GC/MS Profiling and Ex Vivo Antibacterial Activity of Salvadora persica (Siwak) against Enterococcus faecalis as Intracanal Medicament

Introduction. Salvadora persica L. (S. persica, Siwak) has been used for many centuries as oral hygiene tools, particularly in Saudi Arabia. This study aimed to assess the effectiveness of S. persica petroleum ether extract (SPE) as an intracanal bactericidal for endodontic treatment against Enterococcus faecalis. Calcium hydroxide Ca(OH)2 gold standard intracanal medicament was used for comparison. Methods. The gas chromatography mass spectrometry (GC/MS) analysis was carried out to identify the components of SPE. First, the consistency of SPE was accomplished according to ANSI/ADA specification no 57. Forty-five single-rooted mandibular premolars were infected with that of E. faecalis suspension. Colony-forming units (CFU) were counted before the medicaments’ application (CFU-1) and after seven days of their applications (CFU-2). Group I: SPE, Group II: positive control Ca(OH)2, and Group III: saline solution negative control. The microdilution method was applied to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of SPE. Results. Thirty-two compounds were identified (89.09%), with main components of benzyl isothiocyanate (BITC) (33.32%) and steroids (34%). CFU before and after using SPE and Ca(OH)2 recorded a statistically significant reduction in bacterial count ( P = 0.006 ) and ( P = 0.01 ), respectively. There was an insignificant difference between CFU after using SPE and Ca(OH)2 ( P = 0.210 ). On the contrary, comparing both medicaments with the negative control saline group resulted in significant differences, ( P = 0.001 ) and ( P = 0.007 ), respectively. Moreover, the equality of minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of SPE is recorded. Conclusion. This finding could be referred to the high content of bactericidal BITC in synergism with other antimicrobial components, representing 70.71% of SPE. Thus, SPE is a good candidate as an intracanal medicament, which warrants further investigation.

Antifungal Activity of the Essential Oils of Osteophloeum platyspermum (Myristicaceae) against Malassezia spp. and Candida albicans Influenced by Seasonality and Climatic Factors

Aims: Malassezia spp. are involved in a wide range of mammalian skin diseases. The introduction of new drugs is a need. Natural products are known to be effective in the treatment of microbial pathogens. The present study analyzed the O. platyspermum leaf essential oils (EOs) antifungal activity. Study Design: 18 terpenes from 13 O. platyspermum leaf EOs are related to seasonal and climatic variations occurring during the dry (DS) and rainy (RS) seasons in the Amazon Rain-Forest, verified by means of multivariate analyses. Place and Duration of the Study: the study was conducted at the Center for Research in Biodiversity (Microbiology Laboratory and Cell Culture Laboratory), Paulista University, biological activity evaluations occurred between January/2019 and December 2019). Methodology: microdilution broth assay was used in the minimal bactericidal concentration (MBC). Multivariate analyses were used to access the relationship among MBC, seasonality and terpene composition of the EOs. Results: Malassezia pachydermatis showed higher sensitivity to the EOs than M. furfur or C. albicans. The DS EOs were linked to the presence of limonene, myrcene, α-terpineol, linalool, terpinen-4-ol, cubenol-1-epi, influenced by insolation, temperature and evaporation, while β-elemene, γ-elemene, neo-intermedeol, elemol, α-cadinol, spathulenol, isospathulenol, viridiflorol, δ-amorphene and ledol were linked to the RS EOs, and were influenced by precipitation, relative humidity and wind velocity. DS EOs showed better antifungal activity against both Malassezia species, and the presence of the six discriminative terpenes was essential for the antifungal activity. Conclusions: The DS EOs are a potential source of new leads to defeat animal dermatological microbes.

Chemical Composition, Antibacterial, Enzyme-Inhibitory, and Anti-Inflammatory Activities of Essential Oil from Hedychiumpuerense Rhizome

