Indian Journal of Experimental Biology Vol. 50, October 2012, pp. 718-728

Screening, isolation, and fermentation of an producer Streptomyces xinghaiensis from soil capable of acting against resistant strains

Konda Shravan Kumar1, Sriramoju Anuradha1, Gadepalli Rama Sarma2, Yenamandra Venkateshwarlu3 & Veerabrahma Kishan1,* 1University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506 009, India 2Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi, USA 3Natural Product Laboratories, Organic Chemistry Division -1, CSIR-Indian Institute of Chemical Technology, (CSIR-IICT), Hyderabad 500 007, India

Received 14 November 2011; revised 11 July 2012

Linezolid resistant cultures are emerging in hospitals. In the present study 3 soil actinomycetes were isolated in a screening programme having potential to produce antibiotic against linezolid resistant cases. One culture was coded as RK- 46 and further studied. The micromorphology, biochemical tests and 16S ribosomal DNA gene sequence analysis were conducted to know the identity of the culture and was found as a strain of Streptomyces xinghaiensis. The culture produced antibiotic active against five clinical resistant strains. The antibiotic production was tested by cultivating in eleven different media. The fermentation profile was studied in YEME medium supplemented with calcium carbonate. The maximum activity was noticed at 72 h. Antibiotic activity was extracted into ethyl acetate and was subjected to activity guided purification by column chromatography, TLC and HPLC methods. The pure compound was eluted with retention time of 6.8 min and subjected to 1H, 13C NMR and Mass spectral analysis. The acquired data was compared with that in natural products data base, and was found to be a known antibiotic, reductiomycin. The purified compound showed activity against 5 linezolid resistant cultures and on Mycobacterium tuberculosis. This compound is also showing mild anti cancer activity and is biologically permeable as per Lipinksi’s rule.

Keywords: Antibiotic, Linezolid resistance, Reductiomycin, Streptomyces xinghaiensis

The global emergence of resistance among gram- inhibit the formation of the initiation complex positive bacterial species has necessitated the rapid constructed with 70S ribosomes, mRNA, initiation discovery and development of alternative agents to factors, IF2, IF3 and formyl methionyl-tRNA3. the penicillins, and glycopeptides1. The Linezolid has been indicated for the treatment of prevalence of bacterial pathogens resistant to the infections caused by various gram-positive available has been increasing over the past including methicillin-resistant enterococci and several decades. This situation constitutes a major Vancomycin resistant enterococci (VRE)4,5. Linezolid challenge for clinicians and microbiologists and is was proven to be effective in both experimental and particularly acute for treatment of infections caused clinical endocarditis6. However, clinical failure to by gram-positive organisms2. Linezolid is a member treat VRE7 and Staphylococcus aureus with linezolid of new antibacterial agents called the oxazolidinones, was also reported5,8. The aim of this study is to screen which are chemically unrelated to currently available soil from Singareni coalmine, and isolate a microbial agents. Linezolid selectively binds the 50S ribosomal culture capable of producing antimicrobial principle subunits, thereby resulting in the inhibition of active against linezolid-resistant cultures. Further, to bacterial protein synthesis. Oxazolidinones are unique study the morphology, taxonomy, fermentation of the in that they do not inhibit elongation but instead antibiotic producer, biological activities of crude extract and purification of the antibiotic. In addition, ———————— efforts have been made to select medium for *Correspondent author better production of antibiotic and finally obtaining of Telephone: + 91870 2446259 1 13 Fax: + 91870 2453508 H, C NMR and Mass spectral data to know the E-mail: [email protected] structure of antibiotic. KUMAR et al: ISOLATION OF ANTIBIOTIC AGAINST LINEZOLID RESISTANT CULTURES 719

