Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)—ampicillin, oxacillin and penicillin—and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99% ± 7% for S. aureus and up to 94% ± 4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79% ± 5% and MRSA up to 16% ± 2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.     

The Antimicrobial Peptide Temporin G: Anti-Biofilm,Anti-Persister Activities,and Potentiator Effect of Tobramycin Efficacy Against Staphylococcus aureus

Bacterial biofilms are a serious threat for human health, and the Gram-positive bacterium Staphylococcus aureus is one of the microorganisms that can easily switch from a planktonic to a sessile lifestyle, providing protection from a large variety of adverse environmental conditions. Dormant non-dividing cells with low metabolic activity, named persisters, are tolerant to antibiotic treatment and are the principal cause of recalcitrant and resistant infections, including skin infections. Antimicrobial peptides (AMPs) hold promise as new anti-infective agents to treat such infections. Here for the first time, we investigated the activity of the frog-skin AMP temporin G (TG) against preformed S. aureus biofilm including persisters, as well as its efficacy in combination with tobramycin, in inhibiting S. aureus growth. TG was found to provoke ~50 to 100% reduction of biofilm viability in the concentration range from 12.5 to 100 µM vs ATCC and clinical isolates and to be active against persister cells (about 70–80% killing at 50–100 µM). Notably, sub-inhibitory concentrations of TG in combination with tobramycin were able to significantly reduce S. aureus growth, potentiating the antibiotic power. No critical cytotoxicity was detected when TG was tested in vitro up to 100 µM against human keratinocytes, confirming its safety profile for the development of a new potential anti-infective drug, especially for treatment of bacterial skin infections.     

Investigation of the Anti-Methicillin-Resistant Staphylococcus aureus Activity of (+)-Tanikolide- and (+)-Malyngolide-Based Analogues Prepared by Asymmetric Synthesis

Herein, we report antibacterial and antifungal evaluation of a series of previously prepared (+)-tanikolide analogues. One analogue, (4S,6S)-4-methyltanikolide, displayed promising anti-methicillin-resistant Staphylococcus aureus activity with a MIC of 12.5 µg/mL. Based on the antimicrobial properties of the structurally related (−)-malyngolide, two further analogues (4S,6S)-4-methylmalyngolide and (4R,6S)-4-methylmalyngolide bearing a shortened n-nonyl alkyl side chain were prepared in the present study using a ZrCl₄-catalysed deprotection/cyclisation as the key step in their asymmetric synthesis. When these were tested for activity against anti-methicillin-resistant Staphylococcus aureus, the MIC increased to 50 µg/mL.     

Synthesis and Evaluation of Novel Oxyalkylated Derivatives of 2′,4′-Dihydroxychalcone as Anti-Oomycete Agents against Bronopol Resistant Strains of Saprolegnia sp.

A series of novel oxyalkylchalcones substituted with alkyl groups were designed and synthesized, and the antioomycete activity of the series was evaluated in vitro against Saprolegnia strains. All tested O-alkylchalcones were synthesized by means of nucleophilic substitution from the natural compound 2′,4′-dihydroxychalcone (1) and the respective alkyl bromide. The natural chalcone (1) and 10 synthetic oxyalkylchalcones (2–11) were tested against Saprolegnia parasitica and Saprolegnia australis. Among synthetic analogs, 2-hydroxy,4-farnesyloxychalcone (11) showed the most potent activity against Saprolegnia sp., with MIC and MOC values of 125 µg/mL (similar to bronopol at 150 µg/mL) and 175 µg/mL, respectively; however, 2′,4′-dihydroxychalcone (1) was the strongest and most active molecule, with MIC and MOC values of 6.25 µg/mL and 12.5 µg/mL.     

Efficacy of a Topical Wound Agent Methanesulfonic Acid and Dimethylsulfoxide on In Vitro Biofilms

A topical desiccating wound agent containing methanesulfonic acid, dimethylsulfoxide and amorphous silica was evaluated in three in vitro models for its efficacy against biofilms produced by Pseudomonas aeruginosa (ATCC-15442) and Staphylococcus aureus (ATCC-6538). The in vitro biofilm models used were; the MBEC Assay^®, Centre for Disease Control (CDC) Biofilm Reactor^® and a Semi-solid biofilm model. A 30-s exposure of a topical wound desiccating agent was used in each model. A complete eradication of viable cells was demonstrated in all models for both strains (p < 0.0001). Imaging with scanning electron microscopy (SEM) was performed where possible. All three models demonstrated complete eradication of viable cells with a 30 s application of a topical wound desiccating agent.     

