Brazilian Journal of Pharmaceutical Sciences Article http://dx.doi.org/10.1590/s2175-97902019000217249 Antimicrobial activity of actinomycetes and characterization of actinomycin-producing strain KRG-1 isolated from Karoo, South Africa Ivana Charousová 1,2*, Juraj Medo2, Lukáš Hleba2, Miroslava Císarová3, Soňa Javoreková2 1 Apha medical s.r.o., Clinical Microbiology Laboratory, Slovak Republic, 2 Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Slovak Republic, 3 University of SS. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Department of Biology, Slovak Republic In the present study we reported the antimicrobial activity of actinomycetes isolated from aridic soil sample collected in Karoo, South Africa. Eighty-six actinomycete strains were isolated and purified, out of them thirty-four morphologically different strains were tested for antimicrobial activity. Among 35 isolates, 10 (28.57%) showed both antibacterial and antifungal activity. The ethyl acetate extract of strain KRG-1 showed the strongest antimicrobial activity and therefore was selected for further investigation. The almost complete nucleotide sequence of the 16S rRNA gene as well as distinctive matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) profile of whole-cell proteins acquired for strain KRG-1 led to the identification ofStreptomyces antibioticus KRG-1 (GenBank accession number: KX827270). The ethyl acetate extract of KRG-1 was fractionated by HPLC method against the most suppressed bacterium Staphylococcus aureus (Newman). LC//MS analysis led to the identification of the active peak that exhibited UV-VIS maxima at 442 nm and the ESI-HRMS spectrum + + showing the prominent ion clusters for [M-H2O+H] at m/z 635.3109 and for [M+Na] at m/z 1269.6148. This information could be assigned to chromopeptide lactone antibiotic - actinomycin. Our results suggest that unexplored soils could be an interesting source for exploring antibacterial secondary metabolites. Keywords: Actinomycin/antimicrobial activity. Streptomyces. Soil. 16S rRNA. MALDI-TOF MS. INTRODUCTION The actinomycines belong to a family of chromopeptide lactone antibiotics that present antitumor Actinomycetes are free living Gram - positive and cytotoxic properties (Praveen et al., 2008). They bacteria having high G+C content (>55%) in DNA represent an important class of natural products that, (Kämpfer, 2012). The most important and dominant genus despite being discovered more than 70 years ago, continue within Actinobacteria is Streptomyces (Ceylan, Okmen, to be a focus of many research areas, especially in the Ugur, 2008). Members of this group referred to as the biological and medicinal sciences (Kurosawa et al., biological antagonistic types. They are of special interest 2006). Among the actinomycines, actinomycin D has since streptomycetes are the ones that are exploited and been studied most extensively and it is used for treatment their metabolites are used in the manufacture of antibiotics of malignant tumors, such as Wilms’ tumor (Green, (Kekuda, Shobha, Onkarappa, 2010). Streptomyces 1977), and childhood rhabdomyosarcoma (Womer, provides more than half (70%) of the naturally occurring 1997). The biological activity of actinomycin D is related antibiotics (Bérdy, 2005) with high commercial value and to its ability to bind to the DNA duplex, these being continue to be routinely screened for interesting bioactive associated with DNA functionality, leading to RNA and, substances (Takahashi, 2004; Meena et al., 2013). consequently, protein synthesis inhibition (Martinez, Chacon-Garcia, 2005; Boer, Canals, Coll, 2009). The two main mechanisms are intercalation to DNA and the *Correspondence: I. Charousová. Department of Microbiology, Faculty of stabilization of cleavable complexes of topoisomerases I Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic. Tel. +421 37 641 5814. E-mail: and II with DNA, or the drug penetrates to a place in the [email protected] DNA structure where topoisomerase binds with DNA, Braz. J. Pharm. Sci. 2019;55:e17249 Page 1 / 11 I. Charousová, J. Medo, L. Hleba, M. Císarová, S. Javoreková respectively (Koba, Konopa, 2005). Actinomycin binds Pseudomonas aeruginosa (PA14), Chromobacterium to the highest-energy beta-DNA form found within the violaceum (DSM 30191), Candida albicans (DSM boundaries connecting double-stranded B-DNA with 1665), Pichia anomala (DSM 6766)] and Mucor hiemalis single-stranded DNA in the transcription complex (Sobell, (DSM 2656) obtained from the German Collection 2016) and physically obstructs the trascriptional complex of Microorganisms and Cell Cultures, Braunschweig, (Huang et al., 2000). Germany and the American Type Culture