Materials and Methods culture RK-46, after growing for 14 days at 28 oC on Clinical isolates resistant to linezolid namely, the standard media suggested by the International E. faecium CC-X48585, JM-H56842 and SM-T23230 Streptomyces Project (ISP)13 . To examine the spore were obtained from New York Medical Health chain morphology, the isolate RK-46 was incubated Centre, USA and one strain each of Staphylococcus on starch casein nitrate agar for 14 days and examined aureus from MGM Hospital, Warangal and CMC, using light microscopy (x 400) by agar block Vellore respectively. Linezolid was a gift from Hetero method14. Amongst the morphological categories Drugs Ltd, Hyderabad. Mueller- Hinton Agar, starch, suggested by Pridham et al.15, the two categories of casein were purchased from Himedia Labs, Mumbai. Rectiflexibiles and Spirales were employed for Cycloheximide was purchased from Serva evaluation of spore chain morphology. The FeinBiokemica, Heidelberg, Germany. physiological tests like growth at different Assesment of linezolid resistance of clinical temperatures, pH, and the production of acids from isolates—Five colonies, each at least 1 mm in carbohydrates and hydrolysis of casein, gelatin, and diameter, were suspended in 5 mL of sterile 0.9% starch were performed as described in ISP16-18. saline. The turbidity of the suspension was adjusted 16S rDNA sequencing for the genus spectrophotometrically to match the transmittance of Streptomyces—For sequence analysis, bacterial a 0.5 McFarland barium sulfate turbidity standard9. genomic DNA was extracted and amplification of 16S The susceptibility breakpoint for linezolid is rDNA was performed on the isolated DNA using the 4 µg/mL10. Disks of linezolid containing various primers F—5′-GGATGAGCCCGCGGCCTA-3΄and concentrations ranging from 2-30 µg/disk were R—5΄-CGGTGTGTACAAGGCCCGGGAACG-3΄. prepared and placed on Mueller-Hinton agar plate The amplified DNA products were resolved on 1.5% seeded with linezolid resistant cultures, incubated at (w/v) agarose gel and then excised from the gel and 37 oC for overnight and zone of inhibition was noted. purified. The purified products were sequenced using Screening and isolation of antibiotic producers ABI PRISM 377 DNA instrument (Applied against linezolid-resistant cultures from soil—Soil Biosystem Inc., CA, USA). The sequence was samples were collected at a depth of about 100 meters analysed using BLAST (NCBI) to determine the from an incline Singareni coalmine in Adilabad phylogenetic positions and dendrogram was district, Andhra Pradesh. Soil sample (1 g) was constructed using Clustal W software by M/s Bioaxis suspended in 100 mL of sterile distilled water in DNA research center, Hyderabad, India. 250 mL conical flask and incubated at 55 oC for 6 min Antibiotic activity in different fermentation for pretreatment and shaken vigorously on a rotary media—The isolated actinomycete culture RK-46 was shaker for 30 min at room temperature and allowed to cultivated in 11 different nutrient media for the settle for another 10 min. Further 1 mL of supernatant production of bioactive metabolite active against was 10 fold serially diluted with sterile distilled water linezolid resistant cultures. A loopful of the culture and 0.1 mL of the supernatant was spread on was inoculated in 250 mL Erlenmeyer flask starch casein agar plates supplemented with containing 60 mL of starch casein-nitrate medium and cycloheximide11. The plates were incubated at 28 oC was shaken on rotary shaker for 48 h at 28 ºC to for one week. Some of the actinomycetes colonies obtain seed inoculum. Seed inoculum (2mL) were selected based on gross morphological was inoculated in six flasks containing 25 mL of the appearance. The selected cultures were maintained on same medium and fermentation was carried for 120 h starch casein agar slants. The antimicrobial activities at 28 ºC. Samples were withdrawn at an interval of the isolates were determined by a giant colony of 24 h and centrifuged at 4000 rpm for 10 min. technique against linezolid resistant cultures and type About 50 µL of culture filtrate was subjected to agar cultures of both gram-positive and gram-negative diffusion assay by cup plate method on plates seeded bacteria12. All the isolates tested were coded as RK with linezolid resistant cultures and Bacillis subtilis series. The actinomycete strain which produced (MTCC 619). The same procedure was adopted to antibiotic against linezolid resistant culture was remaining ten different media namely MPG medium designated as RK-46. (2% mannitol, 2% peptone and 1% glucose), Nutrient Culture identification—The gross morphological broth19, MDPY medium (2% mannitol, 2% dextrin, observations were made on the antibiotic producing 1% peptone and 0.3% yeast extract), Czapek-Dox 720 INDIAN J EXP BIOL, OCTOBER 2012

medium, Soyabean meal medium (2% soyabean meal, Micro green algae Chlorella vulgaris, Scenedesmus 2% glucose, 0.5% NaCl and 0.3% CaCO3), Tryptone subspicatus and Chlorella sorokiniana were also soya broth ( 17 g pancreatic digest of casein, 3.0 g evaluated in this study. The medium used for papaic digest of soyabean meal, 5.0 g NaCl, 2.5 g antifungal studies was potato dextrose agar (PDA) dipotassium phosphate, 2.5 g dextrose and 1L H2O ), (Difco, USA) and for micro green algae was the Tryptone soya broth plus 2% mannitol, ISP-2 Bold’s Basal medium (BBM)20. The antimicrobial medium, ISP-2 supplemented with 0.5g/L CaCO3 and activity was estimated by measuring the diameter of SM medium (2% soya bean meal, 2% mannitol) zone of inhibition. respectively as shown in Table 4. Antitubercular activity—The effect of the ethyl Fermentation Profile—A 1cm2 piece of Yeast acetate extract was studied on multidrug resistant extract malt extract agar (ISP-2) containing 7 days old Mycobacterium tuberculosis strain C77/08. The strain culture was used to inoculate in six Erlenmeyer flasks was isolated from a patient’s sputum with a known containing 25 mL of medium, composed of malt history of tuberculosis (multidrug resistant) by extract 10 g, yeast extract 4 g, glucose 4 g and CaCl2 Dr Chitra Chandrashekar in Dr Iravatham Clinical 0.5 g/L (pH 7.2 before sterilization). The flasks Laboratory, Hyderabad21. LJ medium (4 mL) was were shaken at 200 rpm on a gyratory shaker poured into a sterile Mcartney bottle, containing (GFL, Germany) at 28 ºC. The study was conducted different concentrations of drug solutions (10, 100 for 5 days. A typical time course of the fermentation and 1000 µg/mL) tightly capped and kept in a slanting is shown in Fig. 3. Flasks were removed from shaker position overnight. The culture was streaked on at 8 h interval and examined for pH, packed cell Lowenstein Jensen medium and incubated at 37 ºC for volume (PCV) and antibiotic production. The growth 3-4 weeks. Antibiotic concentrations of rifampicin was expressed in terms of PCV by centrifuging 25 mL and isoniazid were prepared geometrically in the of fermented broth at 4000 rpm (1636 g) for 20 min. range of 2-256 µg/mL. A positive control and solvent To estimate the optimum time course of antibiotic control (0.1 mL DMSO and 0.9 mL sterile distilled production, culture filtrates at different time intervals water) were included in the study. were subjected to agar diffusion assay by cup plate Cytotoxicity test (Brine shrimp test)—The Artemia technique against 18 h culture of Bacillus subtilis salina test introduced in 1968 by R. F. Brown22 was (MTCC 619). As the nature of antibiotic principle was used to determine the cytotoxicity of active unknown, the concentration of antibiotic principle compounds. A separatory funnel was filled with was expressed in terms of known antibiotic standard, artificial sea water (55 mL), to this Artemia salina sulphate. For this, streptomycin sulphate eggs (200 mg), were added and kept for 3 days with a I.P was taken in different concentrations of known gentle supply of air until larvae emerged. One side of units and their corresponding zones of inhibition the funnel was covered with aluminium foil. After produced against standardized spore suspension of 5 min, the migration of larvae to the bright side of the Bacillus subtilis (MTCC 619) were noted and a funnel was observed due to positive phototaxis. The standard graph was plotted. The concentration of larvae was pipetted out with a Pasteur pipette. About antimicrobial principle produced by isolate RK-46 20-40 larvae were placed in 990 µL of sea water in was expressed using standard units of streptomycin each of the 24 well micro titer plate. The dead larvae sulphate19. were counted under a microscope. DMSO solution Biological activities of crude extract/ culture (10µL) of the ethyl acetate extract (10 µg/mL) was filtrate—The antimicrobial activity studies were added to microwells. The reference preparation was performed by cup plate method. Five linezolid actinomycin D (10 µg/mL). The negative control was resistant cultures were grown on Mueller- Hinton agar treated with only DMSO (10 µg/mL). After medium. The crude extract was dissolved in methanol incubation for 24 h and further storage of micro titer (1 mg/mL) and 50 µL of the samples were placed in plate for 24 h in refrigerator (to ensure immobility), the cups. Fresh culture filtrates (50 µL) were also dead larvae were counted under microscope. placed in the cups and incubated for 24 h at 37 ºC. Fermentation and extraction of antibiotic from Further, crude extracts were also tested on plates fermented broth—Flasks of 500 mL, 1 L and 2 L were containing the cultures of Mucor miehi Tü 284, loaded with ¼ of their capacity with a medium Candida albicans, R. solani, A. cochlioides (fungal). consisting malt extract 10 g, yeast extract 4 g, glucose KUMAR et al: ISOLATION OF ANTIBIOTIC AGAINST LINEZOLID RESISTANT CULTURES 721

