Virulence Factors and Biofilm Formation in Vancomycin Resistant Enterococcus faecalis and Enterococcus faecium Isolates in Brazil

In this work, we evaluated biofilm formation of Vancomycin Resistant of E. faecalis and E. faecium (VRE) in different culture media and adhesion substrate, as well as cellular hydrophobicity and presence of virulence genes. For this, 35 isolates were collected from a public hospital in Recife, Pernambuco, Brazil and identified by the Matrix-Assisted Laser Desorption Ionization - Time-of-flight - Mass Spectrometry (MALDI-TOF-MS) technique. Biofilm formation was analyzed by the Crystal Violet (CV) method and fluorescence microscopy, cellular hydrophobicity by hydrocarbon interaction and the presence of gelE, esp and asa1 genes by Polymerase Chain Reaction (PCR). 12 isolates were identified as E. faecalis and 23 as E. faecium. Most were obtained in Coronary Units (40.0%) and Intensive Care Unit (31.4%). E. faecium isolates were more resistant to the antibiotics tested than E. faecalis;however, E. faecalis stood out as a biofilm producer. Regarding the presence and gene frequency, it was observed that gelE (54.3%) and esp (54.3%) were the most prevalent, followed by asa1 (22.9%). When comparing the gene frequency, it was observed that gelE and esp were predominant (48.6% for both species), while asa1 was more frequent in E. faecalis (20.0%). The data presented here are worrying, because they reveal the virulence potential of isolates VRE, which contributes to the dissemination and persistence of these pathogens in the hospital environment.

HPLC-DAD analysis and antifungal effect of Hyptis martiusii Benth(Lamiaceae) against Candida strains

Objective: To evaluate the anti Candida activity of Hyptis martiusii decoction and its major compound, caffeic acid alone or in the presence of fluconazole, as well as their cytotoxic effect. Methods: The decoction was characterized using high performance liquid chromatography coupled with diode array detector. For the antifungal activity, the minimum inhibitory concentration(MIC) and the potential effect of the decoction with the fluconazole were evaluated by microdilution method using 96-well microtiter trays. The osmotic fragility test was performed using erythrocytes under saline stress. All tests were performed in triplicate. Results: The chemical characterization of the decoction was performed by high performance liquid chromatography and revealed the presence of seven compounds, including caffeic acid as major constituent. The antifungal tests demonstrated that both decoction(DHm) and caffeic acid obtained from Hyptis martiusii presented MIC and MFC ≥4096 μg/mL against Candida albicans and Candida tropicalis strains. However, in the presence of fluconazole, DHm and caffeic acid presented IC_(50) of 2.60 and 2.53 μg/mL respectively, demonstrating significant synergistic effects against Candida strains. The modulator activity of DHm might be due to the presence of caffeic acid. Moreover, DHm and caffeic acid did not cause significant hemolytic effects, indicating that they present low cytotoxicity. Conclusions: These data indicate that DHm potentiates the activity of the fluconazole, without enhancement of the toxicity, encouraging further toxicological, pharmacological and phytochemical studies to provide consistent evidence of the potential of this plant to be used in drug development.

Polyphenolic composition, antibacterial, modulator and neuroprotective activity of Tarenaya spinosa(Jacq.) Raf.(Cleomaceae)

Objective: To evaluate the antibacterial activity and neuroprotective capacity of the ethanolic and aqueous extracts of Tarenaya spinosa(T. spinosa) as well as to determine and quantify some of its polyphenols by high performance liquid chromatography with diode-array detection(HPLC-DAD). Methods: The bacterial Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa strains, grown in Heart Agar Infusion, were tested. The drugs gentamicin, norfloxacin and imipenem were used to evaluate the modulating or antagonistic capacity of the T. spinosa extracts. The extract was analysed by HPLC-DAD to determine the main phenolic compounds. For the cell viability tests, individual heads of the Nauphoeta cinerea arthropod model were removed, homogenized in Trifluoromethyl ketone and centrifuged afterwards. Subsequently, 20 μL of NaN O2 were added to the biological material, except in the control group, to evaluate the protection capacity of the extracts. The homogenate of the insect heads was incubated for 2 h in tubes containing tetrazolium bromide. Results: HPLC-DAD demonstrated that the ethanolic extract of T. spinosa presented caffeic acid as the major compound. The ethanolic extract also showed neuroprotective effects at concentrations ≥ 10 μg/mL, while aqueous extract was shown to have a protective effect only at the concentration of 100 μg/mL. The aqueous extract demonstrated a clinically relevant antibacterial activity against the Staphylococcus aureus multidrug resistant strain-MDR, with MIC 512 μg/m L. However, when the extracts were associated with gentamicin and imipenem, a synergism was detected against Staphylococcus aureus and Escherichia coli MDR strains. Conclusions: Although it does not present an antibacterial action, the extracts of T. spinosa can be used in the pharmaceutical industries since its extracts show modulating action of drugs. Besides, these natural products have neuroprotective capacity.

