Bactericidal Concentration Assays Research Articles

Influence of critical micelle concentration of choline-based long chain fatty acid soaps on their antibacterial activity against Methicillin resistant Staphylococcus aureus

HypothesisAntimicrobial resistance (AMR) is a pressing global health concern. ESKAPEE pathogens, such as Methicillin-resistant Staphylococcus aureus (MRSA) are notable of concern in healthcare settings due to their resistance to critical antibiotics. To combat AMR, the development of alternatives such as bacterial membrane-active agents is crucial. Fatty acids (FAs) have emerged as a sustainable, antibiotic-free solution with inherent antibacterial activity. However, long chain saturated fatty acids (LCFAs) sodium soaps exhibit poorly antibacterial properties in comparison to short chain FAs, believed to be linked to limited solubility in aqueous media. ExperimentsWe employed choline as a chaotropic organic counter-ion to enhance the solubility of LCFAs and investigated their antibacterial effects against MRSA. The optimal medium conditions for micelle formation for LCFAs was first investigated. Then, we determined the critical micelle concentration (CMC), micellar morphology, and aggregation number through surface tension measurements and small angle neutron scattering experiments. Antimicrobial activity was assessed using minimum bactericidal concentration (MBC) assays and time-kill experiments. FindingsWe have identified conditions where LCFAs are effective against MRSA for the first time, providing valuable insights for developing new antibacterial agents to fight AMR. LCFAs need to be used above their Krafft temperatures and CMC to exhibit antibacterial efficacy.

Bioactive compound from marine seagrass Streptomyces argenteolus TMA13: combatting fish pathogens with time-kill kinetics and live-dead cell imaging.

Actinobacteria, pervasive in aquatic and terrestrial environments, exhibit a filamentous morphology, possess DNA with a specific G + C content and production of numerous secondary metabolites. This study, focused on actinobacteria isolated from marine seagrass, investigating their antibacterial activity against fish pathogens. Among 28 isolates, Streptomyces argenteolus TMA13 displayed the maximum zone of inhibition against fish pathogens-Aeromonas hydrophila (10mm), Aeromonas caviae (22mm), Edwardsiella tarda (17mm), Vibrio harveyi (22mm) and Vibrio anguillarum (12mm) using the agar plug method. Optimization of this potent strain involved with various factors, including pH, temperature, carbon source and salt condition to enhance both yield production and antibacterial efficacy. In anti-biofilm assay shows the maximum percentage of inhibition while increasing concentration of TMA13 extract. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) assays with TMA13 crude extract demonstrated potent activity against fish pathogens at remarkably low concentrations. Time-kill kinetics assay showcased growth curve variations over different time intervals for all fish pathogens treated with a 100µg/ml concentration of crude extract, indicating a decline in cells viability and progression into the death phase. Additionally, fluorescence microscopic visualization of bacterial cells exposed to the extracts emitting green and red fluorescence, enabling live-dead cell differentiation was also studied. Further characterization of the crude extract through GC-MS and FT-IR analyses performed and identified secondary metabolites with functional groups exhibiting significant antibacterial activity. This study elucidates the capacity of Streptomyces argenteolus TMA13 to enhance the production of antibiotic compounds effective against fish pathogens.

Design of Thermosensitive Niosomes by Eutectic Mixture of Natural Fatty Acids.

In the current study, a smart release system responsive to temperature was developed to improve the efficiency of tetracycline (TC) in antibacterial therapy. The nanovesicles designed consist of a non-ionic surfactant, SPAN60, cholesterol and a phase change material (PCM) as a thermoresponsive gating material. Niosomes were prepared using an increasing amount of PCM and characterized in terms of size, zeta potential, colloidal stability and thermoresponsive properties. The vesicles that developed were homogenous in size, had good biocompatibility and stability for up to 3 months and demonstrated thermoresponsive behavior. A low drug leakage was observed at 37 °C, while a rapid release occurred at 42 °C, due to the faster diffusion rate of the drug trough the melted PCM. This controllable drug release capacity allows us to avoid premature drug release, minimizing unwanted and toxic effects and ensuring a long retention time in the nanodevice so that it reaches the infected sites. In addition, TC-loaded niosomes were screened to investigate their antibacterial activity against various Gram-positive and Gram-negative bacteria by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. An interesting temperature-dependent antibacterial activity was observed against some bacterial strains: the niosomes activity against S. epidermis, for example, was improved by the temperature increase, as suggested by a reduction in MIC values from 112.81 to 14.10 μM observed at 37 and 42 °C, respectively. Taken together, the thermoresponsive platform developed allows us to use lower antibiotic amounts while ensuring therapeutic efficacy and, so, will advance the development of a novel antibacterial agent in clinical practice.

