Natural biocide cocktails: Combinatorial antibiotic effects of prodigiosin and biosurfactants.
ABSTRACT: Bacterial secondary metabolites are naturally produced to prevail amongst competitors in a shared habitat and thus represent a valuable source for antibiotic discovery. The transformation of newly discovered antibiotic compounds into effective drugs often requires additional surfactant components for drug formulation. Nature may also provide blueprints in this respect: A cocktail of two compounds consisting of the antibacterial red pigment prodigiosin and the biosurfactant serrawettin W1 is naturally produced by the bacterium Serratia marcescens, which occurs in highly competitive habitats including soil. We show here a combinatorial antibacterial effect of these compounds, but also of prodigiosin mixed with other (bio)surfactants, against the soil-dwelling bacterium Corynebacterium glutamicum taken as a model target bacterium. Prodigiosin exerted a combinatorial inhibitory effect with all tested surfactants in a disk diffusion assay which was especially pronounced in combination with N-myristoyltyrosine. Minimal inhibitory and bactericidal concentrations (MIC and MBC) of the individual compounds were 2.56 ?g/mL prodigiosin and 32 ?g/mL N-myristoyltyrosine, and the MIC of prodigiosin was decreased by 3 orders of magnitude to 0.005 ?g/mL in the presence of 16 ?g/mL N-myristoyltyrosine, indicative of synergistic interaction. Investigation of bacterial survival revealed similar combinatorial effects; moreover, antagonistic effects were observed at higher compound concentrations. Finally, the investigation of microcolony formation under combined application of concentrations just below the MBC revealed heterogeneity of responses with cell death or delayed growth. In summary, this study describes the combinatorial antibacterial effects of microbial biomolecules, which may have ecological relevance by inhibiting cohabiting species, but shall furthermore inspire drug development in the combat of infectious disease.
Project description:Gram-negative bacteria of the genus Serratia are potential producers of many useful secondary metabolites, such as prodigiosin and serrawettins, which have potential applications in environmental bioremediation or in the pharmaceutical industry. Several Serratia strains produce prodigiosin and serrawettin W1 as the main bioactive compounds, and the biosynthetic pathways are co-regulated by quorum sensing (QS). In contrast, the Serratia strain, which can simultaneously produce prodigiosin and serrawettin W2, has not been reported. This study focused on analyzing the genomic sequence of Serratia sp. strain YD25T isolated from rhizosphere soil under continuously planted burley tobacco collected from Yongding, Fujian province, China, which is unique in producing both prodigiosin and serrawettin W2.A hybrid polyketide synthases (PKS)-non-ribosomal peptide synthetases (NRPS) gene cluster putatively involved in biosynthesis of antimicrobial serrawettin W2 was identified in the genome of YD25T, and its biosynthesis pathway was proposed. We found potent antimicrobial activity of serrawettin W2 purified from YD25T against various pathogenic bacteria and fungi as well as antitumor activity against Hela cells. Subsequently, comparative genomic analyses were performed among a total of 133 Serratia species. The prodigiosin biosynthesis gene cluster in YD25T belongs to the type I pig cluster, which is the main form of pig-encoding genes existing in most of the pigmented Serratia species. In addition, a complete autoinducer-2 (AI-2) system (including luxS, lsrBACDEF, lsrGK, and lsrR) as a conserved bacterial operator is found in the genome of Serratia sp. strain YD25T. Phylogenetic analysis based on concatenated Lsr and LuxS proteins revealed that YD25T formed an independent branch and was clearly distant from the strains that solely produce either prodigiosin or serrawettin W2. The Fe (III) ion reduction assay confirmed that strain YD25T could produce an AI-2 signal molecule. Phylogenetic analysis using the genomic sequence of YD25T combined with phylogenetic and phenotypic analyses support this strain as a member of a novel and previously uncharacterized Serratia species.Genomic sequence and metabolite analysis of Serratia surfactantfaciens YD25T indicate that this strain can be further explored for the production of useful metabolites. Unveiling the genomic sequence of S. surfactantfaciens YD25T benefits the usage of this unique strain as a model system for studying the biosynthesis regulation of both prodigiosin and serrawettin W2 by the QS system.