Antibacterial activity and inhibition against Staphylococcus aureus NorA efflux pump by ferulic acid and its esterified derivatives

Objective:To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives(methyl,ethyl,propyl,and butyl)against resistance mechanisms in Staphylococcus aureus strains.Methods:Ferulic acid derivatives were obtained by esterification with methanol,ethanol,propanol,and butanol,and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis.The minimum inhibitory concentrations(MIC)of ferulic acid and its esterified derivatives,ethidium bromide,and norfloxacin were obtained using the microdilution test,while the efflux pump inhibition test was conducted by examining reduction in the MICs of norfloxacin and ethidium bromide.Molecular docking was also carried out using the Schrodinger Suite 2015 molecular modeling software.A three-dimensional model of NorA efflux pump was generated using I-TASSER.The best scoring model was used as a receptor for ligand-receptor docking.Results:The methyl and butyl ester derivatives did not demonstrate significant antimicrobial activity.However,a significant synergic effect was evidenced when norfloxacin was combined with the ethyl and propyl esterified derivatives.The docking study demonstrated favorable energy of interaction between ferulate derivatives and NorA,and amino acid residues TYR57,TYR58,and LEU255 were present commonly in stabilizing all complexes.The PCA analysis corroborated the docking hypothesis that the lipophilic character and hydrogen bond interactions were the most relevant characteristics involved with NorA inhibitors.The pharmacokinetic parameters of ferulic acid derivatives showed good ADMET properties,demonstrating that they can be easily absorbed and have no effect or inhibit the cytochrome P450 enzyme complex,revealing their potential as drug candidates.Conclusions:This study provides strong evidence that the molecular basis for this activity is potentially due to the NorA efflux pump.

LC–MS characterization, anti-kinetoplastide and cytotoxic activities of natural products from Eugenia jambolana Lam. and Eugenia uniflora

Objective: To evaluate the trypanocidal, leishmanicidal and cytotoxic activity of Eugenia jambolana(E. jambolana) and Eugenia uniflora(E. uniflora) extracts and fractions.Methods: The products were characterized by LC–MS. Antiparasitic assays were performed and cytotoxicity was evaluated in fibroblastos. In vitro assays were performed using spectrophotometric evaluation. All assays were performed in thrice.Results: The results showed that the extracts and the tannic fraction from E. jambolana inhibited 100% of the epimastigote lines. The ethanolic extract was the most efficient in all concentrations tested against the three parasite strains. In the cytotoxicity assay the flavonoid fraction showed low toxicity. All E. uniflora samples showed cytotoxicity at the highest concentration tested, but the extract showed no toxic effect on the fibroblasts at the lowest concentration. The flavonoid and tannic fractions were more efficient against Leishmania braziliensis promastigotes compared to the extract. However, the extracts and the tannic fraction were more effective against Leishmania infantum strains. The effect on epimastigote cells was observed at all concentrations tested, with all E. uniflora samples. However, the samples were more effective at the highest concentration, where there was inhibition in 100% of the Trypanosoma cruzi strains.Conclusions: The species E. jambolana and E. uniflora presented antiparasitic activity against all tested parasite strains, indicating that these species can serve as an alternative therapy as they were efficient in the tests performed. The E. uniflora extract and the E. jambolana flavonoid fraction presented a low cytotoxicity, opening the floor for new biological studies.

Antibacterial enhancement of antibiotic activity by Enterolobium contortisiliquum(Vell.)Morong

Objective: To identify the main chemical classes of compounds from aqueous extract of Enterolobium contortisiliquum(E. contortisiliquum) seed bark and to evaluate its antibacterial activity, as well as its potential to increase the activity of antibiotics against strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.Methods: Different classes of compounds in the aqueous extract of E. contortisiliquum were evaluated based on the visual changes in the coloration and the formation of precipitate after the addition of specific reagents. The antibacterial activity of the extract and its potential to increase of antibiotic activity of antibiotics drugs, gentamicin and norfloxacin was determined by using the microdilution method.Results: Our results demonstrated that the following secondary metabolites were presented in E. contortisiliquum seed bark: flavones, flavonols, xanthones, flavononols,chalcones, aurones, flavones and catechins. The extract itself had very low antibacterial activity against all bacterial strains tested(MIC ≥ 1024 μg/m L), but there was an increase in the antibiotic activity of gentamicin and norfloxacin when combined in the subinhibitory concentration(i.e., MIC/8).Conclusions: Our data suggests that E. contortisiliquum seed bark may be an alternative source for new drugs with the potential to increase antibiotic activity against different strains of bacteria.