Impact of chlorhexidine and povidone-iodine antiseptic solutions on the cervicovaginal microenvironment during laparoscopic hysterectomies: a pilot study

The use of chlorhexidine gluconate (CHG) as surgical preparation solution has been advocated due to reduced bacterial loads compared with povidone-iodine (PI). We aimed to investigate changes to the vaginal microenvironment among patients who had laparoscopic hysterectomy and were surgically prepped using 4% CHG compared to 7.5% PI. Premenopausal women who underwent laparoscopic hysterectomy for benign conditions and were prepped with either CHG or PI per surgeon’s choice were enrolled. Vaginal swabs and cervicovaginal lavages were collected prior to vaginal preparation and at 4–6 week post-operative visits for microbiome (α and β diversity, bacterial relative abundances, vaginal pH) and immune marker analyses (protein profiles and concentrations). Antimicrobial activity of clinical CHG and PI formulations were tested in vitro using minimal inhibitory and bactericidal concentration assays. Between February 2021 and June 2022, 41 patients were enrolled. Seven patients either withdrew consent or met exclusion criteria for vaginal bleeding. Thirty-four patients had pre-operative samples collected; 13 patients were lost to follow-up. A total of 21 patients with longitudinal samples of pre- and post-operative collection contributed to this study: 13 in the CHG group and 8 in the PI group. Prior to surgery, 75–77% of women in both groups exhibited Lactobacillus dominance. PI did not change overall vaginal microbiome profiles; however, CHG impacted Lactobacillus iners-dominant profiles, shifting to other lactobacilli (50%) or dysbiotic anaerobes (33%). Lactobacillus crispatus-dominant profiles, which are optimal for vaginal health, were not impacted by either antiseptic solution. In vitro assays further confirmed higher susceptibility of L. iners to CHG solution compared to other vaginal lactobacilli species. Pro-inflammatory cytokines or chemokines were not increased in the CHG or PI group. Our study suggests that CHG does not increase the rate of post-operative vaginal dysbiosis, or genital inflammation compared to PI. Species-specific effects of CHG on vaginal lactobacilli and its clinical impact require further investigation.

Analysis of the Molecular Structure of Hydroxychavicol, a Promising Oral Antibacterial

Abstract Objectives In order to better understand hydroxychavicol’s effectiveness as an oral antibacterial, its structural components were analyzed with respect to minimum inhibitory concentrations and minimum bactericidal concentrations against various oral bacteria. These structural components include the free hydroxy groups and allyl chain connected to hydroxychavicol’s benzene core. Methods Six structural analogs of hydroxychavicol were tested against a range of oral bacteria using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. MIC results were obtained using serial microdilution techniques in 96-well plates with resazurin dye as a colorimetric indicator. Aliquots within each MIC concentration range were then placed on appropriate agar medium and the minimum bactericidal concentration was determined as the lowest concentration with no observed colony growth. Key Findings A synergistic interaction was observed between the allyl chain and hydroxy groups on the benzene core of hydroxychavicol, which resulted in lower MICs against the tested oral bacteria. It was also found that a hydroxy group para to the allyl chain on the benzene ring resulted in more effective inhibition, with a MIC of <50 μg/mL against R. dentocariosa. Additionally, analytes possessing free hydroxy groups ortho to one another on the benzene ring resulted in MICs of 200-300 μg/mL or lower, whereas analytes with free hydroxy groups meta to one another on the benzene ring exhibited MICs of >1000 μg/mL. Conclusions This study helps elucidate the structural components responsible for hydroxychavicol’s effectiveness as an oral antibacterial. The findings herein help to understand the mechanism of hydroxychavicol’s antibacterial properties and will be helpful in the design and synthesis of more effective oral antibacterial treatments.

Lippia grata Essential Oil Acts Synergistically with Ampicillin Against Staphylococcus aureus and its Biofilm.