4 g and CaCO3 0.5 g/L (pH 7.2 before sterilization). A Antibacterial activities of isolated compound KSK- 1 cm2 piece of YEME agar from 7 days old culture 1(reductiomycin on clinical resistant strains and was used to inoculate the broth. Based on the Mycobacterium tuberculosis—The antimicrobial fermentation profile the production was carried out at activity of the purified compound KSK-1 was also 28 ºC for 72 h on gyratory shaker at 200 rpm. The studied on linezolid resistant cultures, namely, fermented broth (12 L) was filtered and mycelium E. faecium CC-X48585, E. faecium JM-H56842, was separated. The culture filterate was adsorbed on E. faecium SM-T23230 and one strain each of Amberlite XAD-16 column (5 cm × 35 cm). The Methicillin resistant Staphylococcus aureus (MRSA) Amberlite XAD-16 resin was washed with 4 L of from MGM Hospital, Warangal and Vancomycin deionized water, and the metabolites were eluted with resistant Staphylococcus aureus ATCC 43300 (VRSA) 1 L of methanol. The solution was reduced in vaccuo, by disk diffusion method. The organisms were seeded pH was adjusted to 4 and extracted with ethyl acetate on Mueller-Hinton agar by spread plate technique. (3 × 300 mL) at room temperature23,24. The combined Disks containing various antibiotics namely, Linezolid organic layers were evaporated under reduced (30 µg), Methicillin (5 µg), Vancomycin (30 µg) and pressure to give a brown-red residue (ca. 1.62 g). The two different concentrations of isolated compound (30 and 50 µg/mL) were placed on the plates and incubated biomass was extracted with acetone and it could not at 37 ºC for 24 h. The zones of inhibition for all the show any antimicrobial principle and was discarded. antibiotics were observed. Purification of antibiotic—Crude (1.62 g) of ethyl The antitubercular activity of the compound was acetate extract was loaded onto silica gel 60 (Merck) studied on Mycobacterium tuberculosis strain H37Rv column 3 × 32 cm and the column was eluted with (ATCC 27294), a type strain, according to the method 21 stepwise 100% CHCl3 and 95:5, 90:10, 50:50 CHCl3- of Bikshapathi et al . MeOH and 100% MeOH and obtained five fractions. Anticancer activity of isolated compound—The Fraction 2 was again subjected to silica gel column anticancer activity of the compound was evaluated (2 cm × 20 cm) chromatography and eluted with against three cancer cell lines namely, HT-29 cell line hexane-ethyl acetate gradient (100% hexane to 30:70 (colon cancer), PC-3 cell line (prostrate cancer) and hexane:ethyl acetate) to obtain 0.8 mg of compound B-16 cell line (mouse macrophages cell line) and was 1. Fraction 4 (140 mg) was dissolved in 2 mL of compared with a standard, doxorubicin by MTT methanol and loaded into Sephadex LH-20 column assay25. (3 cm × 40 cm) and eluted with methanol to obtain Studies on the absorption/permeation across 2 fractions. About 43 mg of fraction 4.2 was biological membranes—Computational approach: separated by preparative high-performance liquid Application of Lipinski’s rule— Lipinski’s rule of five chromatography (Jasco 2087, Jasco Co., Essex, UK) is a computational approach developed to predict the equipped with a Develosil C18 column (10 µm, solubility and permeability across biological 26 250 mm × 20 mm; Nomura Chemical Co., Japan) at membranes. According to Lipinski’s rule poor room temperature, solvent 0.1%TFA as solvent A and absorption or permeation is more likely when there 0.1%TFA in acetonitrile as solvent B at a flow rate of are more than 5H-bond donors, more than 10H-bond 20 mL/min and detection was performed using UV acceptors, molecular weight greater than 500 and C detector. The gradient was from 20% B to 100% B log P greater than 5. To evaluate drug likeness better, in 30 min to obtain compound 2 (3.8 mg, Rt 6.8 min) the rules have spawned many extensions, for and compound 3 (5.2 mg, Rt 2.5 min). example: partition coefficient, log P in -0.4 to 5.6 range. Molar refractivity from 40 to 130. Structure and spectroscopic analysis of purified product—The purified compound was subjected to Results and Discussion spectral studies by MS, 1H and 13C NMR and the data The clinical resistant strains of E. faecium CC- were checked in database like Dictionary of Natural X48585, JM-H56842 and SM-T23230 and 1 13 Products, (Chapman and Hall). Both H and C NMR Staphylococcus aureus ATCC 43300 and spectra were recorded on Brucker AVANCE Staphylococcus aureus MGM were examined and DRX 400 MHz NMR spectrometer. EI-MS data found to be resistant to linezolid at 30 mcg were obtained on a WATERS ZQ-LCMS mass concentration and whereas NCCLS standard for spectrometer. linezolid at 8 µg, with a zone of inhibition less than 722 INDIAN J EXP BIOL, OCTOBER 2012

