BioRisk 17: 357-365 (2022) Pete rag ae ei aca rey doi: 10.3897/biorisk.| 7.77484 RESEARCH ARTICLE & B lO R IS k https://biorisk.pensoft.net Comparative determination of antimicrobial activity of the Balkan endemic species Stachys thracica Davidov during the process of ex situ conservation Desislava Mantovska', Detelina Petrova', Lybomira Yocheva’, Zhenya Yordanova' | Department of Plant Physiology, Faculty of Biology, Sofia University “St. Kliment Ohridski’, Sofia, Bulgaria 2 Department of biology, medicinal genetics and microbiology, Faculty of Medicine, Sofia University “St. Kli- ment Ohridski”, Sofia, Bulgaria Corresponding author: Desislava Mantovska (d_mantovska@biofac. uni-sofia.bg) Academic editor: Galina Radeva | Received 1 November 2021 | Accepted 28 December 2021 | Published 21 April 2022 Citation: Mantovska D, Petrova D, Yocheva L, Yordanova Z (2022) Comparative determination of antimicrobial activity of the Balkan endemic species Stachys thracica Davidov during the process of ex situ conservation. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 357-365. https://doi.org/10.3897/biorisk.17.77484 Abstract Stachys thracica Davidov — Thracian woundwort is a Balkan endemic plant included in The Red Data Book of Bulgaria with conservational status “rare”. The plants from genus Stachys have a long history of use to treat various diseases, inflammatory conditions, coughs, ulcers, genital tumors, and infected wounds. Due to its limited distribution the information on the biological activity and chemical composition of S. thracica is rather scarce. The aim of the present research is the comparative determination of the antimi- crobial activity of methanolic extracts obtained from in situ wild, in vitro cultivated and ex vitro adapted S. thracica plants. The in vitro shoot culture of the Thracian woundwort was maintained in hormone-free MS medium under controlled environmental conditions. The methanolic extracts from in situ, in vitro cultivated and ex vitro adapted S. thracica plants were active mainly against Gram-negative bacteria. All three extracts showed equal activity against Acinetobacter calcoaceticus. The establishment of in vitro shoot culture and its subsequent adaptation in ex vitro conditions was an appropriate alternative approach for the ex situ conservation of S. thracica as well as for the study of its biological activity. Keywords Antimicrobial activity, in vitro cultivation, Thracian woundwort Copyright Desislava Mantovska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 358 Desislava Mantovska et al. / BioRisk 17: 357—365 (2022) Introduction Genus Stachys. L, or woundworts, comprises more than 300 herbs and shrubs and is considered one of the largest genera from Lamiaceae family (Tomou et al. 2020). Most of the species are distributed in temperate and tropical regions of the world especially in the Mediterranean. There are 22 species from Stachys genus in Bulgaria, 5 of which are under the protection of the Bulgarian Biodiversity Law. The natural habitats of some of these species are located in some of the Bulgarian national parks and others are within localities included in NATURA 2000. The plants from genus Stachys have a long history of use in ethnomedicine for various diseases, coughs, ulcers, genital tu- mors, inflammatory conditions, and infected wounds (Tundis et al. 2007; Conforti et al. 2009; Goren et al. 2014). It is reported that woundworts exhibit various biological effects such as antioxi- dant, antibacterial, anti-inflammatory, wound healing, cytotoxic, hepatoprotective properties (Khanavi et al. 2005; Vunda¢ et al. 2007; Haznagy-Radnai et al. 2012; Tun- dis et al. 2014; Tomou et al. 2020). According to different phytochemical studies, the plants from the Stachys genus are sources mainly of phenylethanoid glycosides (Karioti et al. 2010; Delazar et al. 2011), iridoids (Murata et al. 2008; Tundis et al. 2014) and phenolic acids (Venditti et al. 2014). Taking into account the research done so far, Stachys species may be considered a favourable subject for exploration and discovery of secondary metabolites with anti- microbial potential. The inconsistent application of antibiotics poses a great risk of antibiotic resistance in most of the microbial species that cause human infections (Ventola et al. 2015). This cre- ates an urgency for the research and discovery of alternative sources of antimicrobial agents. Plants have been used by humanity since ancient times for the treatment of various bacterial infections even without scientific proof of their effectiveness. As a potential source of numerous biologically active substances, plant species have always been po- tential candidates for alternative agents with antimicrobial activity. In recent years the antimicrobial potential of some Stachys species was a great point of interest among