Antimicrobial resistance (AMR) presents a global challenge as microorganisms evolve to withstand the effects of antibiotics. In addition, the improper use of antibiotics significantly contributes to the AMR acceleration. Essential oils have garnered attention for their antimicrobial potential. Indeed, essential oils extracted from plants contain compounds that exhibit antibacterial activity, including against resistant microorganisms. Hence, this study aimed to evaluate the antimicrobial and antibiofilm activity of the essential oil (EO) extracted from Lippia grata and its combination with ampicillin against Staphylococcus aureus strains (ATCC 25923, ATCC 700698, and JKD6008). The plant material (leaves) was gathered in Mossoro, RN, and the EO was obtained using the hydrodistillation method with the Clevenger apparatus. The antimicrobial activity of the EO was assessed through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Antibiofilm activity was evaluated by measuring biomass using crystal violet (CV) staining, viable cell counting, and analysis of preformed biofilms. In addition, the synergistic effects of the EO in combination with ampicillin were examined by scanning electron and confocal microscopy. The EO displayed a MIC value of 2.5mg/mL against all tested S. aureus strains and an MBC only against S. aureus JKD6008 at 2.5mg/mL. L. grata EO caused complete biofilm inhibition at concentrations ranging from 10 to 0.312mg/mL against S. aureus ATCC 25923 and 10 to 1.25mg/mL against S. aureus ATCC 700698 and S. aureus JKD6008. In the viable cell quantification assay, there was a reduction in CFU ranging from 1.0 to 8.0 logs. The combination of EO with ampicillin exhibited a synergistic effect against all strains. Moreover, the combination showed a significantly inhibiting biofilm formation and eradicating preformed biofilms. Furthermore, the EO and ampicillin (individually and in combination) altered the cellular morphology of S. aureus cells. Regarding the mechanism, the results revealed that L. grata EO increased membrane permeability and caused significant membrane damage. Concerning the synergy mechanism, the results revealed that the combination of EO and ampicillin increases membrane permeability and causes considerable membrane damage, further inhibiting bacteria synergistically. The findings obtained here suggest that L. grata EO in combination with ampicillin could be a viable treatment option against S. aureus infections, including MRSA strain.

Fabrication and Therapeutic Process of a Green Silver-Nanoparticle-Embedded Mucilage Microsphere for Pathogenic-Bacteria-Infected Second-Degree Burn and Excision Wounds.

Multidrug-resistant bacteria are a serious problem in biomedical applications that decrease the wound healing process and increase the mortality rate. Therefore, in this study, we have prepared a green-synthesized silver-nanoparticle-encapsulated mucilage microsphere (HMMS@GSNP) from Hibiscus rosa sinensis leaves and applied it to pathogen-infected burn and excision wounds. Biophysical properties like size, polydispersity index, absorbance capacity, and drug release were measured by different techniques like field-emission scanning electron microscopy, dynamic light scattering, swelling ratio, etc. The strong antibacterial activity of a HMMS@GSNP microsphere was measured by minimum inhibitory concentration assay, minimum bactericidal concentration assay, and agar well diffusion methods. The HMMS@GSNP microsphere enhanced the cell viability, cell proliferation, migration, antioxidant, and antiinflammation activity compared to untreated GSNP and HMMS, as quantified by MTT assay, BrdU assay, scratch wound assay, reactive oxygen species scavenging assay, and Western blot analysis, respectively. In the in vivo experiment, we used a methicillin-resistant Staphylococcus aureus bacteria-infected, burn-and-excision-wound-created male BALB/c mice model. The HMMS@GSNP-treated burn-and-excision-wound-infected mice showed significant results compared to other groups (untreated, Silverex Ionic Gel, AgNO3, HMMS, and GSNP), and the mice tissues were utilized for bacteria count, immunoblot analysis, histological studies, and real-time polymerase chain reaction. Thus, the HMM@GSNP microsphere is an excellent therapeutic material that can be used as a topical agent for the management of chronic wound therapy.

Antibiofilm Efficacy and Mode of Action of Etlingera elatior Extracts Against Staphylococcus aureus

