Surface Disinfection and Sanitizing Action of the Alcohol-Free Essential Oil-based Green Formulation.

The surface disinfectant property of a prepared formulation using potential and effective EO (Murraya koenigii), phytochemical (Geraniol), and an amino acid epsilon-L-Poly-Lysine (ɛ-PL) is examined in this present study. To investigate its potential as a surface disinfectant (SD) different tests using multiple bacterial strains were conducted. All tested bacterial strains were inhibited by the SD treatments, with a MIC range of (0.78-3.12%) v/v. Notably, Staphylococcus sp. was found to be more susceptible to the treatment than its gram-negative counterparts. In the test, sterile stainless-steel surfaces were used and externally contaminated with Escherichia sp. Cleaning the surface with the prepared formulation was more effective than the equal concentration of vinegar in terms of bacterial growth reduction. Vinegar was used as a mother solvent in the preparation of the SD due to its proven antibacterial effect. It is worth mentioning, this formulation is also proven to be effective on biofilm-embedded bacterial cells of Pseudomonas aeruginosa PAO1 as found in epifluorescence microscopy staining. Even though the impact of each constituent needs to be further explored, the effectiveness of this formulation may encourage large farms to seek out alternatives that are more environmentally friendly, safe, and effective than conventional products.

Diketopiperazine derivative from marine actinomycetes Nocardiopsis sp. SCA30 with antimicrobial activity against MRSA.

Actinobacteria isolated from marine sources are a potential source of novel natural products. In this study, we report isolation, biological activity and characterization of secondary metabolites from strain Nocardiopsis sp. SCA30, isolated from marine sediments of Havelock Islands, Andaman and Nicobar, India. The ethyl acetate extracts of the isolate on screening for biological activity demonstrated antibacterial potency and antiproliferative activity. The extracts showed anticancer activity in a panel of cell lines, including HCT 15, HT 29, MCF 7 and MDA-MB 468, at concentrations ranging from 62.5 to 1000 µg/ml. A dose-dependent reduction in cell viability was observed in all the tested cell lines. The extract at 15 µg/ml and 30 µg/ml inhibited growth of methicillin-resistant Staphylococcus aureus ATCC NR-46071 and NR-46171 with MIC's of 15.62 and 7.81 µg/ml, respectively. LC-MS and NMR studies revealed that the antibacterial and anticancer compound isolated from Nocardiopsis sp. SCA30 is 1-acetyl-4-4(hydroxyphenyl)piperazine.

Quorum sensing modulatory and biofilm inhibitory activity of Plectranthus barbatus essential oil: a novel intervention strategy.

The essential oil (EO) from the roots of Plectranthus barbatus Andr. (Syn. Coleus forskohlii Briq.) was evaluated for quorum sensing (QS) inhibitory activity. P. barbatus EO was screened for inhibition of QS regulated violacein production in Chromobacterium violaceum (ATCC 12472) wild-type strain. At inhibitory (6.25% v/v) and sub-inhibitory concentrations (3.125% v/v) of the EO, dose-dependent response in the inhibition of violacein production was observed in C. violaceum. Similarly, sub-MIC (6.25% v/v) of P. barbatus EO disrupted QS regulated biofilm formation by 27.87% and inhibited swarming and twitching motility in Pseudomonas aeruginosa PA01 implying its anti-infective and QS modulatory activity. Fluorescence microscopy studies confirmed the disruption of biofilm formation by EO in P. aeruginosa PAO1. Promising antibacterial activity was recorded at concentrations as low as 3.12% v/v for Listeria monocytogenes (ATCC 13932) and at 6.25% v/v for both Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 25241) and Escherichia coli (ATCC 11775). Furthermore, significant dose-dependent inhibition was observed for biofilm formation and motility in all the tested pathogens in different treated concentrations. GC-MS analysis revealed α-pinene, endo-borneol, bornyl acetate, 1-Hexyl-2-Nitrocyclohexane as the major phytoconstituents. P. barbatus EO or its constituent compounds with QS modulatory, antimicrobial and biofilm inhibitory property could be potential new-age dietary source based intervention and preservation technologies.

Culturable diversity of bacterial endophytes associated with medicinal plants of the Western Ghats, India.