Chemical composition, antiparasitic and cytotoxic activities of aqueous extracts of Ziziphus joazeiro Mart

Objective: To compare the in vitro antiparasitic activity of aqueous extracts from Ziziphus joazeiro leaves and stem bark against Trypanosoma cruzi, Leishmania braziliensis, and Leishmania infantum, as well as to evaluate its cytotoxicity in mammalian cells, in addition to identifying the chemical composition of the extracts. Methods: Ziziphus joazeiro leaf and stem bark aqueous extracts were prepared by cold extraction maceration and subjected to ultra-efficient liquid chromatography coupled to a quadrupole/time of flight system. The susceptibility assays used Trypanosoma cruzi CLB5 strains and promastigote forms of Leishmania braziliensis and Leishmania infantum for antiparasitic activity of the extracts. Moreover, mammalian fibroblasts NCTC clone 929 were used for cytotoxicity analysis. Results: Terpenoid compounds, flavonoids and phenolic acid were identified in extracts. The stem bark aqueous extracts presented more significant results in terms of antiparasitic activity compared with the leaf aqueous extracts, especially against Leishmania braziliensis and Leishmania infantum promastigote forms with an IC50 < 500 μg/mL. The cytotoxicity evaluation showed moderate toxicity of the stem bark aqueous extracts, which is relevant information for the rational use of this plant part since it is widely used by the population. Conclusions: These preliminary results may contribute to the formulation of new therapeutic agents against this group of neglected diseases, so further investigations are required to delineate the mechanisms of action mainly of the aqueous extract of stem bark of Ziziphus joazeiro.

Metformin reduces testosterone level in type 2 diabetes mellitus patients and its therapy by traditional medicine

Background:Type 2 Diabetes Mellitus(T2DM)is a chronic metabolic disorder characterized by insulin resistance and hyperglycemia.Metformin,a commonly prescribed treatment for T2DM,is known for its effectiveness in improving glycemic control.Recent studies suggest that metformin may also influence testosterone levels in male patients,a hormone crucial for male reproductive health,muscle mass maintenance,and overall quality of life.Medicinal plants were traditionally used for influencing the testosterone level in the body which may be given as a therapy for the side–effects of metformin tablet.Objective:To assess the impact of metformin treatment on testosterone levels in male patients with T2DM compared to non-diabetic males,and to evaluate the potential implications of traditional medical treatments in managing the consequences of reduced testosterone levels.Methods:This study is a prospective observational investigation conducted among a total of 60 patients,divided into two groups:30 males taking metformin as a hyperglycaemic agent with T2DM and 30 males without any diabetic treatment.This study was be carried out in different outpatient department of Shri Mahant Indiresh Hospital,Patel Nagar,Dehradun to assess the level of testosterone in male patients with T2DM taking metformin and in non-Diabetic participants.Based on these findings,potential traditional medicines methods for reducing testosterone levels were summarized.Results:It was demonstrated that male patients with T2DM undergoing metformin treatment exhibited significant reduction in testosterone serum levels compared to non-diabetic patients.Several plants were examinedand,and clinical studies were conducted on them.It was discovered that these plants have been effective in increasingtestosterone level,suggesting their potential use as therapy for patients.Conclusion:This study shows reduction in the testosterone levels,which may lead to low sex drives and induction of low testosterone induced erectile dysfunction.While metformin is used to treat T2DM,it decreases testosterone levels,which negatively impacts the patients’quality of life.So,there is essential need to further study the effect of metformin on testosterone levels in patients with T2DM.Additionally,plants that may be useful in increasing the testosterone levels should be studied further for their beneficial characteristics and potential therapeutic applications.

Molecular evolution,virology and spatial distribution of HCV genotypes in Pakistan:A meta-analysis

Background:Hepatitis C,caused by the Hepatitis C Virus(HCV),is the second most common form of viral hepatitis.The geographical distribution of HCV genotypes can be quite complex,making it challenging to ascertain the most prevalent genotype in a specific area.Methods:To address this,a review was conducted to determine the prevalence of HCV genotypes across various provinces and as a whole in Pakistan.The scientific literature regarding the prevalence,distribution,genotyping,and epidemiology of HCV was gathered from published articles spanning the years 1996-2020.Results:Genotype 1 accounted for 5.1%of the patients,with its predominant subtype being 1a at 4.38%.The frequencies of its other subtypes,1b and 1c,were observed to be 1.0%and 0.31%respectively.Genotype 2 had a frequency of 2.66%,with the most widely distributed subtype being 2a at 2.11%of the patients.Its other subtypes,2b and 2c,had frequencies of 0.17%and 0.36%respectively.The most prevalent genotype among all isolates was 3(65.35%),with the most frequent subtype being 3a(55.15%),followed by 3b(7.18%).The prevalence of genotypes 4,5,and 6 were scarce in Pakistan,with frequencies of 0.97%,0.08%,and 0.32%respectively.The prevalence of untypeable and mixed genotypes was 21.34%and 3.53%respectively.Estimating genotypes proves to be a productive method in assisting with the duration and selection of antiviral treatment.Different HCV genotypes can exhibit variations in their response to specific antiviral treatments.Different genotypes may have distinct natural histories,including variations in disease progression and severity.Some genotypes may lead to more rapid liver damage,while others progress more slowly.Conclusions:This information can guide screening and testing strategies,helping to identify individuals at higher risk of developing severe complications.Studying the distribution of HCV genotypes in a population can provide valuable insights into the transmission dynamics of the virus.