Staphylococcus aureus represents a major bacterial human pathogen that causes a wide variety of clinical manifestations. Various medicinal plants have been used to control its infection, however, the effect of Etlingera elatior on S. aureus biofilm is still uncertain. Thus, the objective of this study was to evaluate the antibacterial and antibiofilm efficacy of E. elatior extracts against S. aureus. Phytochemical screening was carried out to determine the presence of phenols, tannins, saponins, and alkaloids in different extracts (acetone, methanol, ethanol, and aqueous) of E. elatior. Antibacterial activities were determined by disk diffusion assay, minimum inhibitory concentration assay (MIC), and minimum bactericidal concentration assay (MBC) while antibiofilm activities were determined by crystal violet assay and Fourier-transform infrared (FTIR) spectroscopy. All the extracts were found to contain phenols, tannins, saponins, and alkaloids. Only acetone extract showed a high amount of saponins. Among all the extracts, acetone extract showed the widest inhibition zone (21.23±0.2 mm), lowest MIC (20 mg/mL), and lowest MBC (50 mg/mL) values. The acetone extract also showed the highest antibiofilm activities at all biofilm stages (6 hr: 12%-31%; 12 hr: 20%-36%; 18 hr: 27%-32%; 24 hr: 5%-46%). Further analysis with FTIR spectroscopy revealed spectral changes associated with proteins (1700–1400 cm-1), phospholipids, and polysaccharides (1300–700 cm-1) in S. aureus biofilm following the treatment with 200 mg/mL of E. elatior extracts. In conclusion, E. elatior is a potential source of antibacterial and antibiofilm agents to control S. aureus infections. Changes in the composition of proteins, phospholipids, and polysaccharides may mediate the biofilm inhibition by E. elatior extracts. The acetone extract of E. elatior may be useful for various applications such as antimicrobial topical cream and wound dressing.

1,3,6-Trigalloylglucose: A Novel Potent Anti-Helicobacter pylori Adhesion Agent Derived from Aqueous Extracts of Terminalia chebula Retz.

1,3,6-Trigalloylglucose is a natural compound that can be extracted from the aqueous extracts of ripe fruit of Terminalia chebula Retz, commonly known as "Haritaki". The potential anti-Helicobacter pylori (HP) activity of this compound has not been extensively studied or confirmed in scientific research. This compound was isolated using a semi-preparative liquid chromatography (LC) system and identified through Ultra-high-performance liquid chromatography-MS/MS (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR). Its role was evaluated using Minimum inhibitory concentration (MIC) assay and minimum bactericidal concentration (MBC) assay, scanning electron microscope (SEM), inhibiting kinetics curves, urea fast test, Cell Counting Kit-8 (CCK-8) assay, Western blot, and Griess Reagent System. Results showed that this compound effectively inhibits the growth of HP strain ATCC 700392, damages the HP structure, and suppresses the Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Importantly, it exhibits selective antimicrobial activity without impacting normal epithelial cells GES-1. In vitro studies have revealed that 1,3,6-Trigalloylglucose acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, a critical step in HP infection. These findings underscore the potential of 1,3,6-Trigalloylglucose as a targeted therapeutic agent against HP infections.

Xenomyrothecium tongaense PTS8: a rare endophyte of Polianthes tuberosa with salient antagonism against multidrug-resistant pathogens.

Endophytes refer to microorganisms residing within the endosphere of plants, particularly perennials, without inflicting noticeable injury or inducing obvious morphological variations to their host plant or host organism. Endophytic fungi, although often overlooked microorganisms, have garnered interest due to their significant biological diversity and ability to produce novel pharmacological substances. In this study, fourteen endophytic fungi retrieved were from the stem of the perennial plant Polianthes tuberosa of the Asparagaceae family. These fungal crude metabolites were tested for antagonistic susceptibility to Multi-Drug Resistant (MDR) pathogens using agar well diffusion, Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) assays. The chequerboard test was used to assess the synergistic impact of active extract. In early antibacterial screening using the Agar plug diffusion test, three of fourteen endophytes demonstrated antagonism against Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus (VRE). Three isolates were grown in liquid medium and their secondary metabolites were recovered using various organic solvents. Eight extracts from three endophytic fungi displayed antagonism against one or more human pathogens with diameters ranging from 11 to 24 mm. The highest antagonistic effect was obtained in ethyl acetate extract for PTS8 isolate against two MRSA (ATCC 43300, 700699) with 20 ± 0.27 and 22 ± 0.47 mm zones of inhibition, respectively, among different solvent extracts. The extract had MICs of 3.12 ± 0.05 and 1.56 ± 0.05 μg/mL, and MBCs of 50 ± 0.01 and 12.5 ± 0.04 μg/mL, respectively. Antagonism against VRE was 18 ± 0.23 mm Zone of Inhibition (ZOI) with MIC and MBC of 6.25 ± 0.25 and 25 ± 0.01 μg/mL. When ethyl acetate extract was coupled with antibiotics, the chequerboard assay demonstrated a synergistic impact against MDR bacteria. In an antioxidant test, it had an inhibitory impact of 87 ± 0.5% and 88.5 ± 0.5% in 2,2-Diphenyl-1-Picrylhydrazyl and reducing power assay, respectively, at 150 μg/mL concentration. PTS8 was identified as a Xenomyrothecium tongaense strain by 18S rRNA internal transcribed spacer (ITS) sequencing. To our insight, it is the foremost study to demonstrate the presence of an X. tongaense endophyte in the stem of P. tuberosa and the first report to study the antibacterial efficacy of X. tongaense which might serve as a powerful antibacterial source against antibiotic-resistant human infections.