21 mm resistant was considered as resistant7,10. No grown in ISP media 1, 2, 3, 5 and 6, starch casein zone of inhibition was observed at 30 µg agar, nutrient agar was found to be grey, whereas in concentration (Table 1). The habitat differences glycerol-aspargine agar it was white in colour. The would result in rare type of microorganisms. In the growth was good in all the tested media. Soluble process of screening of soil actinomycetes capable of pigmentation was not observed in ISP and the other producing antibiotics, about 56 actinomycete media (Table 2). The temperature range for isolate colonies, macroscopically different from each other, RK-46 was 15–45 ºC. The biochemical tests revealed were selected and further purified by streak plate that casein hydrolysis and starch hydrolysis were technique and coded as RK-1 to RK-56. The isolates positive, whereas gelatin hydrolysis, citrate utilization were stored on starch casein agar tubes. Three tests, Voges Proskauer, urea hydrolysis, hydrogen cultures showed activity against linezolid resistant sulphide production, nitrate reduction and oxidase cultures but two cultures lost ability to produce the tests were negative. The acid production from antibiotic principles. One culture, RK-46 was found to carbohydrates was observed in arabinose, mesoinositol, be stable and produced antibiotic active against and galactose, whereas in case of other carbon sources linezolid resistant cultures and was studied in detail. it was not observed. The different physiological and The micromorphological studies of RK-46 indicated biochemical properties of Streptomyces aureofaciens that the mycelium with sporophores as rectiflexibili NRRL 220914,16,17 are also shown (Table 3) for type of spore chain as observed by light microscopy comparison. Initially, CSIR-Institute of Microbial (Fig. 1). The colour of the aerial mycelium when Technology (IMTECH), Chandigarh identified the culture RK-46 as a strain of Streptomyces Table 1—Resistance of clinical isolates towards linezolid and aureofaciens. The RK46 was deposited with an other antibiotics accession number of MTCC 8770. S.No Organism Strain no. Resistance Diameter of Phylogenetic analysis of the strain RK-46 using the concentration zone of observed inhibition 16S rDNA sequence—The 1,490 bp sequence (µg/mL) (mm) obtained from the strain RK-46 was aligned with the 1 E. faecium CC-X48585 30 Nil available 16S rDNA gene sequence obtained from 2 E . faecium JM-H56842 30 Nil GeneBank (NCBI) database and it was matched 3 E. faecium SM-T23230 30 Nil exactly with that of a strain of Streptomyces sp S187. 4 S. aureus* ATCC 43300 30 Nil The phylogenetic analysis of the strain RK-46 using 5 S. aureus** MGMa 30 Nil the 16S rDNA gene sequence data (not shown) aIsolated at Mahatma Gandhi Memorial Hospital, Warangal, India suggested that the strain could be Streptomyces resistant to antibiotics. xinghaiensis27 (Fig. 2). * VRSA- Vancomycin resistant culture at 30 µg/disk The antibacterial activity of the RK-46 was tested ** MRSA- Methicillin resistant culture at 5 µg/disk by cultivating in 11 different fermentation media. Based on the relative zones of inhibition, the antibacterial activity of each production medium was determined and showed in (Table 4). Three media showed activities, they being, soyabean mannitol (SM), Malt extract-Yeast extract supplemented with calcium carbonate (M2+ medium) and MPG medium. One of the three media (Malt extract, Yeast extract medium+ CaCO3 (M2+ medium) was used for studying the fermentation profile of the RK-46 culture. The production of antibacterial substance was carried out in shake flask culture. A typical time course of the fermentation is shown in Fig 3. The production started after 32 h and gradually reached maximum at 72 h and decreased gradually until 96 h