different research groups. Published data indicate that different polar extracts, as well as essential oils, show antimicrobial activity against human pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa (Dulger et al. 2005; Aleebra- him-Dehkordy et al. 2016; Cuce et al. 2016). Stachys thracica Davidov (The Plant List) or Thracian woundwort is a Balkan en- demic plant distributed in Bulgaria, Greece and Turkey. In Bulgaria, it is classified as “rare” and some of its localities are within Natura 2000 ecological network. The popu- lations of the Thracian woundwort are comprised of a small number of individuals and are located in the Strandja Mountain, Black Sea coast and Sofia region. There is no available data on ex-situ conservation of the species and its chemical composition and biological activity are not well studied. The aim of the present research is a comparative determination of the antimicro- bial activity of methanolic extracts obtained from in situ wild, in vitro cultivated and ex vitro adapted Stachys thracica plants. Comparative determination of the antimicrobial activity of Stachys thracica Davidov... 359 Materials and methods Plant material S. thracica Davidov plants grew in situ in their natural habitat near the village of Sinemorets, Tsarevo municipality, Bulgaria. A small set of samples from aerial parts of the plants in the period of active blooming (in June) and seeds (in September) were collected with the permission of the Ministry of Environment and Water of Bulgaria. A voucher specimen SO107847 was deposited in the Herbarium of Sofia University “St. Kliment Ohridski”. In vitro shoot culture from S. thracica was induced by sterilisation of seeds with 70% ethanol for 5 min. ‘The sterilised seeds were placed on a germination medium con- taining water and agar (WA) and further, the sprouting seedlings were transferred on MS medium (Murashige and Skoog 1962) supplemented with 3% sucrose and 0.7% agar,without growth regulators. The in vitro collection was maintained under controlled environmental conditions (16 h light/8 h dark, 60 mmol/(m’s) photosynthetic photon flux density, Philips TLD-33, temperature 25 °C and 60-70% relative air humidity). Ex vitro adaptation was performed in three stages with plants having well-devel- oped root systems. At the first step, the regenerated plants were planted in pots and subjected to acclimation in a phytotron chamber for a period of one month. After that, they were transferred to a greenhouse for another month and at a final stage were planted on the experimental field of Sofia University “St. Kliment Ohridski”. Methanolic extracts preparation Three grams (3 g) of finely powdered dry plant material from aboveground parts of in situ grown, in vitro cultivated and ex vitro adapted S. thracica were subjected to triple sonication extraction with 30 ml chloroform (Sigma-Aldrich, Spain) in ultrasonic bath for 10 minutes. In the next step, the dried biomass was extracted three times with methanol for 30 minutes. The final plant extract from each variant was concentrated through a vacuum evaporator (IKA, Germany) and dried to constant dry weight. The yields of extracts from in situ, in vitro cultivated and ex vitro adapted plants were 13.8%, 28.46% and 13.6% respectively. For the current study, each methanolic extract was dissolved in 5% DMSO. Antimicrobial activity Microbial strains The methanolic extracts from in situ, in vitro cultivated and ex vitro adapted S. thracica plants were individually tested against seven Gram-negative microbial strains — Pseu- domonas aeruginosa NBIMCC 3700, Proteus mirabilis NBIMCC 8690, Proteus hau- seri NBIMCC 1393, Enterobacter cloacea NBIMCC 8570, Acinetobacter calcoaceticus NBIMCC 3730, Escherichia coli NBIMCC 8954, Klebsiella pneumoniae NBIMCC 3670 360 Desislava Mantovska et al. / BioRisk 17: 357-365 (2022) and three Gram-positive bacteria — Staphylococcus aureus AVCC 25923, Staphylococcus epidermitis NBIMCC 3360, Enterococcus faecalis NBIMCC 1093 microbial species and the yeast Candida albicans NBIMCC 74. The microbial specimens were purchased from The Bulgarian Collection for Industrial Microorganisms and Cell cultures (NB- CIMCC). The bacterial strains were cultured overnight at 37 °C on Muller-Hinton agar (MHA) and the yeast was cultured on Sabouraud Dextrose Agar (SDA). Disk diffusion assay An initial screening of the antimicrobial activity of the dried methanolic extracts from S. thracica was performed by agar disk diffusion method according to the guidelines of CLSI (Clinical and Laboratory Standards Institute). The dried extracts from in situ, in vitro and ex vitro adapted plants were dissolved in 5% DMSO to a final concentration of 200 mg/ml and filtered by 0.45 um Millipore filters for sterilization. Briefly, 100 ul of each suspension containing 10’ cell/ml was inoculated in 25 ml