Hedychium puerense, a perennial rhizomatous herb, is used as an ornamental, medicinal, and edible plant in Yunnan Province, China. Essential oils from Hedychium plants are widely used in perfumes and traditional medicine, but there are no studies on the constituents and bioactivities of H. puerense essential oil (EO). Therefore, this study was designed to explore the chemical composition, antibacterial, enzyme-inhibitory, and anti-inflammatory activities of H. puerense rhizome EO. The gas chromatography with flame ionization or mass selective detection (GC-FID/MS) results indicated that H. puerense EO was mainly composed of linalool (26.5%), β-pinene (18.6%), γ-terpinene (12.1%), terpinen-4-ol (7.7%), α-pinene (5.8%), sabinene (4.9%), E-nerolidol (4.1%), and p-cymene (3.6%). For biological activities, H. puerense EO displayed broad-spectrum antibacterial properties against Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli with diameter of inhibition zone (DIZ) values ranging from 7.44 to 10.30 mm, a minimal inhibitory concentration (MIC) of 3.13–6.25 mg/m), and a minimal bactericidal concentration (MBC) of 3.13–12.50 mg/mL. Moreover, the EO significantly inhibited acetylcholinesterase (AChE) (IC50 = 0.94 ± 0.02 mg/mL) and butyrylcholinesterase (BChE) (IC50 = 1.32 ± 0.06 mg/mL) activities, and exhibited a moderate inhibitory effect on α-glucosidase (IC50 = 5.42 ± 0.32 mg/mL) and tyrosinase (IC50 = 3.23 ± 0.21 mg/mL). Furthermore, the EO significantly suppressed the secretion of the pro-inflammatory mediator, nitric oxide (NO) (99.23 ± 0.26%), cytokines tumor necrosis factor-α (TNF-α) (97.14 ± 0.11%), and interleukin-6 (IL-6) (82.42 ± 0.16%) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at 250 μg/mL without cytotoxicity. Hence, H. puerense EO can be considered a bioactive, natural product that has great potential for utilization in the fields of food, cosmetics, and pharmaceutics.

Efficacy of Thai Plant Extracts for Antibacterial and Anti-Biofilm Activities against Pathogenic Bacteria

The emergence of drug-resistant bacteria has impacted the outcome of current therapeutics as a threat to global healthcare; novel medicines are urgently needed. Thirteen medicinal plants were collected in Northeastern Thailand, and their crude ethanolic extracts were evaluated for antibacterial activities against Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 using the broth micro-dilution method. Piper betle leaf ethanolic extract showed optimal activity against both representative bacterial strains. Activity was also observed against clinical isolates of methicillin-resistant S. aureus (MRSA) and E. coli, with minimal inhibitory concentration (MIC) ranging from 0.31 mg/mL to 2.5 mg/mL and minimal bactericidal concentration (MBC) ranging from 0.62 mg/mL to 2.5 mg/mL. A time-kill study revealed that the extract activity was time- and dose-dependent, and also bactericidal on the tested bacteria. P. betle extract inhibited biofilm formation and promoted biofilm eradication in both S. aureus and E. coli. 4-Allyl-1,2-diacetoxybenzene and eugenol were identified as the most abundant compounds in the extract and may play major roles in the anti-bacterial and anti-biofilm activity. Results suggest that ethanolic P. betle leaf extract shows promise as an alternative method for the prevention of bacterial diseases.

In Vitro Effect of Copper (I) Complex [Cu(NN1)2](ClO4) on Vibrio harveyi BB170 Biofilm Formation

Biofilm formation in pathogenic bacteria is an important factor of resistance to antimicrobial treatments, allowing them to survive for a long time in their hosts. In the search for new antibiofilm agents, in this work we report the activity of a copper (I) complex, [Cu(NN1)2]ClO4, synthesized with Cu (I) and NN1, an imine ligand 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one, a derivate of natural compound coumarin. The antibacterial and antibiofilm capacity was evaluated in Vibrio harveyi BB170 used as model bacteria. Antibacterial activity was measured in vitro by minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and half-maximal inhibitory concentration (IC50) determination. Antibiofilm capacity of copper (I) complex was analyzed by different concentrations of IC50 values. The results showed that the sub-IC50 concentration, 12.6 µg/mL of the copper (I) complex, was able to reduce biofilm formation by more than 75%, and bacterial viability was reduced by 50%. Inverted and confocal laser scanning microscopy showed that the [Cu(NN1)2]ClO4 complex affected the biofilm structure. Therefore, the copper (I) complex is effective as an antibiofilm compound in V. harveyi BB170.

Antibacterial Activity of Kaolin–Silver Nanomaterials: Alternative Approach to the Use of Antibiotics in Animal Production

According to the search for alternatives to replace antibiotics in animal production suggested in the antimicrobial resistance action plans around the world, the objective of this work was to evaluate the bactericidal effect of kaolin–silver nanomaterial for its possible inclusion as an additive in animal feed. The antibacterial activity of the C3 (kaolin–silver nanomaterial) product was tested against a wide spectrum of Gram-negative and Gram-positive bacteria (including multidrug resistant strains) by performing antibiograms, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), as well as growth inhibition curves against seven strains causing infections in animals. The C3 product generated inhibition halos in all the tested strains, and a higher activity against Gram-negative bacteria was found, with MBC values ranged from 7.8 µg/mL (P. aeruginosa) to 15.6 µg/mL (E. coli and Salmonella). In contrast, it was necessary to increase the concentration to 31.3 µg/mL or 250 µg/mL to eliminate 99.9% of the initial population of S. aureus ATCC 6538 and E. faecium ATCC 19434, respectively. Conversely, the inhibition growth curves showed a faster bactericidal activity against Gram-negative bacteria (between 2 and 4 h), while it took at least 24 h to observe a reduction in cell viability of S. aureus ATCC 6538. In short, this study shows that the kaolin–silver nanomaterials developed in the framework of the INTERREG POCTEFA EFA183/16/OUTBIOTICS project exhibit antibacterial activity against a wide spectrum of bacteria. However, additional studies on animal safety and environmental impact are necessary to evaluate the effectiveness of the proposed alternative in the context of One Health.