Fig.1—Slide culture of Streptomyces xinghaiensis (RK-46) over and afterwards no antibacterial activity was observed. Starch casein agar medium showing Rectus flexibilis type of During fermentation cycle pH was in between 7-9.5. Sporophore, indicated by bold arrow (magnification 400X) During maximum activity period the pH was in KUMAR et al: ISOLATION OF ANTIBIOTIC AGAINST LINEZOLID RESISTANT CULTURES 723

Table 2—Gross morphological characters of isolate RK-46 when grown in different media medium Growth Aerial mycelium— Vegetative Soluble spore color mycelium pigment Tryptone-yeast extract agar (ISP-1)a good light grey white none Yeast extract - malt extract agar (ISP-2)a good grey white none Oatmeal agar (ISP-3)a good grey grey none Inorganic salts-soluble starch agar (ISP-4)a good grey white none Glycerol-aspargine agar (ISP-5)a good white white none Peptone-yeast iron agar (ISP-6)a good whitish grey white none Starch casien nitrate agarb good grey grey none Nutrient agarb good grey grey none a= ref. 13 ; b= ref.19

Table 3—Comparative physiological and biochemical properties of isolate-RK-46 Name of the test RK-46 Streptomyces aureofaciens Streptomyces NRRL 220917, xinghaiensis NRRL B-2467427, Gram's staining Gram +ve Gram +ve Gram +ve Cell shape Mycelial Mycelial Mycelial Spore Chain Morphology Rectus flexibilis RetinaculumApertum Rectus flexibilis Endospore Negative N A Negative Sporangia Bulging Negative N A Negative Motility Negative N A Negative Flourescence Negative N A N A Temperature range for growth(°C) 15 - 45°C 10 - 45°C 10 - 45°C pH range for growth 5 - 10 6 - 9 7 -9 Growth on NaCl 2% Positive Positive Positive Growth on NaCl 4%,6% & 8% Positive Positive Positive Growth on NaCl 10% Positive Negative Negative Growth on Mackonkey Agar Negative N A N A Indole Test Negative N A N A Methyl Red Negative N A N A Voges Proskauer Negative N A N A Citrate Utilization Negative N A N A Casein Hydrolysis Positive Positive Positive Gelatin Hydrolysis Negative Positive Positive Starch Hydrolysis Positive Positive Negative Urea Hydrolysis Negative Positive N A Nitrate Reduction Negative Negative N A Hydrogen sulphide Production Negative Positive N A Catalase Positive NA Positive Oxidase Negative NA N A Acid production from carbohydrates Salicin Negative Positive N A Arabinose Positive Positive Negative Galactose Positive Positive Positive Dextrose Negative Positive Positive Meso-inositol Positive Positive Negative Raffinose Negative Positive (Weak) Negative Rhamnose Negative Positive Positive Fructose Negative Positive (Weak) Positive Mannitol Negative Positive Positive Sorbitol Negative Negative Negative Sucrose Negative Positive Positive Xylose Negative Positive N A * Positive = Utilized; Negative = Not Utilized , NA-= Information not available

724 INDIAN J EXP BIOL, OCTOBER 2012

Fig. 3—Fermentation Profile of RK-46 showing maximum activity at 72 h and biomass at 56 h.

Fig. 2—Dendrogram showing the relationships between Table 5—Antimicrobial activity of crude extract of RK-46 in Streptomyces sp. RK- 46 and other Streptomyces sp. cup/disk plate assay against different cultures

Table 4—Antibiotic activity on different fermentation media Test organism Diameter of zone of inhibition (mm) S. No Name of the media Bioactivity against linezolid resistant E. faecium CC-X48585 19 cultures E .faecium JM-H56842 26 1 Soyabean meal, Mannitol (SM) Active E. faecium SM-T23230 32 medium 10 Staphylococcus aureus ATCC 43300 21

2 ISP-2 +0.5g/L CaCO3 Active Staphylococcus aureus MGM 23 3 Mannitol, peptone and glucose Active Mucor miehi Tü 284 12 medium Candida albicans 12 4 Tryptone-soya Broth + 2% No activity R. solani nil mannitol A. cochlioides nil 5 Mannitol, dextrin- peptone-yeast No activity Chlorella vulgaris, nil extract medium Chlorella sorokiniana nil 6 Starch casein nitrate19 No activity Scenedesmus subspicatus nil 7 Soya bean -meal medium19 No activity