MHA for bacterial strains and SDA for the yest respectively. Sterile paper disks (6 mm diameter) were impregnated with the extracts (8 mg/disk) and allowed to dry under aseptic conditions before placing them on the inoculated agar. DMSO at concentration 5% was used as a negative control. The antibiotics tetracycline and amikacin were used as a positive con- trol for the bacterial strains and nystatin for C. albicans. The samples were incubated at 37 °C for 24 hours and 48 hours for bacterial strains and C. albicans respectively. The antimicrobial activity of the extracts was related to the inhibition zones. Micro-well dilution assay Bacterial strains which were sensitive to the methanolic extracts in the disk diffusion assay were studied for their minimal inhibitory concentration (MIC) using the micro-well dilu- tion assay (Wiegand 2008, EUCAST). For the experiment, the bacterial suspension was prepared in a liquid MH-Muller Hinton medium with a density of 0.5 on the McFarland scale, corresponding to 10’ cells/ml. The 96-well plates were prepared by dispersing 50 ul MH broth in each well. ‘The serial dilutions of each extract were prepared directly in the wells as the starting concentration was 64 mg/ml. Then, 50 pl of each well was transferred to the next and the final dilution of each extract was 2 mg/ml. Finally, 50 ul of the bacte- rial suspension was added to each well and the final volume of each well was 150 ul. Prior to incubation, the absorbance of each microplate was measured using ELISA reader (Usen Kit Inc., China) at 4=630 nm and this was considered the absorption at 0 h. Results In vitro multiplication and ex situ conservation of S. thracica In vitro shoot culture from S. thracica was successfully induced by the sterilisation and subsequent germination of ripe dried seeds. The in vitro regenerated plants Comparative determination of the antimicrobial activity of Stachys thracica Davidov... 361 _ | i. a - 7 = a S. thracica in situ Germinated Multiplicated plants in seed in vitro vitro f3 ao =) 9) thracica exeuiro Ex vitro adapted plants in Ex vitro adapted plants greenhouse in growth chamber Figure I. Ex situ conservation of S. thracica. were further propagated and maintained on hormone-free MS medium and char- acterised with plentiful leaf biomass and well-developed roots. This allowed their further 3-stage acclimatisation — in a phytotron chamber, in a greenhouse and an experimental field. For the current research, the collection of ex vitro adapted S. thracica plants were successfully maintained on the experimental field with 83% survival rate (Fig. 1). Antimicrobial activity The antimicrobial activity of the methanolic extracts from in situ, in vitro cultivated and ex vitro adapted S. thracica plants was evaluated against 11 microorganisms that are frequently related to human infections and are typically present in infected wounds. The results from the preliminary screening by the disk diffusion assay as well as the microdilution assay are presented in Table 1. All tested extracts show ten- dency to be active against Gram-negative bacterial strains rather than Gram-positive strains. Overall, the extracts of S. thracica showed activity against only 4 of the tested microbial strains and no dependency between the type of extract and its ac- tivity was observed (Fig. 2). The most sensitive microbial species appeared to be A. calcoaceticus as all three extracts showed bactericidal zones and MIC values of 8 mg/ ml. The other bacterial strains that were sensitive to either of the extracts were K. pneumoniae, P mirabilis and E. faecalis. The highest MIC value — 16 mg/ml and the smallest inhibitory zone — 7 mm were established against K. pneumoniae. Although the zones in P aeruginosa were seen as bacteriostatic, no activity was detected in the microdilution assay. 362 Desislava Mantovska et al. / BioRisk 17: 357-365 (2022) Discussion In vitro multiplication and ex vitro adaptation of S. thracica The conservational status of the Thracian woundwort and its limited distribution en- forced us to apply an alternative method which would allow simultaneously the con- servation of the species and the determination of its biological activity. The in vitro micropropagation is a reliable method for ex-situ conservation of endemic and threat- ened plant species and it is successfully applied for the investigation of such, without disturbing their natural population and habitats. For the current study we successfully initiated in vitro shoot cultures of S. thracica from sterilised ripe dried seeds. The in vitro culture is successfully grown on MS me- dium without the addition of plant growth regulators and the micropropagated plants are characterised with vigorous growth, plentiful leaf biomass and very well-developed root system. This in turn led to the successful ex vitro acclimation