Antibacterial activity and mechanism of chelerythrine against Streptococcus agalactiae.

Streptococcus agalactiae (S.agalactiae), also known as group B Streptococcus (GBS), is a highly infectious pathogen. Prolonged antibiotic usage leads to significant issues of antibiotic residue and resistance. Chelerythrine (CHE) is a naturally occurring benzophenidine alkaloid and chelerythrine chloride (CHEC) is its hydrochloride form with diverse biological and pharmacological activities. However, the antibacterial mechanism of CHEC against GBS remains unclear. Thus, this study aims to investigate the in vitro antibacterial activity of CHEC on GBS and elucidate its underlying mechanism. The antibacterial effect of CHEC on GBS was assessed using inhibitory zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays, as well as by constructing a time-kill curve. The antibacterial mechanism of CHEC was investigated through techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), measurement of alkaline phosphatase (AKP) activity, determination of Na+ K+, Ca2+ Mg2+-adenosine triphosphate (ATP) activity, observation of membrane permeability, and analysis of intracellular reactive oxygen species (ROS) and mRNA expression levels of key virulence genes. The results demonstrated that the inhibition zone diameters of CHEC against GBS were 14.32 mm, 12.67 mm, and 10.76 mm at concentrations of 2 mg/mL, 1 mg/mL, and 0.5 mg/mL, respectively. The MIC and MBC values were determined as 256 μg/mL and 512 μg/mL correspondingly. In the time-kill curve, 8 × MIC, 4 × MIC and 2 × MIC CHEC could completely kill GBS within 24 h. SEM and TEM analyses revealed significant morphological alterations in GBS cells treated with CHEC including shrinkage, collapse, and leakage of cellular fluids. Furthermore, the antibacterial mechanism underlying CHEC's efficacy against GBS was attributed to its disruption of cell wall integrity as well as membrane permeability resulting in extracellular release of intracellular ATP, AKP, Na+ K+, Ca2+ Mg2+. Additionally CHEC could increase the ROS production leading to oxidative damage and downregulating mRNA expression levels of key virulence genes in GBS cells. In conclusion, CHEC holds potential as an antimicrobial agent against GBS and further investigations are necessary to elucidate additional molecular mechanisms.

Whole-transcriptome analysis reveals mechanisms underlying antibacterial activity and biofilm inhibition by a malic acid combination (MAC) in Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is a highly prevalent bacterial species known for its ability to cause various infections and its remarkable adaptability and biofilm-forming capabilities. In earlier work, we conducted research involving the screening of 33 metabolites obtained from a commercial source against two prevalent bacterial strains, Escherichia coli and Staphylococcus aureus. Through screening assays, we discovered a novel malic acid combination (MAC) consisting of malic acid, citric acid, glycine, and hippuric acid, which displayed significant inhibitory effects. However, the precise underlying mechanism and the potential impact of the MAC on bacterial biofilm formation remain unknown and warrant further investigation. Methods To determine the antibacterial effectiveness of the MAC against Pseudomonas aeruginosa, we conducted minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques were employed to observe bacterial morphology and biofilm formation. We further performed a biofilm inhibition assay to assess the effect of the MAC on biofilm formation. Whole-transcriptome sequencing and bioinformatics analysis were employed to elucidate the antibacterial mechanism of the MAC. Additionally, the expression levels of differentially expressed genes were validated using the real-time PCR approach. Results Our findings demonstrated the antibacterial activity of the MAC against P. aeruginosa. SEM analysis revealed that the MAC can induce morphological changes in bacterial cells. The biofilm assay showed that the MAC could reduce biofilm formation. Whole-transcriptome analysis revealed 1093 differentially expressed genes consisting of 659 upregulated genes and 434 downregulated genes, in response to the MAC treatment. Mechanistically, the MAC inhibited P. aeruginosa growth by targeting metabolic processes, secretion system, signal transduction, and cell membrane functions, thereby potentially compromising the survival of this human pathogen. This study provides valuable insights into the antibacterial and antibiofilm activities of the MAC, a synergistic and cost-effective malic acid combination, which holds promise as a potential therapeutic drug cocktail for treating human infectious diseases in the future.