8 Tryptone-soya broth medium No activity resistant Mycobacterium tuberculosis strain C77/08 9 Nutrient broth19 No activity was found at a concentration of 1000 µg/mL and 10 Malt extract, yeast extract, No activity 100% mortality of Artemia salina larvae was glucose(M 2 medium) observed at a concentration of 100 µg/mL. 11 Czapek–Dox medium19 No activity Identification of antibiotic active against linezolid maintained in between 7.5-9.5 upto 72 h. Later, no resistant cultures—The antibiotic active against change in pH was observed. Biomass gradually linezolid resistant cultures were isolated from the increased and reached maximum at 56 h and then extract of the strain RK-46 using solvent extraction gradually decreased till 104 h and maintained at the and chromatography. The scheme of purification is same level up to 120 h. shown in Fig. 4. The spectral properties of the Biological activities of crude extract / culture compound were studied by LC-MS, 1H and 13C NMR. filtrate of RK-46—The ethyl acetate extract of strain The mass spectra of the compound showed a RK-46 was active against five linezolid resistant molecular ion peak at m/z 292 in negative ion mode cultures, Mucor miehi Tü 284, Candida albicans, and m/z 316 in positive ion mode (M+Na) and m/z Chlorella vulgaris, Scenedesmus subspicatus and 609 (2M+Na). The compound exhibited resonances Chlorella sorokiniana (Table 5). However, no for 15 protons (δ 2.12 (3 H), 2.57- 2.50 (2 H), 2.62 inhibition of growth was observed against plant (2 H), 2.70 (1 H), 3.06 (1 H), 5.77 (1 H), 6.75 (1 H), pathogens, R. solani, A. cochlioides and micro green 6.88 (1 H) 7.45-7.58 (2 H one from NH), 13.64 (1 H) algae, Chlorella vulgaris, Scenedesmus subspicatus (Table 6). 13C NMR (101MHz) data of our compound and Chlorella sorokiniana. Inhibition of multi drug showed resonances for 14 carbons [δ 197.27, s, KUMAR et al: ISOLATION OF ANTIBIOTIC AGAINST LINEZOLID RESISTANT CULTURES 725

C-1, 173.64 (s,C-3), 169.53 (s, 2´-OCOCH3), 165.66 2-propenamide with a molecular formula C14H15NO6 (s, C-1´´), 165.9 (s, C-3), 150.80 (d, C-5´), 135.82 (Fig. 5). Further, this identification was confirmed by (d, C-3´´), 115.35 (d, C-2´´), 115.03 (d, C-2) 114.65 search in database of Dictionary of Natural Products, (s, C-4´) 98.46 (d, C-2´) , 34.17 (t, C- 3´), 32.14 (t, C-5), (Chapman and Hall). 25.58 (t, C-4) 20.97 (q, 2´-OCOCH3)] (Table 7). Antibacterial, antitubercular and anticancer These results were in agreement with the reported activity of pure compound KSK-1—The antimicrobial 28,29 spectral data , suggesting the antibiotic to be activity of the isolated pure compound KSK-1, was reductiomycin (3– [5– (Acetoxy)– 4, 5–dihydro–3 – studied on linezolid resistant cultures namely E. furanyl]-N-(2-hydroxy-5-oxo-1-cyclopenten-1-yl) faecium CC-X48585, E. faecium JM-H56842, E. faecium SM-T23230 and one strain each of Staphylococcus aureus (MRSA) from MGM Hospital, Warangal and Staphylococcus aureus ATCC 43300 (VRSA) by disk diffusion method. The zones of inhibition for all the antibiotics were observed. This indicated that the compound KSK-1 exhibited antibacterial activity against all the five clinical isolates of linezolid resistant cultures at a concentration of 50 µg/mL. Interestingly, Staphylococcus aureus (MRSA) and Staphylococcus aureus (VRSA) were more susceptible when compared to E. faecium strains (Table 8). The crude antibiotic extract showed activity on multi drug resistant Mycobacterium tuberculosis C77/08 on L J medium. The antitubercular activity of

13 Table 7— C NMR data of the compound in CDCl3 Assignments Chemical shift,ppm Literature values of our Compound (Konda Y et al.29) of Reductiomycin

CH3CO 20.97 20.9 C-4 25.58 25.7 C-5 32.14 32.3 C-3´ 34.17 34.4 C-2´ 98.46 98.6 C-4´ 114.65 114.8 Fig. 4—Scheme of isolation of antibiotic from crude extract. C-2 115.03 115.2 C-2´´ 115.35 115.5 1 Table 6— H NMR data of the compound in CDCl3 C-3´´ 135.82 135.6 C-5´ 150.80 150.6 Assignments Chemical shift, ppm Literature values of the compound (Konda Y et al.29) C-1´´ 165.66 165.9 for Reductiomycin CH3CO 169.53 169.4 C-3 173.64 173.9 CH3CO 2.12 (s) 3 H 2.10 (s) 3H C-1 197.27 197.2 4-CH2, 5-CH2 2.50- 2.62 4H 2.45-2.68 m, 4H

H- 3´ 2.70 (dt) 1H 2.68 (dt) 1H H- 3´ 3.06 (dd) 1H 3.06 (ddd) 1H H-2´´ 5.77 (d) 1H 5.86 (d) 1H H-2´ 6.75 (dd) 1H 6.71 (dd) 1H H-5´ 6.88 (t) 1H 6.84 (t) 1H

H-3´´ 7.45 (d) 1H 7.48 (d) 1H NH 7.58 (s) 1H 7.84 (s)1H Fig. 5—Chemical structure of the isolated compound known as Enolic OH 13.64 (s) 1H 13.71 (s) 1H Reductiomycin. 726 INDIAN J EXP BIOL, OCTOBER 2012