of the Thracian woundwort with 83% survival rate. Table |. Antimicrobial activity of methanolic extracts from in situ wild, in vitro cultivated and ex vitro adapted Stachys thracica plants. Test microorganisms Stachys thracica methanolic extracts Antibiotics In situ In vitro Ex vitro Amicacine ‘Tetracycline Nystatine DD? MIC DD: MIC DD* MIC DD? DD: NA Acinetobacter calcoaceticus 9 8 8 8 7.5 8 11 12 NA Enterobacter cloacea - - - - - - 8 21 NA Proteus mirabilis 6* - 8* - 8* 8 13 8 NA Proteus hauseri - NA - NA - NA 20 19 NA Staphylococcus aureus - NA - NA - NA 20 28 NA Staphylococcus epidermitis - NA - NA - NA 20 12 NA Klebsiela pneumoniae M, 16 9 4 - NA 15 29 NA Pseudomonas aeruginosa 15* - 12* - 10* - 25 12 NA Escherichia coli - NA - NA - NA 12 22 NA Enterococcus feacalis - NA 9* 4 - NA 8 25 NA Candida albicans - NA - NA - NA NA NA 18 (-) — no antimicrobial activity; (*) — bacteriostatic zone; aDD — disc diffusion method; Inhibition zones (mm); bMIC — minimal inhibitory concentration (mg/ml); NA — not tested. . Figure 2. Antimicrobial activity of methanolic extracts from in situ ', in vitro ? and ex vitro * S. thracica plants measured by the disk diffusion assay AA. calcoaceticus B P mirabilis C K. pneumoniae D E. faecalis. Comparative determination of the antimicrobial activity of Stachys thracica Davidov... 363 Similar to our results, the successfully initiated in vitro culture from the Balkan endemic S. maritima was maintained on a hormone-free MS medium and the plants showed an excellent regeneration rate (Panayotova et al. 2008). Antimicrobial activity There is a high possibility that different growth conditions would affect the biological activity of plants extracts. S. thracica is a source of pharmacologically active secondary metabolites such as phenylethanoid glycosides (Bankova et al. 1999). To evaluate the changes in the antimicrobial activity of methanolic extracts from S. thracica, a compari- son between the in situ grown, in vitro cultivated and ex vitro adapted plants was made. The antimicrobial activity was evaluated against 10 bacterial strains and 1 yeast strain — C. albicans. The disk diffusion method was used for preliminary study of the antibacterial activity and the microdilution assay was applied afterwards for determi- nation of MIC and verification of the results obtained by the initial screening. All the extracts were more active against Gram-negative bacteria which may be due to the different structure of the cell wall of gram-negative and gram-positive bacteria. We ob- served no visible trend in the antimicrobial activity of the methanolic extracts obtained from in situ, in vitro cultivated and ex vitro adapted plants with the exception that all the extracts were equally active against A. calcoaceticus showing inhibitory zones of 8 mm and MIC values — 8 mg/ml. Typically, A. calcoaceticus is a soil bacterium but it is very frequently associated with infections within hospitals due to its ability to form a complex with another species — Acinetobacter baumanii and it is usually used in laboratory testing instead of Acineto- bacter baumanii (Mancilla-Rojano et al. 2020). Ebrahimabadi et al. (2010) reported that the polar fraction of Stachys inflata Benth. was active against only two microbial species which in parts overlaps with our results. In another study, Dulger et al. (2004) demonstrated that methanolic extracts from Stachys species were active against E. coli, P aeruginosa, S. aureus and B. cereus but no activity was established against the tested yeast cultures — C. albicans, K. fragilis and R. rubra. Contrary to our results, Ciice et. al. (2017) reported that methanolic and hexane extracts from in situ and in vitro cultivated S. annua plants were active against S. aureus and the methanolic extract showed activity against P aeruginosa. Conclusions The initiated in vitro culture from S. thracica was successfully maintained on hormone- free MS medium under controlled environmental conditions and the micropropagated plants continued to form plenty of biomass and well-developed roots. The methanolic extracts from the Thracian woundwort showed activity mostly against Gram-negative bacteria and the most sensitive bacterial strain was A. calcoaceticus against which all three different extracts exhibit equal antimicrobial activity. Further research on the 364 Desislava Mantovska et al. / BioRisk 17: 357-365 (2022) chemical profile would be necessary in order to reveal which compounds are responsi- ble for the antimicrobial activity of S. thracica. The established in vitro and ex vitro plant cultures serve as an effective alternative approach for the preservation of the rare S. thracica and at the same time represents a model system for the study of its biological activity and pharmacological potential. 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