The Effect of a Silver Nanoparticle Polysaccharide System on Streptococcal and Saliva-Derived Biofilms

In this work, we studied the antimicrobial properties of a nanocomposite system based on a lactose-substituted chitosan and silver nanoparticles: Chitlac-nAg. Twofold serial dilutions of the colloidal Chitlac-nAg solution were both tested on Streptococcus mitis, Streptococcus mutans, and Streptococcus oralis planktonic phase and biofilm growth mode as well as on saliva samples. The minimum inhibitory and bactericidal concentrations of Chitlac-nAg were evaluated together with its effect on sessile cell viability, as well as both on biofilm formation and on preformed biofilm. In respect to the planktonic bacteria, Chitlac-nAg showed an inhibitory/bactericidal effect against all streptococcal strains at 0.1% (v/v), except for S. mitis ATCC 6249 that was inhibited at one step less. On preformed biofilm, Chitlac-nAg at a value of 0.2%, was able to inhibit the bacterial growth on the supernatant phase as well as on the mature biofilm. For S. mitis ATCC 6249, the biofilm inhibitory concentration of Chitlac-nAg was 0.1%. At sub-inhibitory concentrations, the Streptococcal strains adhesion capability on a polystyrene surface showed a general reduction following a concentration-dependent-way; a similar effect was obtained for the metabolic biofilm activity. From these results, Chitlac-nAg seems to be a promising antibacterial and antibiofilm agent able to hinder plaque formation.     

Silver Nanoparticles from Oregano Leaves’ Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses

The oregano leaves’ extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl–methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV–Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.     

Antibacterial Activity of New Oxazolidin-2-One Analogues in Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is one of the most common causes of nosocomial infections. The purpose of this study was the synthesis and in vitro evaluation of antimicrobial activity of 10 new 3-oxazolidin-2-one analogues on 12 methicillin resistant S. aureus (MRSA) clinical isolates. S. aureus confirmation was achieved via catalase and coagulase test. Molecular characterization of MRSA was performed by amplification of the mecA gene. Antimicrobial susceptibility was evaluated via the Kirby-Bauer disc diffusion susceptibility test protocol, using commonly applied antibiotics and the oxazolidinone analogues. Only (R)-5-((S)-1-dibenzylaminoethyl)-1,3-oxazolidin-2-one (7a) exhibited antibacterial activity at 6.6 μg. These results, allow us to infer that molecules such as 7a can be potentially used to treat infections caused by MRSA strains.     

Essential Oils Biofilm Modulation Activity,Chemical and Machine Learning Analysis. Application on Staphylococcus aureus Isolates from Cystic Fibrosis Patients

Bacterial biofilm plays a pivotal role in chronic Staphylococcus aureus (S. aureus) infection and its inhibition may represent an important strategy to develop novel therapeutic agents. The scientific community is continuously searching for natural and “green alternatives” to chemotherapeutic drugs, including essential oils (EOs), assuming the latter not able to select resistant strains, likely due to their multicomponent nature and, hence, multitarget action. Here it is reported the biofilm production modulation exerted by 61 EOs, also investigated for their antibacterial activity on S. aureus strains, including reference and cystic fibrosis patients’ isolated strains. The EOs biofilm modulation was assessed by Christensen method on five S. aureus strains. Chemical composition, investigated by GC/MS analysis, of the tested EOs allowed a correlation between biofilm modulation potency and putative active components by means of machine learning algorithms application. Some EOs inhibited biofilm growth at 1.00% concentration, although lower concentrations revealed different biological profile. Experimental data led to select antibiofilm EOs based on their ability to inhibit S. aureus biofilm growth, which were characterized for their ability to alter the biofilm organization by means of SEM studies.     

Pereskia aculeata Muller (Cactaceae) Leaves: Chemical Composition and Biological Activities

The aims of this work were to study the chemical composition of the essential oil from the leaves of Pereskia aculeata and to evaluate some biological activities of three leaf extracts. The phenolic content, antioxidant activity, and in vitro antimicrobial and antifungal activities were determined. The methanol extract showed antioxidant activity (EC₅₀ 7.09 mg/mL) and high polyphenols content (15.04 ± 0.31 mg gallic acid equivalents (GAE)/g). The petroleum ether extract exhibited potent antibacterial activity against Escherichia coli, whereas the chloroform extract showed inhibitory activity against Bacillus cereus and Staphylococcus aureus. The petroleum ether and methanol extracts were more effective in inhibiting the growth of Aspergillus versicolor. The possible cytotoxicity of extracts on neuroblastoma SH-SY5Y cancer cell line and the influence on adenylate cyclase (ADCY) expression was also studied. P. aculeata chloroform extract showed antiproliferative activity with an IC₅₀ value of 262.83 µg/mL. Treatments of SH-SY5Y neuroblastoma cells with 100 µg/mL of methanol extract significantly reduced ADCY1 expression.     