Tetraclinis articulata (Vahl) Mast. essential oil as a promising source of bioactive compounds with antimicrobial, antioxidant, anti-inflammatory and dermatoprotective properties: In vitro and in silico evidence

Tetraclinis articulata is a known traditional medicinal plant used to manage various ailments, such as diabetes, rheumatism and infectious diseases. This study aims to determine the chemical constituents of T. articulata essential oil (EO) and to evaluate its in vitro antibacterial, anti-candidal, antioxidant, anti-inflammatory and dermatoprotective properties. In addition, a computational docking approach was used to predict the potential antioxidant, antibacterial, antifungal, anti-inflammatory, and cytotoxic properties of the identified compounds. The volatile oil obtained by hydrodistillation was characterized using gas chromatography-mass spectrometry (GC-MS). The antioxidant activity of T. articulata EO was investigated using three complementary assays: DPPH, ABTS and FRAP. Lipoxygenase (5-LOX) and tyrosinase enzymes were used to assess the anti-inflammatory and dermatoprotective effects of this oil. Moreover, disc-diffusion technique, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were employed for the antimicrobial screening. The GC-MS analysis revealed that bornyl acetate (41.80 %), α-pinene (17.97 %) and camphor (15.97 %) are the major components of the studied EO. Moreover, T. articulata EO has exhibited promising antioxidant effect on FRAP, DPPH, and ABTS experiments. It also significantly inhibited 5-LOX (IC50 = 67.82 ± 0.03 μg/mL) and tyrosinase (IC50 = 211.93 ± 0.02 μg/mL). The results of MIC and MBC assays indicated that T. articulata EO is able to inhibit the growth of all tested bacteria (Gram + and Gram -) and Candida species. The ratio of tolerance level indicated that the tested oil was bactericidal against the Gram + bacteria and Candida species, whereas it has a bacteriostatic behavior against the Gram− bacteria. In light of these findings, T. articulata EO may be suggested as a potential pharmaceutical agent to prevent inflammation and skin problems and may serve as a natural antimicrobial and antioxidant alternative for sustainable application in food products.

Electrospun PVA Nanofibers Loaded with Antimicrobial Herbal Extracts for Healing the Infectious Wound

Background: Low-cost, soluble polyvinyl alcohol (PVA) polymers enhance the efficacy of herbal formulations with established antimicrobial properties. Objectives: This study investigates the healing process of Staphylococcus aureus (S. aureus)-infected wounds using PVA loaded with herbal extracts, including Arnebia euchroma, Allium sativum, and Echinacea purpurea. Methods: minimal bactericidal concentration (MBC) and minimal inhibitory concentration (MIC) assays, Disk Diffusion Method (DDM) tests, and Gas Chromatography-Mass Spectrometry (GC/MS) analyses were conducted on herbal extract samples. Twelve male Wistar rats were divided into G1: Negative control group (healthy mice), G2: Wound model + S. aureus (Positive control), G3: wound model + S. aureus + Povidone-iodine, and G4: Wound model + S. aureus + PVA/formulation. Hematoxylin-eosin and immunofluorescent staining were employed to assess wound healing. Results: The ethanolic herbal extract exhibited potent antibacterial activity against S. aureus, with MIC and MBC values in the 1.87 mg/mL range. The PVA/formulation displayed a zone of inhibition with a diameter of 7 mm against S. aureus. Histopathological investigations indicated that the infected wound treated with Nanofibers extract experienced a significant reduction in inflammation between days 7 and 14. Notably, the increased CD3 expression at this site was remarkable for the healing process. Conclusions: Consequently, this experimental study suggests combining PVA and herbal extracts enhances antibacterial properties and promotes CD3 expression and re-epithelialization effects.

Excretory-secretory products from adult helminth Nippostrongylus brasiliensis have in vitro bactericidal activity.

