux pumps and to determine its toxicity against a eukaryotic model (Drosophila melanogaster). IS-58, K2068 and K4414 S. aureus strains were used in the study. Isoeugenol minimum inhibitory concentration (MIC) and antibiotic modulation were evaluated in efflux pump inhibitory tests as well as in ethidium bromide (EtBr) assays. Toxicity tests against D. melanogaster assessed mortality and negative geotaxis. Isoeugenol obtained a relevant MIC result and a synergism was observed when isoeugenol was associated with the antibiotics, mainly with ciprofloxacin. Isoeugenol was able to affect all three efflux pumps tested, especially in strain K4414. The mortality of D. melanogaster caused by isoeugenol administration started after 12 h of exposure, being volume dependent and having an LC50 of 81.69 µL/L. In the negative geotaxis test, a statistical difference was observed after 24h of exposure compared to the control, demonstrating that damage to the locomotor apparatus had occurred. Based on the results, isoeugenol is a putative efflux pump inhibitor, becoming an alternative in blocking these proteins, and demonstrated acute toxicity against D. melanogaster.

Chemical composition, antifungal activity and potential anti-virulence evaluation of the Eugenia uniflora essential oil against Candida spp.

The development of fungal resistance to antifungal drugs has been worsening over the years and as a result research on new antifungal agents derived from plants has intensified. Eugenia uniflora L. (pitanga) has been studied for its various biological actions. In this study the chemical composition and antifungal effects of the E. uniflora essential oil (EULEO) were investigated against Candida albicans (CA), Candida krusei (CK) and Candida tropicalis (CT) standard strains. The essential oil obtained through hydro-distillation was analyzed by gas chromatography coupled to mass spectrometry (GC-MS). To determine the IC50 of the oil, the cellular viability curve and the inhibitory effects were measured by means of the oil's association with Fluconazole in a broth microdilution assay with spectrophotometric readings. The Minimum Fungicidal Concentration (MFC) was determined by solid medium subculture with the aid of a guide plate while the assays used to verify morphological changes emerging from the action of the fractions were performed in microculture chambers at concentrations based on the microdilution. Two major oil constituents stand out from the chemical analysis: selina-1,3,7(11)-trien-8-one (36.37%) and selina-1,3,7(11)-trien-8-one epoxide (27.32%). The concentration that reduced microorganismal growth was ≥8,192 µg/mL while the IC50 varied, this being between 1892.47 and 12491.80 µg/mL (oil), 10.07 - 80.78 µg/mL (fluconazole) and 18.53 - 295.60 µg/mL (fluconazole + oil). The combined activity (fluconazole + oil) resulted in indifference and antagonism. A MFC of the oil in association with fluconazole was recorded at the concentration of 8,192 µg/mL against CA and CK. The oil caused the inhibition of CA and CT morphological transition. In view of the results obtained, additional research is needed to elucidate the activity of the E. uniflora oil over genetic and biochemical processes regarding its effect on Candida spp. virulence.

Toxicity against Drosophila melanogaster and antiedematogenic and antimicrobial activities of Alternanthera brasiliana (L.) Kuntze (Amaranthaceae).

Bioactive phytocompounds are studied by several bioactivities demonstrated, as their cytotoxic effects. The aim of this work was to evaluate the phytochemical profile, the toxic effect using the Drosophila melanogaster animal model and the anti-inflammatory and antimicrobial effect of the Alternanthera brasiliana (EEAB) ethanol extract. The phytochemical profile was performed using HPLC. The cytotoxic effect was evaluated in vivo using D. melanogaster. The anti-inflammatory effect was determined by neurogenic and antiedematogenic assays, and the antimicrobial activity was assayed using a microdilution method to determine the minimum inhibitory concentration (MIC) of the EEAB alone and in association with antibiotics. The main compound identified on the EEAB was luteolin (1.93%). Its cytotoxic effect was demonstrated after 24 h in the concentrations of 10, 20 and 40 mg/mL. The extract demonstrated an antiedematogenic effect, with a reduction of the edema between 35.57 and 64.17%. The MIC of the extract was ≥1.024 µg/mL, thus being considered clinically irrelevant. However, when the EEAB was associated with gentamicin, a synergism against all bacterial strains assayed was observed: Staphylococcus aureus (SA10), Escherichia coli (EC06) and Pseudomonas aeruginosa (PA24). Due to these results, the EEAB demonstrated a low toxicity in vivo and anti-inflammatory and synergistic activities. These are promising results, mainly against microbial pathogens, and the compounds identified can be a source of carbon backbones for the discovery and creation of new drugs.