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3–H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC₅₀ = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC₅₀ = 12 µM by visual CPE score; EC₅₀ = 8.3 µM by TMS score; MCC > 100 µM, CC₅₀ = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC₅₀ in the range 14.5–97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at β-lactamase, thus protecting the antibiotic from undesirable biotransformation.     

New Coumarins and Anti-Inflammatory Constituents from the Fruits of Cnidium monnieri

The fruit of Cnidium monnieri is commercially used as healthcare products for the improvement of impotence and skin diseases. Three new coumarins, 3''-O-methylmurraol (1), rel-(1''S,2''S)-1''-O-methylphlojodicarpin (2), and (1''S,2''S)-1''-O-methylvaginol (3), have been isolated from the fruits of C. monnieri, together with 14 known compounds (4–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4–12, and 14–17 exhibited inhibition (IC₅₀ ≤ 7.31 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 7, 9–11, 15, and 17 inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤7.83 µg/mL. This investigation reveals that bioactive isolates (especially 6, 7, 14, and 17) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.     

Discovery of a Potent Anti-Yeast Triterpenoid Saponin,Clematoside-S from Urena lobata L.

Urena lobata has been used as a traditional medicinal plant in India and China. In this study, we investigated the antimicrobial activity and isolated the active compound from the leaves of U. lobata. The 80% ethanol extract from U. lobata leaves showed an effective anti-yeast activity against Saccharomyces cerevisiae (S. cerevisiae) strains. Using a combination of chromatographic methods, (−)-trachelogenin (1) and clematoside-S (2) were isolated from this plant for the first time, and their chemical structure was identified by mass spectrometry (MS) and extensive nuclear magnetic resonance (NMR) data analysis. In addition, 1 was found to be inactive against all of the test microorganisms in the antimicrobial assay, whereas 2 exhibits a specific anti-yeast activity against S. cerevisiae strains with diameter of inhibition zones in the range from 11 to 20 mm. Furthermore, the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of 2 against S. cerevisiae strains were detected to be in the ranges of 0.61 to 9.8 μg/mL and 2.42 to 9.8 μg/mL, respectively. This is the first report of 2 with a specific anti-yeast activity. The above result suggests the potential application of U. lobata to be used as a natural anti-yeast agent in food preservation.     

Essential Oil of Cymbopogon nardus (L.) Rendle: A Strategy to Combat Fungal Infections Caused by Candida Species

Background: The incidence of fungal infections, especially those caused by Candida yeasts, has increased over the last two decades. However, the indicated therapy for fungal control has limitations. Hence, medicinal plants have emerged as an alternative in the search for new antifungal agents as they present compounds, such as essential oils, with important biological effects. Published data demonstrate important pharmacological properties of the essential oil of Cymbopogon nardus (L.) Rendle; these include anti-tumor, anti-nociceptive, and antibacterial activities, and so an investigation of this compound against pathogenic fungi is interesting. Objective: The aim of this study was to evaluate the chemical composition and biological potential of essential oil (EO) obtained from the leaves of C. nardus focusing on its antifungal profile against Candida species. Methods: The EO was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). Testing of the antifungal potential against standard and clinical strains was performed by determining the minimal inhibitory concentration (MIC), time-kill, inhibition of Candida albicans hyphae growth, and inhibition of mature biofilms. Additionally, the cytotoxicity was investigated by the IC₅₀ against HepG-2 (hepatic) and MRC-5 (fibroblast) cell lines. Results: According to the chemical analysis, the main compounds of the EO were the oxygen-containing monoterpenes: citronellal, geranial, geraniol, citronellol, and neral. The results showed important antifungal potential for all strains tested with MIC values ranging from 250 to 1000 μg/mL, except for two clinical isolates of C. tropicalis (MIC > 1000 μg/mL). The time-kill assay showed that the EO inhibited the growth of the yeast and inhibited hyphal formation of C. albicans strains at concentrations ranging from 15.8 to 1000 μg/mL. Inhibition of mature biofilms of strains of C. albicans, C. krusei and C. parapsilosis occurred at a concentration of 10× MIC. The values of the IC₅₀ for the EO were 96.6 μg/mL (HepG-2) and 33.1 μg/mL (MRC-5). Conclusion: As a major virulence mechanism is attributed to these types of infections, the EO is a promising compound to inhibit Candida species, especially considering its action against biofilm.     

In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (K_d) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.     