Project description:This study is aimed to determine the distribution, diversity and bioprospecting aspects of marine pigmented bacteria (MPB) isolated from pristine Andaman Islands, India. A total of 180 samples including seawater, sediment, marine plants, invertebrates, and vertebrates were collected and investigated for isolating pigmented bacteria. Results revealed that sediment, invertebrates, and seawater samples were colonized with a greater number of pigmented bacteria pertains to 27.9 × 103 CFU/mL, 24.1 × 103 CFU/mL and 6.7 × 103 CFU/mL respectively. Orange (21.6 × 103 CFU/mL) and red (8.0 × 103 CFU/mL) MPB were predominant than other pigmented bacteria. Fourteen potential MPB were selected based on their intense pigmentation and tested for bioactive nature and food colorant applications. Out of 14, two red pigmented strains BSE6.1 & S2.1 displayed potential multifaceted applications, such as antibacterial, antioxidant, food colorant, and staining properties. Brown pigmented strains CO8 and yellow pigmented strain SQ2.3 have displayed staining properties. Chemical characterization of red pigment using TLC, HP-LC, GC-MS, FT-IR and 1H-NMR analysis revealed prodigiosin as a main chemical constituent. Pure form of prodigiosin compound fractions obtained from both the strains displayed effective antibacterial activity against different human pathogens. MIC and MBC assays revealed that S2.1 requires 300 µg and 150 µg, respectively, and BSE6.1 require 400 µg concentrations of pigment compound for complete inhibition of S. aureus subsp. aureus. On the basis of 16S rRNA sequence analysis, strains S2.1 and BSE6.1 were identified as Zooshikella sp. and Streptomyces sp. and assigned under the GenBank accession numbers: MK680108 and MK951781 respectively.
Project description:BACKGROUND:The quest for novel sources of antibacterial compounds have necessitated the inclusion of ericoid mycorrhizal fungi (ERM) commonly found within the root of ericaceous plants. Agar-well diffusion method was used to detect antibacterial activity and was followed by the microbroth diffusion method [minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)]. RESULTS:The results of the phytochemical screening indicated that only alkaloids, flavonoids, phenols, saponins, cardiac glycosides and terpenoids were present, while steroids and tannins were absent. The MIC of the extracts ranged between 2 and 16?mg/mL, and the lowest MIC was obtained with Staphylococcus aureus. Also, the result of the MBC study indicated that the fungal extract was most active at concentrations of 2 and 4?mg/mL against Bacillus subtilis and S. aureus, respectively. CONCLUSIONS:This bioassay showed, for the first time, antibacterial activity of L. incrustata against some bacterial species. Subsequently, ERM fungi should be given attention when searching for antimicrobial agents because they could provide a solution to solve problems associated with conventional disease treatments (i.e. pathogenic microorganisms resistance).
Project description:Herein we report the design, synthesis, computational, and experimental evaluation of the antimicrobial activity of fourteen new 3-amino-5-(indol-3-yl) methylene-4-oxo-2-thioxothiazolidine derivatives. The structures were designed, and their antimicrobial activity and toxicity were predicted in silico. All synthesized compounds exhibited antibacterial activity against eight Gram-positive and Gram-negative bacteria. Their activity exceeded those of ampicillin and (for the majority of compounds) streptomycin. The most sensitive bacterium was S. aureus (American Type Culture Collection ATCC 6538), while L. monocytogenes (NCTC 7973) was the most resistant. The best antibacterial activity was observed for compound 5d (Z)-N-(5-((1H-indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-hydroxybenzamide (Minimal inhibitory concentration, MIC at 37.9-113.8 ?M, and Minimal bactericidal concentration MBC at 57.8-118.3 ?M). Three most active compounds 5d, 5g, and 5k being evaluated against three resistant strains, Methicillin resistant Staphilococcus aureus (MRSA), P. aeruginosa, and E. coli, were more potent against MRSA than ampicillin (MIC at 248-372 ?M, MBC at 372-1240 ?M). At the same time, streptomycin (MIC at 43-172 ?M, MBC at 86-344 ?M) did not show bactericidal activity at all. The compound 5d was also more active than ampicillin towards resistant P. aeruginosa strain. Antifungal activity of all compounds exceeded those of the reference antifungal agents bifonazole (MIC at 480-640 ?M, and MFC at 640-800 ?M) and ketoconazole (MIC 285-475 ?M and MFC 380-950 ?M). The best activity was exhibited by compound 5g. The most sensitive fungal was T. viride (IAM 5061), while A. fumigatus (human isolate) was the most resistant. Low cytotoxicity against HEK-293 human embryonic kidney cell line and reasonable selectivity indices were shown for the most active compounds 5d, 5g, 5k, 7c using thiazolyl blue tetrazolium bromide MTT assay. The docking studies indicated a probable involvement of E. coli Mur B inhibition in the antibacterial action, while CYP51 inhibition is likely responsible for the antifungal activity of the tested compounds.
