Antibacterial Properties of Bacterial Endophytes Isolated from the Medicinal Plant Origanum heracleoticum L.

BACKGROUND: Bacterial endophytic communities associated with medicinal plants synthesize a plethora of bioactive compounds with biological activities. Their easy isolation and growth procedures make bacterial endophytes an untapped source of novel drugs, which might help to face the problem of antimicrobial resistance. This study investigates the antagonistic potential of endophytic bacteria isolated from different compartments of the medicinal plant O. heracleoticum against human opportunistic pathogens. METHODS: A panel of endophytes was employed in cross-streaking tests against multidrug-resistant human pathogens, followed by high-resolution chemical profiling using headspace-gas chromatography/mass spectrometry. RESULTS: Endophytic bacteria exhibited the ability to antagonize the growth of opportunistic pathogens belonging to the Burkholderia cepacia complex (Bcc). The different inhibition patterns observed were related to their taxonomic attribution at the genus level; most active strains belong to the Gram-positive genera Bacillus, Arthrobacter, and Pseudarthrobacter. Bcc strains of clinical origin were more sensitive than environmental strains. Cross-streaking tests against other 36 human multidrug-resistant pathogens revealed the highest antimicrobial activity towards the Coagulase-negative staphylococci and Klebsiella pneumoniae strains. Interestingly, strains of human origin were the most inhibited, in both groups. Concerning the production of volatile organic compounds (VOCs), the strain Arthrobacter sp. OHL24 was the best producer of such compounds, while two Priestia strains were good ketones producers and so could be considered for further biotechnological applications. CONCLUSIONS: Overall, this study highlights the diverse antagonistic activities of O. heracleoticum-associated endophytes against both Bcc and multidrug-resistant (MDR) human pathogens. These findings hold important implications for investigating bacterial endophytes of medicinal plants as new sources of antimicrobial compounds.

The Cultivable Bacterial Microbiota Associated to the Medicinal Plant Origanum vulgare L.: From Antibiotic Resistance to Growth-Inhibitory Properties.

The insurgence of antibiotic resistance and emergence of multidrug-resistant (MDR) pathogens prioritize research to discover new antimicrobials. In this context, medicinal plants produce bioactive compounds of pharmacological interest: some extracts have antimicrobial properties that can contrast different pathogens. For such a purpose, Origanum vulgare L. (Lamiaceae family) is a medicinal aromatic plant, whose essential oil (EO) is recognized for its antiseptic, antimicrobial and antiviral activities. The cultivable bacteria from different compartments (i.e., flower, leaf, stem and soil) were isolated in order to: (i) characterize the bacterial microbiota associated to the plant, determining the forces responsible for the structuring of its composition (by evaluation of cross inhibition); (ii) investigate if bacterial endophytes demonstrate antimicrobial activities against human pathogens. A pool of plants belonging to O. vulgare species was collected and the specimen chemotype was defined by hydrodistillation of its essential oil. The isolation of plant associated bacteria was performed from the four compartments. Microbiota was further characterized through a culture-independent approach and next-generation sequencing analysis, as well. Isolates were molecularly typed by Random Amplified Polymorphic DNA (RAPD) profiling and taxonomically assigned by 16S rRNA gene sequencing. Antibiotic resistance profiles of isolates and pairwise cross-inhibition of isolates on agar plates (i.e., antagonistic interactions) were also assessed. High level of diversity of bacterial isolates was detected at both genus and strain level in all different compartments. Most strains were tolerant against common antibiotics; moreover, they produced antagonistic patterns of interactions mainly with strains from different compartments with respect to that of original isolation. Strains that exhibited high inhibitory properties were further tested against human pathogens, revealing a strong capacity to inhibit the growth of strains resistant to several antibiotics. In conclusion, this study regarded the characterization of O. vulgare L. chemotype and of the bacterial communities associated to this medicinal plant, also allowing the evaluation of antibiotic resistance and antagonistic interactions. This study provided the bases for further analyses on the possible involvement of endophytic bacteria in the production of antimicrobial molecules that could have an important role in clinical and therapeutic applications.

Volatile profile of Echinacea purpurea plants after in vitro endophyte infection.

The differences in volatile profile of Echinacea purpurea plants not-inoculated (EpC) and inoculated with their endophytes from roots (EpR) and stem/leaves (EpS/L) were analysed and compared by GC-FID/GC-MS in an in vitro model. Non-terpenes and sesquiterpene hydrocarbons were the most abundant classes with an opposite behaviour of EpS/L showing a decreased emission of sesquiterpenes and an increase of non-terpene derivatives. The main compounds obtained from EpS/L were (Z)-8-dodecen-1-ol and 1-pentadecene, while germacrene D and ß-caryophyllene were the key compounds in EpC and EpR. For the first time, this work indicates that bacterial endophytes modify the aroma profiles of infected and non-infected in vitro plants of the important medicinal plant E. purpurea. Therefore, our model of infection could permit to select endophytic strains to use as biotechnological tool in the production of medicinal plants enriched in volatile bioactive compounds.

The influence of Echinacea purpurea leaf microbiota on chicoric acid level.