Table 8—Antimicrobial activity of isolated compound (KSK-1) against linezolid and other antibiotic resistant cultures Isolated compound average zone of inhibition (mm) Name of the culture 30 µg/mL 50 µg/mL Linezolid Vancomycin Methicillin Methanolsolvent Conc Conc. (30µg/disk) (30 µg/disk) (5 µg/disk) (control) E. faecium Nil 12 Nil NT NT Nil CC-X48585 E. faecium Nil 13 Nil NT NT Nil JM-H56842 E. faecium Nil 12 Nil NT NT Nil SM-T23230 S. aureus (MRSA) Nil 17 Nil NT Nil Nil S. aureus (VRSA) Nil 18 Nil Nil NT Nil NT = Not Tested

the pure isolated KSK-1 (reductiomycin) was also Table 9—Properties of isolated compound with their calculated studied on Mycobacterium tuberculosis strain H37Rv and desirable values as per Lipinski’s rule and its extension (ATCC 27294) at a concentration of 128 µg/mL and Properties Calculated Desirable compared with rifampicin and isoniazid as standards. values values The Mycobacterium tuberculosis H37Rv (ATCC Molecular weight 293.00 < 500 27294), was inhibited at 128 µg/mL indicating, H-bond acceptor 6 < 10 anti-tubercular activity of the pure compound, whereas H-bond donor 2 < 5 the inhibition of growth by isoniazid and rifampicin Log P (molecule based) - 0.047 -0.4 to 5.6 was found to be at 0.25 and 8 µg/mL respectively. This Molar refractivity 70.402 40 to 130 indicated that the KSK-1 was relatively less active. The micromorphology, biochemical and physiological The cytotoxicity of the compound was evaluated tests were performed. Initially the culture was against three cancer cell lines namely, HT-29 (Human identified as a strain of Streptomyces aureofaciens 17 colon cancer), PC-3 (Human prostrate cancer) and B-16 upon comparison with Streptomyces aureofaciens , (Mouse Macrophages) and was compared with a standard, but the 16S ribosomal DNA gene sequence analysis doxorubicin by MTT assay. The IC-50 (Inhibitory 50% of RK-46 confirmed the culture as a strain of concentration) was insignificant when compared to the Streptomyces xinghaiensis. The antibiotic activity was standard. However, this observation can be an indicator studied in 11 different media. About three media of very low toxicity of the compound tested. could yield some activity. Fermentation profile of the Further, the reductiomycin was subjected to RK-46 in YEME medium supplemented with 0.5 g/L Christopher Lipinski’s rule of five to predict the calcium carbonate was studied and found to produce solubility and permeability across biological maximum activity at 72 h. The antibiotic principles membranes. Properties of compound with their were extracted by ethyl acetate and the crude extract calculated values and desirable values as per showed activity. The purification was conducted by Lipinski’s rule of five was compared26 (Table 9). column chromatography and HPLC methods. The identified compound (reductiomycin) was Presently, one active metabolite was isolated with expected to have good permeability. Even the retention time of 6.8 min. The 1H, 13C NMR and Mass extensions of Lipinski’s rule about molar refractivity data of the compound was compared with that of the supported this view since its molar refractivity was reported for natural compounds in data bases. The less than 130. All these desirable characters of search indicated that the compound found in this reductiomycin indicated good absorption or study was already reported as reductiomycin. The permeability across biological membranes. isolated compound showed activity against five linezolid resistant cultures, Mycobacterium tuberculosis, Conclusion less active compared with existing drugs and was not An actinomycete, RK-46 was isolated from the highly active against cancer cell lines. The compound coalmine at the depth of 100 meters from the ground. has absorption and permeation characteristics in KUMAR et al: ISOLATION OF ANTIBIOTIC AGAINST LINEZOLID RESISTANT CULTURES 727