Introduction. Intestinal helminths and microbiota share the same anatomical niche during infection and are likely to interact either directly or indirectly. Whether intestinal helminths employ bactericidal strategies that influence their microbial environment is not completely understood.Hypothesis. In the present study, the hypothesis that the adult hookworm Nippostrongylus brasiliensis produces molecules that impair bacterial growth in vitro, is tested.Aim. To investigate the in vitro bactericidal activity of Nippostrongylus brasiliensis against commensal and pathogenic bacteria.Methodology. The bactericidal effect of somatic extract and excretory-secretory products of adult Nippostrongylus brasiliensis on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae) bacteria was assessed using growth assays. Minimum inhibitory concentration and minimum bactericidal concentration assays were performed using excretory-secretory products released from the pathogen.Results. Broad-spectrum in vitro bactericidal activity in excretory-secretory products, but not somatic extract of adult Nippostrongylus brasiliensis was detected. The bactericidal activity of excretory-secretory products was concentration-dependent, maintained after heat treatment, and preserved after repeated freezing and thawing.Conclusion. The results of this study demonstrate that helminths such as Nippostrongylus brasiliensis release molecules via their excretory-secretory pathway that have broad-spectrum bactericidal activity. The mechanisms responsible for this bactericidal activity remain to be determined and further studies aimed at isolating and identifying active bactericidal molecules are needed.

Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Antibacterial and antibiofilm activity of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles and thiazoles in multidrug-resistant pathogens.

To find novel antibiotic drugs, six 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H derivatives named 1b, 1d (pyrazoles), 2a, 2b, 2c, and 2d (thiazoles) were evaluated in silico and in vitro. The in silico analyses were based on ADME pharmacokinetic parameters (absorption, distribution, metabolism, and excretion). The in vitroantibacterial activity was evaluated in Gram-positive and Gram-negative species (Staphylococcus aureus ATCC® 25904, Staphylococcus epidermidis ATCC® 35984, Klebsiella pneumoniae ATCC® 700603, and Acinetobacter baumannii ATCC® 19606), by determination of minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), kinetics curve, and antibiofilm assays. As results, the azoles have activity against the Gram-negative species K. pneumoniae ATCC® 700603 and A. baumannii ATCC® 19606. No antibacterial activity was observed for the Gram-positive bacteria evaluated.Thus, the azoles were evaluated against clinical isolates of K. pneumoniae carbapenemase (KPC) and A. baumannii multidrug-resistant (Ab-MDR). All azoles have antibacterial activity against Ab-MDR isolates (Gram-negative) with MIC values between 512μg/mL and 1,024μg/mL. Against KPC isolates theazoles1b, 1d, and 2d present antibacterial activity (MIC = 1,024μg/mL). In the kinetics curve assay, the 1b and 1d pyrazoles reduced significantly viable cells of Ab-MDR isolates and additionally inhibited 86.6 to 95.8% of the biofilm formation. The in silico results indicate high possibility to permeate the blood-brain barrier (2b) and was predicthuman gastrointestinal absorption (all evaluated azoles). Considering that the research and development of new antibiotics is a priority for drug-resistant pathogens, our study revealed the antibacterial and antibiofilm activity of novel azoles against K. pneumoniae and A. baumannii pathogens.

Antimicrobial and Antibiofilm Activity of Synthetic Peptide [W7]KR12-KAEK Against Enterococcus faecalis Strains.

The emergence of infections caused by microorganisms in the oral cavity and increasing concerns regarding the use of antibiotics have resulted in the development of novel antimicrobial molecules, such as antimicrobial synthetic peptides. The purpose of this study was to evaluate the antimicrobial and antibiofilm activities of the native peptide KR-12 and its derivative, the synthetic peptide [W7]KR12-KAEK, against planktonic and biofilms Enterococcus faecalis strains. The methods used to evaluate the antimicrobial activity in planktonic cultures include minimum inhibitory concentration and minimum bactericidal concentration assays. The effects of [W7]KR12-KAEK on biofilm formation and mature biofilms were evaluated by quantifying biomass (crystal violet staining) and counting colony-forming units. Structural assessments of the biofilms and cellular morphological changes were performed using scanning electron microscopy. Peptide [W7]KR12-KAEK showed potential antimicrobial activity against planktonic cells. Interestingly, the native peptide KR-12 showed no antimicrobial activity. Moreover, it inhibited biofilm formation and disrupted the mature biofilms of E. faecalis strains. These results suggest that [W7]KR12-KAEK may be a potential molecule for the development of auxiliary antimicrobial therapies against oral infections.

