An antibacterial compound pyrimidomycin produced by Streptomyces sp. PSAA01 isolated from soil of Eastern Himalayan foothill

Selective isolation of soil Actinobacteria was undertaken to isolate a new class of antibiotics and bioactive molecules. A Streptomyces sp. PSAA01 (= MTCC 13,157), isolated from soil of Eastern Himalaya foothill was cultivated on a large scale for the production of the antimicrobial SM02. It has been found that the maximum amount of SM02 produced while PSAA01 was grown in ISP-2 medium (pH 7.0) for 7 days at 30 °C in shaking (180 rpm) condition. A significant zone of inhibition against Staphylococcus aureus MTCC 96 has been found with the crude cell-free culture media (50 µL) of 7 days grown PSAA01. After the purification and chemical structural characterization, we found that SM02 is a new antimicrobial having 746 dalton molecular weight. The compound SM02 contains pyrimidine moiety in it and is produced by a species of Streptomyces and thus we have named this antibiotic pyrimidomycin. The antimicrobial spectrum of pyrimidomycin has been found to be restricted in Gram-positive organisms with a MIC of 12 µg/mL. SM02 was found active against Mycobacterium sp. and also multi-drug resistant Gram-positive bacteria with similar potency and found to disrupt the bacterial cell wall. Pyrimidomycin also showed significant impairment in the biofilm formation by S. aureus. Furthermore, pyrimidomycin showed synergy with the most used antibiotic like ampicillin, vancomycin and chloramphenicol. Pyrimidomycin did not have cytotoxicity towards human cell lines indicating its limited activity within bacteria.

www.nature.com/scientificreports/ resistant against most of the antibiotics and making them multi drug-resistant pathogens (MDR pathogens) 9,10 . There are several strategies to overcome the antibiosis by antimicrobial agents such as enzymatic inactivation, target modification, influx impairment and excessive efflux are being followed by most of the MDR pathogens 10,11 . The most common strategy of the MDR pathogens to be resistant against the antimicrobial agent is biofilm formation 12 . The problem of the exponential rise of antimicrobial resistance can be mitigated with discovery of new antimicrobial agents produced by other microbes, as the majority of clinically relevant antibacterial agents (75%) are natural products of the microbial origin or their analogs 10,11,13 . There are many types of antibiotics which are having different targets for the inhibitory effects on the microorganisms. Formation of DNA, cell wall, proteins are the major targets for most of the antibiotics to inhibit the organisms. Beta-lactam drugs like, carbenicillin, penicillin G, cefuroxime, aztreonam, cefoperazone, oxacillin, ampicillin and a glycopeptide drug, vancomycin target the cell wall synthesis of the organisms and inhibit them 14,15 . On the other hand, oleandomycin, clindamycin, lincomycin, tobramycin, chloramphenicol, gentamycin, streptomycin, etc. block cellular protein synthesis to inhibit the microorganisms [16][17][18][19] . Besides these, another category of antibiotics which includes levofloxacin, novobiocin, rifampicin, ofloxacin, nitrofurantoin, etc. target nucleic acid synthesis 20 . However, some antibiotics may affect even the eukaryotic systems by interfering with several cellular functions 21 and thus it is very crucial to check the toxic effect on human cells.
In this study, an antimicrobial producing Streptomyces sp. PSAA01 has been isolated along with 25 other actinobacteria from the soil sample collected from Manas National Park, Assam, India. The antimicrobial production has been optimized taking various media, culture time and media pH. The chemical structural elucidation of the antibiotic molecule has also been revealed using CHNS/O analysis, mass spectrometry, FTIR, 1 H NMR, 13 C NMR, UV-Visible and fluorescence spectroscopy. The antimicrobial and antibiofilm properties of the molecule have been investigated using multiple test pathogens. The cytotoxicity of the molecule has further been checked against the human cell line and probable mode of action has been deciphered.