Bacterial endophytes are found in the internal tissues of plants and have intimate associations with their host. However, little is known about the diversity of medicinal plant endophytes (ME) or their capability to produce specialised metabolites that may contribute to therapeutic properties. We isolated 75 bacterial ME from 24 plant species of the Western Ghats, India. Molecular identification by 16S rRNA gene sequencing grouped MEs into 13 bacterial genera, with members of Gammaproteobacteria and Firmicutes being the most abundant. To improve taxonomic identification, 26 selected MEs were genome sequenced and average nucleotide identity (ANI) used to identify them to the species-level. This identified multiple species in the most common genus as Bacillus. Similarly, identity of the Enterobacterales was also distinguished within Enterobacter and Serratia by ANI and core-gene analysis. AntiSMASH identified non-ribosomal peptide synthase, lantipeptide and bacteriocin biosynthetic gene clusters (BGC) as the most common BGCs found in the ME genomes. A total of five of the ME isolates belonging to Bacillus, Serratia and Enterobacter showed antimicrobial activity against the plant pathogen Pectobacterium carotovorum. Using molecular and genomic approaches we have characterised a unique collection of endophytic bacteria from medicinal plants. Their genomes encode multiple specialised metabolite gene clusters and the collection can now be screened for novel bioactive and medicinal metabolites.

Anti-quorum sensing and biofilm inhibitory activity of Apium graveolens L. oleoresin.

Apium graveolens L. (Apiaceae) is a dietary herb used as a spice, condiment and medicine. A. graveolens (Celery) has been studied for its antimicrobial property and for its application as flavours in food industry. The present study investigated the Apium graveolens oleoresin as an anti-quorum sensing and antibiofilm agent. The quorum sensing and biofilm inhibition study was carried out using biosensor strains Chromobacterium violaceum CV12472 and Pseudomonas aeruginosa PAO1. The MIC of celery oleoresin against C. violaceum CV12472 and P. aeruginosa PAO1 was 10 and 25% v/v, respectively. Inhibition of violacein and biofilm formation was tested at concentrations of oleoresins ranging from 1.56 and 50% v/v. The oleoresins showed a concentration dependent QS inhibitory activity and at sub-MIC of 6.25 and 12.5% v/v, the oleoresins significantly inhibited violacein production and biofilm formation (p < 0.05). Similarly, the celery oleoresin had significant QS modulatory effect on swimming, swarming and twitching motility in P. aeruginosa PAO1 at 12.5% v/v (p < 0.05). The major phytoconstituents present in celery oleoresin as analysed by GC-MS were eicosadiene, benzenemethanol and methyl ester which have not been previously reported. The findings suggest that celery has QS and biofilm inhibitory potential against gram negative pathogens and can find application as food intervention techniques.

Diversity and Bioactive Potential of Actinobacteria from Unexplored Regions of Western Ghats, India.

The search for novel bioactive metabolites continues to be of much importance around the world for pharmaceutical, agricultural, and industrial applications. Actinobacteria constitute one of the extremely interesting groups of microorganisms widely used as important biological contributors for a wide range of novel secondary metabolites. This study focused on the assessment of antimicrobial and antioxidant activity of crude extracts of actinobacterial strains. Western Ghats of India represents unique regions of biologically diverse areas called "hot spots". A total of 32 isolates were obtained from soil samples of different forest locations of Bisle Ghat and Virjapet situated in Western Ghats of Karnataka, India. The isolates were identified as species of Streptomyces, Nocardiopsis, and Nocardioides by cultural, morphological, and molecular studies. Based on preliminary screening, seven isolates were chosen for metabolites extraction and to determine antimicrobial activity qualitatively (disc diffusion method) and quantitatively (micro dilution method) and scavenging activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS(2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals. Crude extracts of all seven isolates exhibited fairly strong antibacterial activity towards MRSA strains (MRSA ATCC 33591, MRSA ATCC NR-46071, and MRSA ATCC 46171) with MIC varying from 15.62 to 125 µg/mL, whereas showed less inhibition potential towards Gram-negative bacteria Salmonella typhi (ATCC 25241) and Escherichia coli (ATCC 11775) with MIC of 125-500 µg/mL. The isolates namely S1A, SS5, SCA35, and SCA 11 inhibited Fusarium moniliforme (MTCC 6576) to a maximum extent with MIC ranging from 62.5 to 250 µg/mL. Crude extract of SCA 11 and SCA 13 exhibited potent scavenging activities against DPPH and ABTS radicals. The results from this study suggest that actinobacterial strains of Western Ghats are an excellent source of natural antimicrobial and antioxidant compounds. Further research investigations on purification, recovery, and structural characterization of the active compounds are to be carried out.