Antimicrobial, anti-inflammatory and anti-arthritic activity of hemolymph lectin (NagLec) isolated from the freshwater crab, Oziotelphusanaga

Lectins are non-immune glycoproteins or proteins having a unique capacity to interact with carbohydrate ligands found on the surface of their host cells. In the present investigation, the lectin was purified from the hemolymph of freshwater crab, Oziotelphusa naga and its antimicrobial, anti-inflammatory and anti-arthritic activity was analysed. The preliminary characterization of the hemagglutinin was carried out to identify the erythrocyte and sugar specificity, optimum pH and temperature and cation dependency. The agglutinin was found to be highly specific to rabbit erythrocyte and inhibited by fetuin and α-lactose. Maximum hemagglutination activity was noted at pH 7.5–8 and temperature 20–40 °C. An O-acetyl sialic acid specific 75 kDa hemolymph lectin, designated as NagLec was isolated from the freshwater crab, Oziotelphusa naga by affinity chromatography on fetuin coupled Sepharose 4 B, with a purification fold of 185. The bacteria Staphylococcus aureus, Proteus mirabilis and fungus Candida albicans had the greatest zone of inhibition when treated with NagLec. The results of the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assays showed that the purified lectin inhibited the growth of Staphylococcus aureus at 0.031 and 0.065 μg/ml, which proved the bactericidal property of NagLec. NagLec generated alterations on the bacterial cells and led to protein leakage, which was dosage (24 and 48 μg/ml) and time dependent (10–40 min). COX and LOX enzyme was inhibited to 49.43% and 61.81% with 100 μg/ml concentration of NagLec respectively, demonstrating NagLec's ability to reduce inflammation. Furthermore, NagLec (500 μg) suppressed protein denaturation up to 77.12% whereas diclofenac sodium (a standard drug) was inhibited by 89.36%. The results indicate that NagLec, a sialic acid specific lectin isolated from the freshwater crab O. naga could be formulated as a nano drug in future owing to its antimicrobial, anti-inflammatory and anti-arthritic potential that could be targeted to specific pathogenic microbes and treat arthritis.

Epiberberine inhibits Helicobacter pylori and reduces host apoptosis and inflammatory damage by down-regulating urease expression

Ethnopharmacological relevanceWith dramatically increasing antibiotic resistance in Helicobacter pylori (H. pylori), it is urgent to find alternative therapeutic agents. Rhizoma Coptidis is a traditional Chinese medicine for gastrointestinal diseases and shows excellent anti-H. pylori effect. Epiberberine (EPI), as one of the major alkaloids of Rhizoma Coptidis, has been reported to have urease-inhibiting activity, but its scavenging effect on H. pylori and the potential mechanism remain unclear. Aim of the studyTo investigate the inhibitory effect of EPI on H. pylori and explore its multi-action on Helicobacter pylori urease (HPU). Materials and methodsUsing minimum inhibitory concentration (MIC) assay, minimum bactericidal concentration (MBC) assay, growth inhibition kinetics assay, bacterial resistance development, transmission electron microscope (TEM) assay, and animal experiments to investigate the inhibitory effect of EPI on H. pylori in vitro and in vivo. Using the Berthelot method, molecular docking and thermal displacement experiments to verify that EPI inhibits urease activity by interacting with HPU. Using transcriptome data, Real-Time PCR (RT-PCR) experiments to investigate the alterations in the expression of urease subunit ureB gene after EPI treatment. Using MTT cell viability assay, Hoechst 33342 staining method, JC-1 reagent detection method, western blot experiments, and Griess method to investigate the anti-apoptosis and anti-inflammation actions of EPI on gastric epithelial cells (GES-1) induced by HPU. ResultsIn vitro experiments proved that EPI has significant anti-H. pylori activity without drug resistance, induces H. pylori fragmentation and apoptosis. In vivo experiments showed that EPI has a certain clearance effect of H. pylori, and can reduce gastric inflammation caused by H. pylori infection. Transcriptome data, RT-PCR experiments, and other experiments demonstrate that EPI has a triple effect: (1) inhibiting the expression of HPU subunits ureB, (2) directly inhibiting urease activity by interacting with HPU, and (3) inhibiting HPU-induced apoptosis and inflammation in GES-1. ConclusionsEPI is an excellent anti-H. pylori agent and reduces host apoptosis and inflammation by inhibiting the activity of urease and down-regulating the expression of ureB.