Antimicrobial and Immunomodulatory Activity of Herb Extracts Used in Burn Wound Healing: “San Huang Powder”

“San Huang Powder,” a nonsterile milled herb powder, is frequently used to treat burn wounds in traditional Chinese herbal medicine. However, treating a wound with a nonsterile dressing or reagent is not compatible with the current guidelines in modern medicine. Therefore, we investigated the bactericidal and anti-inflammatory activities of four herb extracts used in “San Huang Powder” in vitro. Meanwhile, an in vivo porcine model with superficial second-degree burns was used for the experiments since the size and skin composition of pigs are the closest to that of the human body. The minimal bactericidal concentration (MBC) of the herb extracts was determined. The in vitro assay indicated that Rhubarb and Phellodendron bark extracts decreased the levels of inflammatory cytokines, IL-8, and GM-CSF on LPS-induced HMEC-1 cells. In accordance with this result, the histopathological evaluation results showed that the efficacy of “San Huang Powder” containing both herb materials was much better than the group without Rhubarb. Our results not only provide a basis to understand why “San Huang Powder” has been used to clinically treat wounds without sterilization directly since ancient times but also show the advantages of using multiple herb materials simultaneously on wound sites to prevent infection during treatment. Rhubarb is the recommended ingredient involved in the preparation of “San Huang Powder” to ensure the healing efficacy of burn wounds.

A new phenothiazine derivate is active against Clostridioides difficile and shows low cytotoxicity

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 μg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

Antibiotic susceptibility of Staphylococcus aureus with different degrees of biofilm formation

Staphylococcus aureus is one of the most common pathogens in biofilm-associated chronic infections. S. aureus living within biofilms evades the host immune response and is more resistant to antibiotics than planktonic bacteria. In this study, we generated S. aureus with low and high levels of biofilm formation using the rbf (regulator of biofilm formation) gene and performed a BioTimer assay to determine the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of various types of antibiotics. We showed that biofilm formation by S. aureus had a greater effect on MBC than MIC, probably due to the different growth modes between planktonic and biofilm bacteria. Importantly, we found that the MBC for biofilm S. aureus was much higher than that for planktonic cells, but there was little difference in MBC between low and high levels of biofilm formation. These results suggest that once the biofilm is formed, the bactericidal activity of antibiotics is significantly reduced, regardless of the degree of S. aureus biofilm formation. We propose that S. aureus strains with varying degrees of biofilm formation may be useful for evaluating the anti-biofilm activity of antimicrobial agents and understanding antibiotic resistance mechanisms by biofilm development.

Minimal Inhibitory Concentration and Minimal Bactericidal Concentration of Peppermint (Mentha piperita) extracts against standard microorganisms

Different plant extracts are considerably safe from infectious agents and may be used for medical purposes. The present research was conducted against the six standard microorganisms to quantify the antimicrobial activities of peppermint (Mentha piperita) extracts. The traditional approaches of minimum bactericidal concentration (MBC) and minimal inhibitory concentration (MIC) were used to approximate the antibacterial activities of ethanol, methanol, and chloroform peppermint extracts. The inhibitory function of the ethanol extract was comparable to that of chloroform (10 to 80mg/ml) and methanol (10 to 80mg/ml) against all gram-negative microorganisms. The minimum value of MIC was recorded for Streptococcus pyogenes (5mg/ml for extract of ethanol), followed by E. coli (10mg/ml for extract of ethanol) and then by Enterococcus faecalis (15mg/ml for extract of ethanol). With respect to the standard microorganisms, the MBC values were higher for both extracts than the corresponding MIC values. This work demonstrated the possible efficacy of antibacterial action on M. Piperita extracts from normal microorganisms (A. Baumenii, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa and Klebsiella pneumoniae), particularly ethanol extract. In summary, the peppermint ethanol extract had important growth-inhibiting effects on observed standard micro-organisms, followed by chloroform and methanol extracts. Further to in vitro and in vivo studies on a wide variety of natural microorganisms and therapeutic isolates are required to investigate and standardize the inhibitory activity of peppermint extracts against the most dangerous human pathogenic agents.