The controversial anti-proliferative effects of Echinacea purpurea (L.) Moench (Asteraceae) might be related to different plant metabolites contained in plant samples, extracts and products. The influence of bacterial endophytes on the synthesis of bioactive compounds in the medicinal plants has been previously demonstrated but there are only few studies addressing anticancer effects and mechanisms of E. purpurea extracts following endophytic colonization. The present study aimed to test and compare the lactate dehydrogenase (LDH) inhibition potential of n-hexane and methanol extracts from in vitro endophyte non-inoculated and inoculated E. purpurea plants. An in vitro model was previously set up to perform the infection of axenic E. purpurea plants with bacterial endophytic strains isolated from E. purpurea aerial part. Only methanol extracts showed LDH5 inhibition, in particular the richest in chicoric acid and most strongly inhibiting extract was obtained from inoculated stem and leaves of E. purpurea (IC50 = 0.9 mg/ml). Chicoric acid showed an IC50 value (66.7 µM) in enzymatic assays better than that of the reference compound galloflavin. Modeling studies were carried out to suggest the putative interaction mode of chicoric acid in the enzyme active site. This in vitro model on plant-bacterial interaction may lead to obtain extracts from plants enriched in bioactive compounds and it is a new approach for the discovery of novel anticancer compounds.

Tissue specificity and differential effects on in vitro plant growth of single bacterial endophytes isolated from the roots, leaves and rhizospheric soil of Echinacea purpurea.

BACKGROUND: Echinacea-endophyte interaction might affect plant secondary metabolites content and influence bacterial colonization specificity and plant growth, but the underlying mechanisms need deepening. An in vitro model, in which E. purpurea axenic plants as host species and E. angustifolia and Nicotiana tabacum as non-host species inoculated with single endophytes isolated from stem/leaf, root and rhizospheric soil, were used to investigate bacterial colonization. RESULTS: Colonization analysis showed that bacteria tended to reach tissues from which they were originally isolated (tissue-specificity) in host plants but not in non-host ones (species-specificity). Primary root elongation inhibition as well as the promotion of the growth of E. purpurea and E. angustifolia plants were observed and related to endophyte-produced indole-3-Acetic Acid. Bacteria-secreted substances affected plant physiology probably interacting with plant regulators. Plant metabolites played an important role in controlling the endophyte growth. CONCLUSIONS: The proposed in vitro infection model could be, generally used to identify novel bioactive compounds and/or to select specific endophytes contributing to the host metabolism properties.

Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model.

The influence of the interaction(s) between the medicinal plant Echinacea purpurea (L.) Moench and its endophytic communities on the production of alkamides is investigated. To mimic the in vivo conditions, we have set up an infection model of axenic in vitro E. purpurea plants inoculated with a pool of bacterial strains isolated from the E. purpurea stems and leaves. Here we show different alkamide levels between control (not-inoculated) and inoculated plants, suggesting that the alkamide biosynthesis may be modulated by the bacterial infection. Then, we have analysed the branched-chain amino acids (BCCA) decarboxylase gene (GenBank Accession #LT593930; the enzymatic source for the amine moiety formation of the alkamides) expression patterns. The expression profile shows a higher expression level in the inoculated E. purpurea tissues than in the control ones. These results suggest that the plant-endophyte interaction can influence plant secondary metabolism affecting the therapeutic properties of E. purpurea.

Is the plant-associated microbiota of Thymus spp. adapted to plant essential oil?

We examined whether the microbiota of two related aromatic thyme species, Thymus vulgaris and Thymus citriodorus, differs in relation to the composition of the respective essential oil (EO). A total of 576 bacterial isolates were obtained from three districts (leaves, roots and rhizospheric soil). They were taxonomically characterized and inspected for tolerance to the EO from the two thyme species. A district-related taxonomic pattern was found. In particular, high taxonomic diversity among the isolates from leaves was detected. Moreover, data obtained revealed a differential pattern of resistance of the isolates to EOs extracted from T. vulgaris and T. citriodorus, which was interpreted in terms of differing chemical composition of the EO of their respective host plants. In conclusion, we suggest that bacterial colonization of leaves in Thymus spp. is influenced by the EO present in leaf glandular tissue as one of the selective forces shaping endophytic community composition.

Linking Bacterial Endophytic Communities to Essential Oils: Clues from Lavandula angustifolia Mill.

Endophytic bacteria play a crucial role in plant life and are also drawing much attention for their capacity to produce bioactive compounds of relevant biotechnological interest. Here we present the characterisation of the cultivable endophytic bacteria of Lavandula angustifolia Mill.-a species used since antiquity for its therapeutic properties-since the production of bioactive metabolites from medical plants may reside also in the activity of bacterial endophytes through their direct production, PGPR activity on host, and/or elicitation of plant metabolism. Lavender tissues are inhabited by a tissue specific endophytic community dominated by Proteobacteria, highlighting also their difference from the rhizosphere environment where Actinobacteria and Firmicutes are also found. Leaves' endophytic community resulted as the most diverse from the other ecological niches. Overall, the findings reported here suggest: (i) the existence of different entry points for the endophytic community, (ii) its differentiation on the basis of the ecological niche variability, and (iii) a two-step colonization process for roots endophytes. Lastly, many isolates showed a strong inhibition potential against human pathogens and the molecular characterization demonstrated also the presence of not previously described isolates that may constitute a reservoir of bioactive compounds relevant in the field of pathogen control, phytoremediation, and human health.

Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia.

In this work we analyzed the composition and structure of cultivable bacterial communities isolated from the stem/leaf and root compartments of two medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC.) Hell, grown in the same soil, as well as the bacterial community from their rhizospheric soils. Molecular PCR-based techniques were applied to cultivable bacteria isolated from the three compartments of the two plants. The results showed that the two plants and their respective compartments were characterized by different communities, indicating a low degree of strain sharing and a strong selective pressure within plant tissues. Pseudomonas was the most highly represented genus, together with Actinobacteria and Bacillus spp. The presence of distinct bacterial communities in different plant species and among compartments of the same plant species could account for the differences in the medicinal properties of the two plants.