Project description:Usnic acid, a dibenzofuran, was originally isolated from lichens producing secondary metabolites, and is well known as an antibiotic, but is also endowed with several other interesting properties. Thus, the goal of this paper is the design of new usnic acid derivatives and evaluation of their antimicrobial activity. All newly synthesized compounds possess good antibacterial activity with MIC ranging from 1.02-50.93 × 10-2 mmol mL-1 and MBC from 2.05-70.57 × 10-2 mmol mL-1. The most sensitive bacterial species was Staphylococcus aureus, while Pseudomonas aeruginosa and Escherichia coli were the most resistant among the ATCC strains, and MRSA was the most resistant among all tested bacteria (ATCC and clinical isolates). Their antifungal activity was very strong (MIC = 0.35-7.53 × 10-2 mmol mL-1 and MFC = 0.70-15.05 × 10-2 mmol mL-1) - better than those of reference compounds and usnic acid itself. The most sensitive fungal species was Trichoderma viride, while Penicillium versicolor var. cyclopium appeared to be the most resistant. It should be mentioned that in general most of the compounds showed weaker antibacterial activity, but better antifungal properties than usnic acid itself. The results allow us to conclude that the title compounds are good lead compounds for novel more active antibacterial drugs. On the other hand, these compounds are very promising as antifungals.
Project description:Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (-63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (-33%). KS8 supernatant also caused a 0.97-log reduction (-89%) and a 2-log reduction (-99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments.
Project description:In the present study, the antibacterial potential of chitosan grafted with phenolics (CPCs) such as caffeic acid (CCA), ferulic (CFA), and sinapic acid (CSA) were evaluated against foodborne pathogens like Pseudomonas aeruginosa (PA) and Listeria monocytogenes (LM). The geometric means of minimum inhibitory concentration (MIC range 0.05-0.33 mg/ml), bactericidal concentration (MBC range 0.30-0.45 mg/ml), biofilm inhibitory concentration (BIC range 0.42-0.83 mg/ml), and biofilm eradication concentration (BEC range 1.71-3.70 mg/ml) of CPCs were found to be lower than the MIC (0.12-1.08 mg/ml), MBC (0.17-1.84 mg/ml), BIC (4.0-4.50 mg/ml), and BEC (17.4-23.0 mg/ml) of unmodified chitosan against PA and LM. CPCs attenuated the biofilms of PA and LM by increasing the membrane permeability of bacteria embedded within the biofilms. Further, sub MIC of CPCs (0.5 × MIC) significantly reduced the biofilm adhesion (p < 0.001) by representative strains of LM (CCA: 72.2 ± 3.5, CFA: 79.3 ± 0.9, and CSA: 74.9 ± 1.5%) and PA (CCA: 64 ± 1.1, CFA: 67.8 ± 0.8, and CSA: 65.7 ± 4.9%). These results suggested the antibacterial and anti-biofilm potential of CPCs that can be exploited to control foodborne pathogenic infections.