Collection, Actinomycin D is produced by a range of Manassas, VA 20110, USA. Primary screening for Streptomyces species as part of a mixture of actinomycines antibiotic activity of the isolates was done by using the (Kurosawa et al., 2006; Praveen et al., 2008). agar plug method (Eccleston, Brooks, Kurtböke, 2008). Exploring new habitats is one of the most promising Determination of activity was carried out by preparing ways of isolating actinomycete producers of antibiotics 4-6 h cultures of tested bacteria followed by dilution with endowed with antimicrobial activity (Zitouni et al., 2005; Mueller- Hinton (MH) broth (Merck, Germany) to obtain Khanna, Solanki, Lal, 2011; Wadetwar, Patil, 2013). Thus, 0.05 McFarland standard turbidity and 4-6 h culture we report here the antimicrobial activity of actinomycetes of yeasts diluted with Mycosel broth (Cazin, Wiemer, isolated from soil sample collected in Karoo, South Africa Howard, 1989) to obtain 0.01 McFarland turbidity. Spores and characterization and identification of actinomycin- of Mucor hiemalis were collected in sterile distilled water producing strain KRG-1. and then adjusted to a spore density of approximately 104 spores/mL. Plates were incubated at 30 °C and the MATERIAL AND METHODS zones of inhibition were determined after 24 h (bacteria and yeasts) and after 48 h (fungus) with the Haloes Caliper Sample collection and processing (IUL Instruments, USA). Soil sample was collected from Aquilla Safari Extraction of antimicrobial compounds and in the Southern Karoo, South Africa (33° 21’ 5.569”S; secondary screening 19° 56’ 8.23”E) from 10 - 15 cm depth and passed through a 2 mm sieve to remove debris and plants, in July 2015. The selected antagonistic actinomycete isolates were Then the sample was dried for 45 min at 60 °C to eliminate inoculated into liquid 5254 medium (glucose: 15.0 g/L, the bacterial and fungal growth and stored in 4 °C until soymeal: 15.0 g/L, corn steep: 5.0 g/L, CaCO3: 2.0 g/L, examination. NaCl: 5.0 g/L, deionized water: 1000 mL, pH 7) supporting metabolite production and incubated at 30 °C in a shaker at Isolation of actinomycetes and maintenance 250 rpm for five days. After incubation period, the broths were mixed with ethyl acetate (Sigma Aldrich, USA) in Isolation and enumeration of actinomycetes were ratio 1:1 and centrifuged 10 min at 9000 rpm. Supernatants performed by the serial dilution method (10-2 - 104) and were transferred to bottom flasks and evaporated in rotary the spread plate technique on starch-casein medium evaporator (Stuart, UK) at 40 °C. Finally, the extracts were (Poosarla, Ramana, Krishna, 2013) supplemented with dissolved in 1 mL of ethylacetate:acetone:methanol (1:1:1) cycloheximide (1 mg/mL). The plates were incubated solution, resulting in raw extracts of 1:100 concentration. at 30 °C for 7 days. Powdered colonies were picked up, The antimicrobial activities of those extracts were tested transferred to ISP2 medium (Shirling, Gottlieb, 1966) and using the broth-microdillution method according to several times purified. Morphologically different isolates Wiegand, Hilpert, Hancock (2008) methodology against were named as KRG-1 - KRG-35 and stored in presence the same test microorganisms in 96-well microplates of 30% (v/v) glycerol at - 20 °C. (BRAND, Germany). Dilution stages of raw extracts were determined by inhibited wells (A-H). Preliminary screening of actinomycetes for antimicrobial activity by agar plug method Taxonomy and characterization of the most potent strain KRG-1 The actinomycete isolates were tested against eleven test microorganisms i.e. Bacillus subtilis (DSM 10), The morphological, cultural, physiological and Micrococcus luteus (DSM1790), Staphylococcus aureus biochemical characterization as well as molecular and (Newman), Mycobacterium smegmatis (ATCC 700084), protein spectra identification of the isolate was carried out. Escherichia coli (DSM 1116), Escherichia coli (TolC), Page 2 / 11 Braz. J. Pharm. Sci. 2019;55:e17249 Antimicrobial activity of actinomycetes and characterization of actinomycin-producing strain KRG-1 isolated from Karoo Morphological characteristics Genomics, Korea. Gaps and unidentified base positions Morphological signs were examined according to were edited using BioEdit (Hall, 1999). methods given by the International Streptomyces Project (ISP) (Shirling, Gottlieb, 1966). ISP2-ISP7 media were Phylogenetic analysis used to determine the speed growth, reverse colors, Phylogenetic analysis was performed using the colors of aerial mycelium and colors of soluble pigments. Maximum-Likelihood method (Felsenstein, 1981) using Melanin pigment production was determined on ISP6 and PhyML