Isolation and Heterologous Expression of a Polygalacturonase Produced by Fusarium oxysporum f. sp. cubense Race 1 and 4

Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The K_m and V_max values of PGC3 from FOC4 were determined to be 0.70 mg·mL⁻¹ and 101.01 Units·mg·protein⁻¹·min⁻¹, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3–7 and >50% activity in 10–50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris.     

Staphylococcal Enterotoxin Genes in Coagulase-Negative Staphylococci—Stability,Expression, and Genomic Context

In the current study, we screened a collection of coagulase-negative staphylococci (CoNS) isolates for orthologues of staphylococcal enterotoxins (SEs) involved in S. aureus-related staphylococcal food poisoning (SFP). The amplicons corresponding to SEs were detected in S. chromogenes, S. epidermidis, S. haemolyticus, S. borealis, S. pasteuri, S. saprophyticus, S. vitulinus, S. warneri, and S. xylosus. All amplicons were sequenced and identified as parts of known S. aureus or S. epidermidis SE genes. Quantitative real-time PCR allowed determining the relative copy number of each SE amplicon. A significant portion of the amplicons of the sea, seb, sec, and seh genes occurred at low copy numbers. Only the amplicons of the sec gene identified in three isolates of S. epidermidis displayed relative copy numbers comparable to sec in the reference enterotoxigenic S. aureus and S. epidermidis strains. Consecutive passages in microbiological media of selected CoNS isolates carrying low copy numbers of sea, seb, sec, and seh genes resulted in a decrease of gene copy number. S. epidermidis isolates harbored a high copy number of sec, which remained stable over the passages. We demonstrated that enterotoxin genes may occur at highly variable copy numbers in CoNS. However, we could identify enterotoxin genes only in whole-genome sequences of CoNS carrying them in a stable form at high copy numbers. Only those enterotoxins were expressed at the protein level. Our results indicate that PCR-based detection of enterotoxin genes in CoNS should always require an additional control, like analysis of their presence in the bacterial genome. We also demonstrate S. epidermidis as a CoNS species harboring SE genes in a stable form at a specific chromosome site and expressing them as a protein.     

Syngonanthus nitens Bong. (Rhul.)-Loaded Nanostructured System for Vulvovaginal Candidiasis Treatment

Herbal-loaded drug delivery nanotechnological systems have been extensively studied recently. The antimicrobial activity of medicinal plants has shown better pharmacological action when such plants are loaded into a drug delivery system than when they are not loaded. Syngonanthus nitens Bong. (Rhul.) belongs to the Eriocaulaceae family and presents antiulcerogenic, antioxidant, antibacterial, and antifungal activity. The aim of this study was to evaluate the antifungal activity of Syngonanthus nitens (S. nitens) extract that was not loaded (E) or loaded (SE) into a liquid crystal precursor system (S) for the treatment of vulvovaginal candidiasis (VVC) with Candida albicans. The minimal inhibitory concentration (MIC) was determined by the microdilution technique. Additionally, we performed hyphae inhibition and biofilm tests. Finally, experimental candidiasis was evaluated in in vivo models with Wistar female rats. The results showed effective antifungal activity after incorporation into S for all strains tested, with MICs ranging from 31.2 to 62.5 μg/mL. Microscopic observation of SE revealed an absence of filamentous cells 24 h of exposure to a concentration of 31.2 μg/mL. E demonstrated no effective action against biofilms, though SE showed inhibition against biofilms of all strains. In the in vivo experiment, SE was effective in the treatment of infection after only two days of treatment and was more effective than E and amphotericin B. The S. nitens is active against Candida albicans (C. albicans) and the antifungal potential is being enhanced after incorporation into liquid crystal precursor systems (LCPS). These findings represent a promising application of SE in the treatment of VVC.     

Rapid Bioassay-Guided Isolation of Antibacterial Clerodane Type Diterpenoid from Dodonaea viscosa (L.) Jaeq.

Plant extracts are complex matrices and, although crude extracts are widely in use, purified compounds are pivotal in drug discovery. This study describes the application of automated preparative-HPLC combined with a rapid off-line bacterial bioassay, using reduction of a tetrazolium salt as an indicator of bacterial metabolism. This approach enabled the identification of fractions from Dodonaea viscosa that were active against Staphylococcus aureus and Escherichia coli, which, ultimately, resulted in the identification of a clerodane type diterpenoid, 6β-hydroxy-15,16-epoxy-5β, 8β, 9β, 10α-cleroda-3, 13(16), 14-trien-18-oic acid, showing bacteriostatic activity (minimum inhibitory concentration (MIC) = 64–128 µg/mL) against test bacteria. To the best of our knowledge, this is the first report on antibacterial activity of this metabolite from D. viscosa.     

Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis

The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays.