Purification and characterization of actinomycins from Streptomyces strain M7 active against methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus

The increased rate of resistance among two highly concerned pathogens i.e. methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) necessitates the discovery of novel anti-MRSA and anti-VRE compounds. In microbial drug discovery, Streptomyces are well known source of two-thirds of natural antibiotics used clinically. Hence, screening of new strains of streptomycetes is the key step to get novel bioactive compounds with antimicrobial activity against drug resistant bacteria. In the present study, Streptomyces antibioticus strain M7, possessing potent antibacterial activity against different pathogenic bacteria, was isolated from rhizospheric soil of Stevia rebudiana. 16S rRNA sequence of M7 (1418 bp) showed 96.47–100% similarity with different Streptomyces spp. and the maximum similarity (100%) was observed with Streptomyces antibioticus NBRC 12838T (AB184184). Phylogenetic analysis using neighbor joining method further validated its similarity with Streptomyces antibioticus NBRC 12838 T (AB184184) as it formed clade with the latter and showed high boot strap value (99%). Antibacterial metabolites isolated from the fermentation broth were characterized using NMR, FT-IR and LC-MS as actinomycins V, X2 and D. The purified actinomycins exhibited potent antibacterial activities against test bacteria viz. B. subtilis, K. pneumoniae sub sp. pneumoniae, S. aureus, S. epidermidis, S. typhi, E. coli, MRSA and VRE. Among these actinomycins, actinomycin X2 was more effective as compared to actinomycins D and V. The minimum inhibitory concentration values of purified compounds against a set of test bacterial organisms viz. VRE, MRSA, E. coli (S1-LF), K. pneumoniae sub sp. pneumoniae and B. subtilis ranged between 1.95 and 31.25 μg/ml. This study demonstrates that actinomycins V, X2 and D produced by S. antibioticus strain M7 hold the potential to be used against multidrug resistant bacteria, particularly VRE and MRSA.


Background
Antimicrobial resistance among microbial pathogens is a significant public health issue, as infections caused by multidrug resistant bacteria take the lives of many people in every year all over the world [1]. Among Gram-positive pathogens, a global pandemic of resistant Staphylococcus aureus and Enterococcus species currently pose the biggest threat. A single pathogen i.e. methicillin-resistant Staphylococcus aureus (MRSA), which was first discovered in 1961, has become a major source of nosocomial and community associated MRSA infections [2,3]. Clinical isolates of MRSA have high rate of morbidity and mortality as compared to the methicillin susceptible Staphylococcus aureus [4,5]. Also, Enterococcus faecium associated with human infections has been developed as multidrug resistant pathogen to vancomycin, ampicillin, and high-levels of aminoglycosides [6,7].
Vancomycin was the most potent antibacterial drug used against infections caused by MRSA and Enterococcus. However, the first case of MRSA exhibiting resistance to vancomycin was reported from Japanese patient in 1996 [8]. According to CDC (Centers for Disease Control and Prevention) April 2013 report, 30% of hospital-acquired infections responsible for 1300 deaths per year were due to vancomycin-resistant Enterococcus (VRE) pathogens [9]. While powerful antimicrobial drugs such as synercid, linezolid and daptomycin (lipopeptide) are being used to combat the MRSA and VRE, but some reports showed that these pathogens also have emerged resistance to these effective drugs [10][11][12][13]. Because each new antibiotic eventually develops resistance within few years after it is promoted there is always a necessity to find new antimicrobial agents to control antibiotic resistant strains of pathogenic microorganisms.
Recent advances in medical science have sparked to discover the potent therapeutic drugs from the microbial sources. Among microbes, actinobacteria, especially Streptomyces spp. are of immense importance as they are known prolific producers of many novel compounds with diverse biological activities [14][15][16][17]. Although, nearly two third of the naturally occurring marketed antibiotics are obtained from Streptomyces spp. but it is just the tip of the iceberg that have been explored [18]. Therefore, to combat with drug resistance and to discover new therapeutic compounds, we need to screen novel streptomycetes from unexplored resources. Keeping this in mind, we isolated an actinobacterium from rhizospheric soil, exhibiting potent antibacterial activity against multidrug resistant bacteria. The present study reports identification of potent actinobacterium as well as purification and characterization of antibacterial compounds, active against MRSA and VRE, produced by it.

Sample collection
The soil sample was collected into a sterile glass screw cap bottle from the rhizosphere of Stevia rebudiana grown in the fields of Palampur, Himachal Pradesh, India.

Isolation and screening of actinobacteria
Soil sample was air-dried and given the pre-treatment by heating at 100°C for 1 h to create favorable conditions to accomplish the isolation of actinobacteria. Serial dilution of the treated soil was done up to 10 − 6 . Aliquots of 0.1 ml from 10 − 2 , 10 − 3 , and 10 − 4 were spread on the surface of SCNA (starch casein nitrate agar) plates. The medium was supplemented with cycloheximide (50 μg/ml) and nalidixic acid (50 μg/ml) to inhibit the growth of fungi and other bacteria, respectively. Plates were then incubated at 28°C for 7-21 days. Isolated colonies of actinobacteria were subcultured and purified on SCNA plates. The isolates were preserved in 20% glycerol at − 20°C as stock for future use.

Screening for antibacterial activity
Primary screening was performed by modified method of Kirby Bauer antibiotic susceptibility test using dual culture technique [19]. In this, 6 mm plugs of actinobacteria, grown on SCNA plates, were placed on Mueller Hinton Agar medium (MHA) already seeded with test bacteria. The plates were then incubated at 37°C. The results as zone of inhibition (mm) were obtained after 24 h of incubation. Isolates which displayed broad spectrum antibacterial activity in primary screening were subjected to secondary screening using Kirby Bauer agar well diffusion assay [19]. Erlenmeyer flasks (250 ml) containing 50 ml of starch casein nitrate broth were inoculated with 7 days old culture and incubated at 28°C for 7 days at 180 rpm. The MHA plates seeded with test bacteria (OD equivalent to McFarland standard 0.5) were punctured with sterile cork borer to make wells of 6 mm in size. After addition of culture supernatant (50 μl) to each well, the plates were kept in refrigerator for 1 h for diffusion of active metabolites followed by incubation at 37°C for 24 h. The results were observed in terms of inhibition zones around the wells. Out of 12 active isolates, strain M7 was selected based on its strong and broad spectrum antibacterial activity.

Morphological, physiological and biochemical characterization
The culture characteristics of strain M7 were determined according to the International Streptomyces Project (ISP) based on the mycelium growth and color, as well as the soluble pigment at 28°C for 7 days [20]. Melanin production was detected by growing on ISP6 and ISP7 media. Morphological characteristics of the strain, grown on SCNA at 28°C for 4 days, were observed using bright field light and scanning electron microscopy [21]. Physiological and biochemical tests, like growth at different temperatures, pH, salt concentration, and ability to produce different hydrolytic enzymes were performed as per standard protocols [22][23][24]. Analysis of the sugar components in whole cell hydrolysate and isomer of diaminopimelic acid (DAP) in the cell wall was done according to the method given by Lechevalier and Lechevalier [25]. Assimilation of sugars as carbon sources was studied according to Shirling and Gottlieb [20].

Antibacterial activity profile of Streptomyces strain M7
Production of active metabolites was done by carrying out fermentation in Erlenmeyer flasks (250 ml), containing 50 ml of production medium (SCN broth) inoculated with 2% inoculum, at 28°C for 10 days under agitation at 180 rpm. After every 24 h, the flasks were harvested and the biomass was separated from the culture broth by centrifugation at 10,000 rpm for 20 min. The biomass was dried at 60°C for 2 days, weighed and expressed in mg on dry weight basis. The remaining cell free culture supernatant was used to check the antibacterial activity against test bacterial cultures using agar well diffusion assay.

Extraction of active compounds
For the recovery of antibacterial metabolites, 96 h old culture supernatant was extracted twice with different organic solvents viz. ethyl acetate, chloroform, hexane, butanol and diethyl ether using solvent-solvent extraction technique. The separated organic phase was concentrated using the rotary evaporator and redissolved in respective solvent and checked for its antibacterial activity against B. subtilis.

Bioautography
For the analysis of antibacterial metabolites, the ethyl acetate extract was separated by thin layer chromatography (TLC) using ethyl acetate: hexane (9:1, v/v) as solvent system and the developed chromatogram was observed under UV light and in iodine chamber. TLC strips were then aseptically placed on the surface of MHA already seeded with the test bacterium. Then, the plates were kept at 4°C for 1 h to allow diffusion of the active metabolites from the TLC strips. After that the plates were incubated at 37°C for 24 h and observed for the presence of inhibition zones which indicate the number of active compounds in the solvent extract.

Purification of the active compounds
To purify the antibacterial compounds, ethyl acetate extract (150 mg) was subjected to silica gel chromatography. The column (35 × 1.0 cm) was packed with silica gel (60-120 mesh) using hexane as solvent and eluted step-wise with 100% hexane, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 (v/v) of hexane: ethyl acetate, 100% ethyl acetate (200 ml each) at a flow rate of 2 ml/min. A total of 88 fractions of 25 ml each were collected, concentrated and redissolved in the same solvent ratio from which they were recovered. Fractions showing antibacterial activity against B. subtilis were pooled and further purified using size exclusion chromatography with Toyopearl resin HW-40 and methanol as an eluent. A total of 65 fractions were collected and screened for antibacterial activity against B. subtilis. Active fractions were further fractionated using preparative RP-HPLC: Shimadzu MicrosorbMV, 100 mm × 10 mm ID, 10 μm, at a flow rate of 3 ml/min, with mobile phase of acetonitrile: H 2 O (55%) in 30 min and UV detection at 440 nm. The peaks of the chromatogram were collected by using a fraction collector attached with the HPLC system, concentrated and then screened for antibacterial activity.

Structure elucidation of the purified compounds
The structures of the bioactive compounds were elucidated using various spectroscopic techniques. Physicochemical properties such as appearance, color, odor and solubility were determined according to the standard procedures [30]. The UV-Visible spectrum was recorded qualitatively on UV-Visible Spectrophotometer (Shimadzu) in the range of 200-800 nm using chloroform as reference solvent. The mass spectrometry (MS) was done with Bruker MICROTOF II spectrometer, Fourier transformation infrared spectroscopy (FT-IR) was recorded with Perkin-Elmer Spectrum RX-IFTIR spectrophotometer in the range 400-4000 cm − 1 and nuclear magnetic resonance (NMR) spectroscopy was recorded in chloroform-d [99.8 atom% D, containing 0.1% (v/v) tetramethylsilane (TMS)] at 25°C on 500 MHz AVANCE III Bruker spectrometer equipped with a 5 mm double channel solution state probe [31,32].

Antibacterial activity of purified compounds
The antibacterial activity of the purified compounds was assayed using the standard Kirby-Bauer disc diffusion method. Petri plates containing MHA were swabbed with test bacteria and then discs loaded with 25 μg of purified compounds were placed on the surface of the medium followed by compound diffusion at refrigeration temperature for 30 min. The plates were incubated overnight at 37°C and the zones of inhibition were measured in millimetres.

Bioautography of purified compounds
The pure compounds (30 μg) were loaded onto precoated TLC plates and separated using ethyl acetatehexane (9:1, v/v). The dried TLC plates were placed onto the medium seeded with B. subtilis. These plates were kept in refrigerator for 1 h for diffusion. Thereafter, these plates were incubated overnight at 37°C for 24 h, observed for clear zones and Retardation factor (Rf ) values of purified compounds calculated.

Minimum inhibitory concentration (MIC) of purified compounds
Minimum inhibitor concentrations of the purified compounds were determined by 96 well microtiter plate dilution assay. The different concentrations of the purified compound (0.96, 1.97, 3.95, 7.56, 15.12, 31.25, 62.5, 125 μg/ml) were prepared in sterile water [33] and added to the test bacteria viz. VRE, MRSA, S1-LF, K. pneumoniae sub sp. pneumoniae and B. subtilis grown to logarithmic phase (between 0.3 to 0.5 . MIC values were calculated by comparing the growth in wells containing extract to the growth in control wells and are the lowest concentration that resulted in 90% inhibition in growth compared to the growth in control well.

Isolation and screening
Out of 20 different actinobacteria isolates recovered from the soil, 12 isolates showed activity against one or more test bacteria in the primary screening. Among these, 7 isolates displayed antibacterial activity in fermentation broth with varying degree of inhibition against different test bacteria. Isolate M7 exhibiting potent antibacterial activity against all the test bacteria was selected for further studies.

Identification and characterization of strain M7
The actinobacterial strain M7 grew well on all the ISP media and SCNA with different cultural characteristics (Additional file 1: Table S1). The strain produced yellow colored diffusible pigment on SCNA (Fig. 1a, b) and brown colored pigment on ISP6 and ISP7 media. Microscopic studies showed the formation of branched substratum mycelium and rectiflexibilis-type spore chains, bearing 25-38 smooth cylindrical spores on aerial mycelium (Fig. 1c, d). Chemotaxonomic analysis of cell wall and whole cell hydrolysates revealed the presence of type 1 cell wall, containing LL-DAP as the diagnostic amino acid and no characteristic sugar. The physiological and biochemical characteristics of the strain are shown in Table 1. The strain M7 was able to grow at temperature between 25 and 45°C (optimum at 28°C), pH 5 to 10 (optimum at pH 7.0) and could tolerate NaCl upto 5%. M7 strain utilized different tested carbon sources: starch, glycerol, D-glucose, sucrose,    (Fig. 2).

Antibacterial activity profile of Streptomyces strain M7
In vitro bioassay demonstrated strong antibacterial activity of Streptomyces strain M7 against tested bacteria. It showed pronounced inhibition against pathogenic bacteria viz. VRE, MRSA and M. smegmatis with inhibition zones of 23-21 mm. Moderate to weak activity was observed against B. subtilis, K. pneumoniae sub sp. pneumoniae, S. epidermidis, S. typhi, E. coli, S1-LF and S. aureus with inhibition zones of 15-20 mm. This suggests that MRSA, VRE and M. smegmatis are more susceptible as compared to other test bacteria. The production of active metabolites in SCN culture broth started after 24 h of incubation, reached the maximum after 96 h and declined slightly as the incubation was further extended (Fig. 3).

Recovery, separation and bioautography of bioactive metabolites
Among all the solvents used, ethyl acetate was found to be the best solvent to achieve the maximum recovery of active metabolites from fermentation broth of pH 5.0. The extracted metabolites in ethyl acetate were concentrated under reduced pressure using rotary evaporator and resulted orange colored dried extract was redissolved in ethyl acetate. Separation of antibacterial metabolites present in crude solvent extract was carried out by thin layer chromatography using ethyl acetate: hexane (9:1, v/v) as solvent system (Additional file 2: Figure S2a). Bioautography of purified actinomycins also confirmed the three antibacterial compounds with Rf values of 0.25 (compound P1), 0.52 (compound P2), and 0.48 (compound P3) (Additional file 2: Figure  S2b).

Purification of antibacterial compounds from S. antibioticus strain M7
For purification of antibacterial compounds, fermentation was carried out in SCN broth for 4 days at 28°C. After 4th day of incubation, culture broth was centrifuged at 10,000 rpm and then extracted twice using ethyl acetate (1:2, v/v). The obtained orange color crude extract was subjected to silica gel column chromatography for isolation of active compounds. Twenty six fractions , eluted with hexane: ethyl acetate (10:90, v/v) showed antibacterial activity. These were pooled together based on their similar TLC pattern and concentrated. The pooled fraction was further fractionated on size exclusion chromatography using toyopearl resin HW-40. Nine fractions (27)(28)(29)(30)(31)(32)(33)(34)(35) which showed activity were again pooled and finally subjected to semi-preparative HPLC. Individual peaks were collected, and antibacterial activity was detected in two peaks with retention times of 10.524 and 15.443 min (Additional file 2: Figure S1a). Active peak with retention time of 15.443 was further fractionated by performing HPLC using acetonitrile: water (95:5, v/ v) as gradient and resulted in separation of compounds P2 and P3 with retention times of 5.814 and 6.548, respectively (Additional file 2: Figure S1b). The collected peaks were further chromatographed using acetonitrile: water (55:45) and single peaks with retention times of 10.628, 15.318, and 15.999 min were obtained which indicated the purity of the compounds [Additional file 2: Figure S1 (c-e)].