accordance with Lipinski’s ruleof five. It has the published ‘general breakpoints’, J Antimicrob Chemother, 48 potential for antibiotic drug development. (2001) 445. 11 Pirouz T, Karbasian M A & Goodfellow M, Isolation of some aerobic Actinomycetes species from the soil of Zahedan Acknowledgement county, South-east of Iran, Irn J Med Sci, 24 (1999) 65. Thanks are due to Dr. Kenneth Inglima of 12 Cassida J E, Screening, in Industrial Microbiology (New Age New York Medical Center, USA for linezolid International, New Delhi) 1968, 65. resistant cultures. One of the authors (KSK) is 13 Shirling E B & Gottlieb D, Methods for characterization of Streptomyces species, Int J Syst Bacteriol, 16 (1966) 313. thankful to AICTE, New Delhi for QIP doctoral 14 Cross T, in The Bergy’s manual of determinative fellowship, to Prof. H. Laatsch, Goettingen bacteriology: The actinomycetes, edited by J G Holt University, Germany for allowing the lab facilities (Williams &Wilkins Company, Baltimore) 2000, 605. and guidance during his stay, to Dr. Chitra 15 Pridham T G, Hesseltine C W & Benedict R G, A guide for Chandrasekhar, Dr Iravatham Clinical Laboratory, classification of Streptomycetes according to selected group placement of strains in morphological sections, Appl Hyderabad for anti tubercular tests and to Frau Microbiol, 6 (1958) 52. Fredricke Lissy, Goettingen University, Germany for 16 Benedict R G, Pridham T G, Lindenfelser L A, Hall H H & technical assistance. Jackson R W, Further studies in the evaluation of Carbohydrate utilization tests as aids in the differentiation of References species of Streptomyces, Appl Microbiol, 3 (1955) 1. 1 Ballow C H, Biedenbach D J, Rossi F & Jones R N, 17 Groth I, Schutze B, Boettcher T, Pullen C B, Rodriguez C, Multicenter assessment of the linezolid spectrum and activity Leistner E & Goodfellow M, Kitasatospora putterlickiae sp. using the disk diffusion and E test methods: Report of the Nov. isolated from rhizosphere soil, transfer of Streptomyces Zyvox antimicrobial potency study in Latin America kifunensis to the genus Kitasatospora as Kitasatospora (LA-ZAPS), The Brazilian J Infect Dis, 6 (2002) 100. kifunensis comb. Nov., and emended description of 2 Jacqueline C, Navas D, Batard E, Miegeville A, Mabecque V Streptomyces aureofaciens Duggar 1948, Int J Syst Evol L, Kergueris M, Bugnon D, Potel G & Caillon J, In vitro Microbiol, 53 (2003) 2033. and in vivo synergistic activities of linezolid combined 18 Pridham T G & Gottlieb D, The utilization of carbon with subinhibitory concentrations of imipenem against compounds by some Actinomycetales as an aid for species methicillin- resistant Staphylococcus aureus, Antimicrob determination, J Bacteriol, 56 (1948) 107. Agents Chemother, 49 (2005) 45. 19 Saisivam S & Kishan V, Taxanomy, Fermentation, and 3 Prystowsky J, Siddiqui F, Chosay J, Shinabarger D L, biological activities of a new strain of Streptomyces luridus Millichap J, Peterson L R & Noskin G A, Resistance to from Indian soil, Ind J Microbiol, 46 (2006) 153. linezolid: Characterization of mutations in rRNA 20 Sajid I, Yao C B F F, Shabaan K A, Hasnain S & Laatsch H, and comparision of their occurrences in vancomycin- Antifungal and antibacterial activities of indigenous resistant Enterococci, Antimicrob Agents Chemother, 45 Streptomyces isolates from saline farmlands: Prescreening, (2001) 2154. ribotyping and metabolic diversity, World J Microbiol 4 Gupta N, Aparna, Saini S, Kumar B & Arora D R, In vitro Biotechnol, 25 (2009) 601. activity of linezolid in Staphylococcus aureus , Ind J Med 21 Bikshapathi D V R N, Krishna D R & Kishan V, Anti-HIV, Microbiol, 21 (2003) 289. anti-tubercular and mutagenic activities of borrelidin, Ind J 5 Potoski B A, Mangino J E & Goff D A, Clinical failures of Biotechnol, 9 (2010) 265. the linezolid and implications for the clinical microbiology 22 Suleimenov E M, Components of Peusedanum morisonii and laboratory, Emerging Infect Dis, 8 (2002) 1519. their antimicrobial and cytotoxicity activity, Chem Nat 6 Dailey C F, Pagano P J, Buchanan L V, Paquette J A, Comp, 45 (2009) 710. Haas J V & Gibson J K, Efficacy of linezolid plus rifampin 23 Antal N, Fiedler H P, Stackebrant E, Beil W, Ströch K in an experimental model of methicillin- susceptible & Zeeck A, Retymicin, Galtamycin B, Saquayamycin Z Staphylococcus aureus Endocarditis, Antimicrob Agents and Ribofuranosyllumichrome, Novel secondary metabolites Chemother, 47 (2003) 2655. from Micromonospora. sp. Tü 6368, J Antibiot, 58 7 Dibo I, Pillai S K, Gold H S, Baer M R, Wetzler M, Slack J (2005) 95. L, Hazamy P A, Ball D, Hsiao C B, McCarthy Jr P L & 24 Fiedler H P, Nega M, Pfefferle C, Groth I, Kempter C, Segal B H, Linezolid resistant Enterococcus faecalis isolated Stephan H & Metzger J W, Kanchamycins, new polyol from a cord blood transplant recipient, J Clin Microbiol, 42 antibiotics produced by Streptomyces olivaceus Tű (2004) 1843. 4018. Taxonomy, fermentation, isolation and biological 8 Tsiodras S, Gold H S, Sakoulas S, Eliopoulos G M, activities, J Antibiot, 48 (1996) 758. Wennersten C, Venkatraman L, Moellering Jr R C & Ferraro 25 Alley M C, Scudiero D A, Monks A, Hursley M L, M J, Linezolid resistance in clinical isolate of Czerwinski M J, Fine D L, Abbot B J, Mayo J G, Shoemaker Staphylococcus aureus, The Lancet, 358 (2001) 207. R H & Boyd M R, Feasibility of drug screening with panels 9 Andrews J M, Determination of minimum inhibitory of human tumor cell lines using a microculture tetrazolium concentrations, J Antimicrob Chemother, 48 (2001) 5. assay, Canc Res, 48 (1988) 589. 10 Livermore D M, Mushtaq S & Warner M, Susceptibility 26 Lipinski C A, Lombardo F, Beryl W D & Feeney P J, testing with linezolid by different methods, in relation to Experimental and computational approaches to estimate 728 INDIAN J EXP BIOL, OCTOBER 2012

solubility and permeability in drug discovery and 28 Shimizu K, & Tamura G, Reductiomycin, A new antibiotic. development settings, Adv Drug Delivery Rev, 23 (1997) 3. II. Structure elucidation by spectroscopic studies, J Antibiot, 27 Zhao XQ, Li W J, Jiao W C, Li Y, Yuan W J, Zhang Y Q, 34 (1981) 654. Klenk H P, Suh J W & Bai F W, Streptomyces xinghaiensis. 29 Konda Y, Onda M, Hinotozawa K & Omura S, sp. Nov. isolated from marine sediment, Int J Syst Evol Structure of antitumor alkaloid AM-6201, J Antibiot, 34 Microbiol, 59 (2009) 2870. (1981) 1222.