Quorum quenching activity of AiiA lactonase KMMI17 from endophytic Bacillus thuringiensis KMCL07 on AHL- mediated pathogenic phenotype in Pseudomonas aeruginosa.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation to disrupt quorum-sensing in these bacteria could pave the way for the new development in decreasing resistance strains and are of significant interest for clinical, agricultural, and industrial applications. Isolated endophytic Bacillus thuringiensis strain KMCL07 showing quorum quenching activity on Pseudomonas aeruginosa PAO1 has been studied. AiiA lactonase KMMI17 identified belongs to metallo- ß-lactamase superfamily preserving conserved regions of 106HXDH-59 amino acids-H169-21 amino acids-D191 motif, significantly inhibits the biofilm formation and attenuates virulence factor pyocyanin production of PAO1. Insilico molecular docking analysis of lactonase KMMI17 using alternative catalytic site (PDB entry: 3DHA) with the AHL-based QS system regulators of PAO-1, C4 AHL, C6 AHL and 3-oxo-C12 AHL molecules showed good binding affinity between the protein and ligands, Phe111 and Tyr198 residues plays an important role in binding them. Crude enzyme extract was found to have Km value for C6-HSL: 134.2702 ±â€¯34.83 µM-1, C4-HSL: 308.217 ±â€¯139.9 µM-1 and 3-oxo-C12-HSL: 760.463 ±â€¯251.3 µM-1. LCMS analysis confirms the degradation activity of lactonase KMMI17 on AHL molecules and its hydrolytic process, which indicates the potential application of lactonase KMMI17 as a biocontrol agent or an anti-pathogenic drug.

Isolation, characterization, and structural elucidation of 4-methoxyacetanilide from marine actinobacteria Streptomyces sp. SCA29 and evaluation of its enzyme inhibitory, antibacterial, and cytotoxic potential.

Marine actinobacteria are less explored than their terrestrial counterparts as potential source of natural products. The present study was aimed to elucidate the bioactive potential of metabolites produced by marine-derived actinobacterial strain Streptomyces sp.SCA29 isolated from Havelock Island, Andaman and Nicobar Islands, India. The potential isolate SCA29 was identified as Streptomyces sp. by phenotypic, genotypic (16S-rRNA) and phylogenetic analyses. The crude bioactive compound was extracted using organic solvents. The compounds were subjected to separation and purification by column chromatography which yielded six fractions. Each fraction was assayed for inhibition of α-glucosidase and α-amylase enzymes, antagonistic activity against bacterial pathogens, and cytotoxic activity against various cell lines. The fraction F3c was considered to be highly active owing to its significant inhibition potential against α-glucosidase and α-amylase enzymes with IC50 values as 44.26 and 53.19 µg/mL, respectively. The active fraction showed antibacterial activity against test bacterial pathogens with the MIC value ranged from 3.90 to 31.25 µg/mL. The compound also exhibited concentration-dependent cytotoxicity on various cell lines without significant effect against human normal cells. The bioassay-guided fractionation of extract led to the identification of 4-methoxyacetanilide, an acetamide derivative. The structure of the bioactive compound was confirmed by HR-MS, NMR (1H and 13C) and FT-IR spectra, and by comparison with literature data.

Endophytic Paenibacillus amylolyticus KMCLE06 Extracted Dipicolinic Acid as Antibacterial Agent Derived via Dipicolinic Acid Synthetase Gene.