Project description:The design and synthesis of a novel tert-butyl-calixarene functionalized at 1, 3 positions of the lower rim with two terminal 2-hydroxybenzeledene-thiosemicarbazone moieties is reported. The new ligand with multi-dentate chelating properties was fully characterized by several techniques: ESI-Mass spectroscopy, FT-IR, 1H-NMR, and single crystal X-ray diffraction. The solid state structure confirms that the calixarene macrocycle has the expected open cone conformation, with two opposite phenyl rings inclined outwards with large angles. The conformation of the two alkoxythiosemicarbazone arms produces a molecule with a C2 point group symmetry. An interesting chiral helicity is observed, with the two thiosemicarbazone groups oriented in opposite directions like a two-blade propeller. A water molecule is encapsulated in the center of the two-blade propeller through multiple H-bond coordinations. The antibacterial, antifungal, anticancer, and cytotoxic activities of the calixarene-thiosemicarbazone ligand and its metal derivatives (Co2+, Ni2+, Cu2+, and Zn2+) were investigated. A considerable antibacterial activity (in particular against E. coli, MIC, and MBC = 31.25 ?g/mL) was observed for the ligand and its metal derivatives. Significant antifungal activities against yeast (C. albicans) were also observed for the ligand (MIC = 31.25 ?g/mL and MBC = 125 ?g/mL) and for its Co2+ derivative (MIC = 62.5 ?g/mL). All compounds show cytotoxicity against the tested cancerous cells. For the Saos-2 cell line, the promising anticancer activity of ligand L (IC50 < 25 ?g/mL) is higher than its metal derivatives. The microscopic analysis of DAPI-stained cells shows that the treated cells change in morphology, with deformation and fragmentation of the nuclei. The hemo-compatibility study demonstrated that this class of compounds are suitable candidates for further in vivo investigations.
Project description:The Helicobacterpylori bacterium is one of the main causes of chronic gastritis, peptic ulcers, and even gastric cancer. It affects an average of half of the world population. Its difficult eradication depends upon multi-drug therapy. Since its classification as a group 1 carcinogenic by International Agency for Research on Cancer (IARC), the importance of H. pylori eradication has obtained a novel meaning. There is considerable interest in alternative therapies for the eradication of H. pylori using compounds from a wide range of natural products. In the present study, we investigated the antibacterial property of the isocoumarin paepalantine against H. pylori and it exhibited significant anti-H. pylori activity at a minimum inhibitory concentration (MIC) of 128 ?g/mL and at a minimum bactericidal concentration (MBC) of 256 ?g/mL. The scanning electron microscopy (SEM) revealed significant morphological changes of the bacterial cell as a response to a sub-MIC of paepalantine, suggesting a penicillin-binding protein (PBP) inhibition. Computational studies were carried out in order to study binding modes for paepalantine in PBP binding sites, exploring the active and allosteric sites. The data from the present study indicates that paepalantine exhibits significant anti-H. pylori activity, most likely by inhibiting membrane protein synthesis.
Project description:Considering the impact of dental caries on public health and the wide biological potential described for species belonging to the genus Pinus, here we investigate the antibacterial activity of the P. elliottii and P. tropicalis resins and of the diterpene dehydroabietic acid (DHA) against cariogenic bacteria. For this purpose, we have determined the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the resins and of the diterpene. We have also investigated the biofilm inhibition ability (through MBIC50 determination), as well as the synergistic effect (through fractional inhibitory concentration index) and the cytotoxic potential (through XTT assay) of the P. elliottii and P. tropicalis resins. The resins gave promising MIC and MBC values, which ranged from 12.5 to 400 μg/mL; DHA provided MIC and MBC values ranging from 25 to 400 μg/mL. The MICB50 values ranged from 0.78 to 400 μg/mL for the resins. Neither additive nor synergistic effects emerged for the combinations of one of the resins with chlorhexidine. The cytotoxic activity was ≥312.5 and ≥156.3 μg/mL for the P. elliottii and P. tropicalis resins, respectively. The resins showed antibacterial activity against planktonic and sessile cariogenic bacteria. These data are relevant and encourage further research into these plants, which may contribute to the discovery of new substances that can inhibit the growth of cariogenic microorganisms and reduce the incidence of dental caries.