Antibacterial activity of purified compounds
The purified compounds exhibited potent antibacterial activity against a range of both Gram negative and Gram positive bacteria viz. B. subtilis, K. pneumoniae sub sp. pneumoniae, S. aureus, S. epidermidis, S. typhi, E. coli, S1-LF, MRSA and VRE. In case of MRSA and VRE, the compound P2 (Actinomycin X 2 ) was more effective with inhibition zones in the range of 18 and 26 mm as compared to compound P3 (Actinomycin D) (17 and 25 mm) and compound P1 (Actinomycin V) (14 and 24 mm). The compounds showed significant activity against drug resistant strains which are resistant to methicillin (MRSA) (10 μg/disc) and vancomycin (30 μg/disc) (VRE) (Fig. 6, Additional file 1: Table S3).

MIC values of purified compounds
The MIC values of the purified compounds were determined by 96 well plate method. The Actinomycin X 2 was found to be more potent with lowest MICs as compared to actinomycins D and V. The MIC values of purified actinomycin X 2 against test bacteria ranged between 1.95 and 15.62 μg/ml whereas those for actinomycins V and D ranged between 2.25 and 31.25 μg/ml, and 2.0 and 15.0 μg/ml, respectively. All the three actinomycins were found to be more effective against MRSA and VRE with MICs of 1.95-2.25 μg/ml and 3.5-4.0 μg/ml, respectively than against K. pneumoniae sub sp. pneumoniae, S1-LF and B. subtilis with MIC values of 15.0-31.5 μg/ml, 14.23-15.90 μg/ml and 8.0-15.62 μg/ml, respectively. (Additional file 1: Table S3).

Discussion
The emergence and spread of multidrug resistant bacteria cause an array of health problems due to various interconnected factors, many of which are related to over and misuse of antimicrobial drugs and acquisition of resistance genes [34][35][36]. The rising levels of antibiotic resistance have complicated the treatment therapy for HAI (health care-associated infections) MRSA and VRE infections [37]. In this biological arm race, humans appear to be helpless as pathogens continue to develop resistance against each new drug introduced in the market. Hence, there is an urgent need to search new antimicrobial agents against these pathogens.
Actinomycins are cytotoxic compounds which exhibit potential cytotoxicity against various cancer cell lines but low toxicity against normal human cell lines [50]. They are one of the oldest anticancer drugs used in the treatment of various sarcomas. However, antimicrobial activities of actinomycins against pathogenic bacteria, especially MRSA and VRE have gained very little attention. Khieu [52].
Our study demonstrated strong antibacterial activity of actinomycins D, X 2 and V isolated from Streptomyces strain M7 against VRE, MRSA, B. subtilis, K. pneumoniae sub sp. pneumoniae, S. epidermidis, S. typhi, E. coli, S1-LF and S. aureus. The MIC values of actinomycins against VRE were 1.95-2.0 μg/ml, which are higher than those reported in earlier studies [49][50][51]. However, actinomycins were found to be more effective against E. coli and K. pneumoniae (MIC values 15.65-64 μg/ml) as compared to actinomycins D and X 2 (> 128 μg/ml) reported by Wang et al. [51]. The findings of the present investigation also support the extended application of

Availability of data and materials
All data generated or analysed during this study are included in this published article and additional supplementary files.
Authors' contributions RM as research supervisor of MS was involved in the design and planning of research work; analysis and interpretation of data; drafting as well as critical editing of the manuscript for intellectual subject matter. MS was involved in the planning and execution of the research work; analysis and interpretation of the data; manuscript writing following the suggestions of the research supervisor. All authors have read and approved the manuscript.
Ethics approval and consent to participate Not Applicable.