Bioactive natural compounds play pivotal roles in drug discovery and the emergence of multi-drug resistance pathogens demands the development of better/new drugs. Paenibacillus amylolyticus KMCLE06 endophytic bacterium isolated from the medicinal plant Coix lachryma-jobi were analyzed for the potential bioactive secondary metabolite compounds and its gene responsible within polyketide synthases (PKS) clusters. Ethyl acetate extraction of P. amylolyticus KMCLE06 showed significant antibacterial activity which was further processed to partial purification and characterization for bioactive compound. The foremost bioactive component in extraction was found to be dipicolinic acid (DPA). The antibacterial activity showed remarkable activity compared to the commercial standard DPA against both gram-positive and gram-negative pathogens. The MIC and MBC concentrations for partially purified extracted DPA ranged from 62.5 to 125 µg/ml and MBC from 208 to 250 µg/ml, respectively. Sequence analysis of gene amplified using degenerative primer, amplified 543 bp DNA region, revealing conserved putative open reading frame for dipicolinic acid synthetase (DpsA) key gene to produce DPA in most endospore forming bacteria. A search in the structural database for DpsA revealed significant homologous match with enoyl reductase one of the PKS type 1 module protein. This emphasizes endophytic P. amylolyticus KMCLE06 bacteria has presence of spoVF operon producing DPA via dipicolinic acid synthetase and lacks the polyketide synthase type 1 module cluster gene in its genome. And the bioactive compound DPA extracted acts as a stable remarkable antibacterial agent which can be potent compound for multi-resistance pathogens.

Evaluation of Antimicrobial, Enzyme Inhibitory, Antioxidant and Cytotoxic Activities of Partially Purified Volatile Metabolites of Marine Streptomyces sp.S2A.

In the present study, marine actinobacteria Streptomyces sp.S2A was isolated from the Gulf of Mannar, India. Identification was carried out by 16S rRNA analysis. Bioactive metabolites were extracted by solvent extraction method. The metabolites were assayed for antagonistic activity against bacterial and fungal pathogens, inhibition of α-glucosidase and α-amylase enzymes, antioxidant activity and cytotoxic activity against various cell lines. The actinobacterial extract showed significant antagonistic activity against four gram-positive and two gram-negative pathogens. Excellent reduction in the growth of fungal pathogens was also observed. The minimum inhibitory concentration of the partially purified extract (PPE) was determined as 31.25 µg/mL against Klebsiella pneumoniae, 15.62 µg/mL against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus. The lowest MIC was observed against Micrococcus luteus as 7.8 µg/mL. MIC against fungal pathogens was determined as 62.5 µg/mL against Bipolaris maydis and 15.62 µg/mL against Fusarium moniliforme. The α-glucosidase and α-amylase inhibitory potential of the fractions were carried out by microtiter plate method. IC50 value of active fraction for α-glucosidase and α-amylase inhibition was found to be 21.17 µg/mL and 20.46 µg/mL respectively. The antioxidant activity of partially purified extract (PPE) (DPPH, ABTS, FRAP and Metal chelating activity) were observed and were also found to have significant cytotoxic activity against HT-29, MDA and U-87MG cell lines. The compound analysis was performed using gas chromatography-mass spectrometry (GC-MS) and resulted in three constituents; pyrrolo[1⁻a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl)-, being the main component (80%). Overall, the strain possesses a wide spectrum of antimicrobial, enzyme inhibitory, antioxidant and cytotoxic activities which affords the production of significant bioactive metabolites as potential pharmacological agents.

Purification and characterisation of a quorum quenching AHL-lactonase from the endophytic bacterium Enterobacter sp. CS66.

The quorum quenching (QQ) activity of endophytic bacteria associated with medicinal plants was explored. Extracts of the Gram-negative Enterobacter sp. CS66 possessed potent N-acylhomoserine lactone (AHL) hydrolytic activity in vitro. Using degenerate primers, we PCR-amplified an open reading frame (denoted aiiE) from CS66 that was 96% identical to the well-characterised AHL-lactonase AiiA from Bacillus thuringiensis, but only 30% was identical to AHL-lactonases from other Gram-negative species. This confirms that close AiiA homologs can be found in both Gram-positive and Gram-negative bacteria. Purified AiiE exhibited potent AHL-lactonase activity against a broad range of AHLs. Furthermore, aiiE was able to reduce the production of secreted plant cell wall-degrading hydrolytic enzymes when expressed in trans in the economically important plant pathogen, Pectobacterium atrosepticum. Our results indicate the presence of a novel AHL-lactonase in Enterobacter sp. CS66 with significant potential as a biocontrol agent.

Inhibition of biofilm formation and quorum sensing mediated phenotypes by berberine in Pseudomonas aeruginosa and Salmonella typhimurium.

Quorum sensing is involved in biofilm formation and modulates virulence factor production in pathogenic bacteria. Quorum sensing inhibitors can be used as novel intervention strategies for attenuating bacterial pathogenicity. Berberine is an isoquinoline alkaloid with pharmacological properties. The present study investigated the sub-inhibitory concentrations of berberine for inhibiting biofilm formation and quorum sensing regulated phenotypes in the bacterial pathogens Pseudomonas aeruginosa PA01 and Salmonella enterica serovar Typhimurium. Berberine inhibited quorum sensing regulated violacein production in C. violaceum. It reduced the pigment production in the wild type strain at 1.6 mg mL-1 by 62.67%. In the opportunistic pathogen, P. aeruginosa PA01, at sub-MIC, it showed significant antibiofilm activity in by reducing biomass by 71.70% (p < 0.05). It prevented biofilm formation and inactivated biofilm maturation in bacterial pathogens at the concentration ranging from 0.019 to 1.25 mg mL-1. In silico studies showed that berberine interacted with the quorum sensing signal receptors, LasR and RhlR. Furthermore, its anti-infective properties in S. Typhimurium were studied. At sub-inhibitory concentrations of 0.019 mg mL-1, it reduced biofilm formation in S. Typhimurium by 31.20%. It significantly prevented invasion and adhesion of Salmonella invasion in the colonic cell, HT 29 by 55.37% and 54.68%, respectively. It was capable of reducing in vivo virulence in Caenorhabditis elegans infected with Salmonella at 0.038 mg mL-1 by 65.38%. Our results suggest that berberine, previously recognised for its antimicrobial activity, could find potential application as an anti-biofilm and anti-infective agent based on its quorum sensing inhibitory activity.

Endophytic Peptides - A Source of Therapeutic Agents.

Endophytic peptides have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to the conventional antibiotics. The world human population is increasingly facing different types of health issues. For instance, emerging wide array of the drug-resistant pathogens is a health concern. Research on bioactive agents and other natural products of microbes is essential to combat antimicrobial resistance. It is necessary to find new potential antibiotics to address this problem. The use of therapeutic plants in traditional medicine is well known. The medicinal plants contain endophytes which are repositories of bioactive compounds. The natural therapeutic agents produced by endophytes do have several potential applications in the field of pharmaceutical industry. Ecomycins, Pseudomycins, Munumbicins and Xiamycins are the antibacterial, antimycotic and antiplasmodial endophytic derived bioactive agents. Among these, some are having significant antiviral properties and inhibitory activity against plant and human bacterial pathogens. Therefore, to deal with emerging drug-resistant pathogens endophytic peptides could serve as a potential source of novel antibiotics. This review focuses on the peptides derived from plant endophytes, their biological and pharmaceutical applications, and their mechanisms of action.

In vitro antibiofilm activity of Murraya koenigii essential oil extracted using supercritical fluid CO2 method against Pseudomonas aeruginosa PAO1.

The antibiofilm activity of Murraya koenigii essential oil (EO) against Pseudomonas aeruginosa PAO1 was investigated in this study. A decrease in the production of rhamnolipid, extracellular polymeric substance and swarming motility was observed by the EO treatment (0.3% v/v). The static microtitre plate assay revealed 80% reduction in biofilm formation by P. aeruginosa PAO1 on M. koenigii EO treatment. Fluorescence microscopy and scanning electron microscopy analyses confirmed the reduction of biofilm formation in P. aeruginosa PAO1 when treated with M. koenigii EO. Gas chromatography-mass spectrometry analysis of the EO revealed the presence of well-known antibiofilm agents such as spathulenol (5.85%), cinnamaldehyde (0.37%) and linalool (0.04%). Cinnamaldehyde has not been previously reported in M. koenigii EO. The potent antibiofilm properties of M. koenigii EO may be effectively exploited in food and pharmaceutical industries as well as in controlling Pseudomonas biofilms on indwelling medical devices.