Methods
Selective isolation of actinomycetes. The soil samples were collected from Manas National Park, Chirang and Baksa (26° 65′ N-91 E; altitude: 200 ft; annual rainfall: 333 cm and a temperature range from 15 to 37 °C). In order to enrich and selectively isolate the actinobacteria, the soil samples were subjected to pretreatment with CaCO 3 and incubated for 7 days at room temperature followed by incubation for 2 h at 65 °C in a hot air oven 22,23 . 1 g of the pre-treated soil was dissolved in 1 mL of 0.9% NaCl and diluted sequentially up to 10 −6 . Each 0.1 mL of the diluted sample was spread on a starch casein medium. The medium was supplemented with cycloheximide (50 μg/mL) and nystatin (50 μg/mL) to inhibit undesired fungal growth. After incubation for 3-4 days at 30 °C, the inoculated plates showed different actinobacterial colonies (25). Each of the colonies having different and unique features has been selected and streaked on fresh ISP-2 (International Streptomyces Project) plate 22,25 . Identification of the PSAA01. An isolate, PSAA01 with differential mycelia (aerial and substrate mycelium) and diffusible pigments, was incubated for 14 days on ISP-2 medium 25 . Various colony morphology like colony size, shape, texture, margin, optical properties was observed on 14 days of incubation on ISP-2 medium. The cellular morphology has been observed under Scanning Electron Microscope following the standard protocol 26 . The 16S rDNA was amplified with the help of 8F and 1492R universal primers followed by sequencing of the amplicon and further sequence analysis using BLASTn program (https:// blast. ncbi. nlm. nih. gov) and EZbiocloud server 27 . 16S rDNA sequences of the closest type strains were obtained from the EZbiocloud server 27 and the phylogenetic analyses were performed using Neighbour Joining (NJ) 28 and Maximum Likelihood (ML) 29 algorithms. To reveal the evolutionary status of PSAA01, the distance-based and character-based phylogenetic trees were constructed. The 16S rDNA sequence of the strain was deposited in NCBI and the accession number is MT829328.
Preliminary screening for antimicrobial property. Antimicrobial activity of the strain PSAA01 was examined in Mueller-Hinton (MH) agar media following the standard agar-diffusion method 30 . In this method, 5-7 days old colony of PSAA01 (10 mm in diameter) was picked and inoculated on the plate which is just spread with exponentially grown test organisms such as Staphylococcus aureus MTCC 96, Bacillus cereus MTCC 1272 (Gram-positive), Escherichia coli MTCC 1687, and Pseudomonas aeruginosa MTCC 424 (Gram-negative). For a homogenous distribution of antimicrobial compound, already produced in the inoculum, the plates were kept at 4 °C for 2 h followed by the incubation at 37 °C for further 24 h 26 .
Culture conditions for SM02 production. The optimal production of the antimicrobial compound produced by PSAA01 has been explored with various media conditions, incubation time and the media pH. Five media AIA 25 , ISP-2 31 , ISP-3 31 , tryptic soya broth (TSB) 32 , and starch casein (composition: soluble starch-10 g, K 2 HPO 4 -2 g, KNO 3 -2 g, casein-0.3 g, MgSO 4 .7H 2 O-0.05 g, CaCO 3 -0.02 g, FeSO 4 .7H 2 O-0.01 g, agar-15 g, water-1000 mL and pH-7.0 ± 0.1) were selected by virtue of the differences in their composition 24 while growth parameters like temperature (30 °C), pH (8), shaking speed (180 rpm), time (7 days), and inoculum (1%) were kept constant. The medium which showed the maximum antibiotic production (derived comparing the efficacy of its antibiosis) was selected for the all-downstream experiments. The timepoint and pH for the maximum antibiotic production in the selected medium were also investigated. The medium which allows the maximum production of antibiotics was checked by measuring the wet weight of the cell mass of the strain PSAA01 inoculated in five different media. and compared with the standard antibiotics like ampicillin (10 μg) and chloramphenicol (4 μg). To determine the antimicrobial efficacy of the compound, 0.1 mL culture of 0.1 OD (A 600 nm) cell of test organisms were seeded on MH agar medium and the 50 μL of the cell-free extract was applied in the well made in each plate along with the standard antibiotic in independent well 26 . The plates were incubated for 24 h for all the test organisms and 48 h for M. smegmatis mc 2 155. After the incubation the zone of inhibition was observed.
Production, extraction, and purification. The strain PSAA01 was inoculated in a freshly prepared sterile ISP-2 medium (volume: 3 L) and incubated at 30 °C for 7 days in shaking (180 rpm) condition. After the incubation, the medium was centrifuged for 15 min at 13,000 rpm to collect the supernatant. An equal volume of ethyl acetate with the supernatant was added to extract the active compounds. After extraction, the active organic phase was collected and dried by a rotary evaporator. The crude mixture was subjected to load on a preparative thin layer chromatography (TLC) plate (Silica gel 60 F 254 ) using PET:CHCl 3 (1:1,v/v), R f = 0.30. The collected TLC-spot with desired activity was then run into flush column chromatography consisting of silica gel bed (60-120 mesh) with the same eluent ratio of the solvents to purify the compound. The pure product was obtained as light brown solid after the evaporation of the solvent from the eluted fractions through the rotary evaporator. We obtained 40 mg pure product from the 3L extract solutions. The purified product was then used further for its chemical and functional characterization. fetal bovine serum (Invitrogen), penicillin (100 μg/mL), and streptomycin (100 μg/mL). Cells were initially propagated in a 75 cm 2 polystyrene, filter-capped tissue culture flask in an atmosphere of 5% CO 2 and 95% air at 37 °C in a CO 2 incubator. When the cells reached the logarithmic phase, the cell density was adjusted to 1.0 × 10 5 per/well in culture media. The cells were then used to inoculate in a glass-bottom dish, with 1.0 mL (1.0 × 10 4 cells) of cell suspension in each dish. After cell adhesion, the culture medium was removed. The cell layer was rinsed twice with phosphate-buffered saline (PBS) (pH 7.0). Cells were centrifuged at 1200 rpm for 10 min after treatment with trypsin. The supernatant has been discarded and the fresh medium was added to the pellet to adjust 10 4 cells in 100 μL medium followed by incubation overnight. All tests have been repeated atleast three times. Overnight grown cells were treated with different concentrations of SM02 compound and further incubated overnight. Upon incubation 20 μL MTT reagents (5 mg/mL) were added in each set and incubated at shaking condition for 4 h. The supernatants were discarded and 150 μL DMSO was added to dissolve formazan,  Analytical experiments to characterize SM02. The solvents used were distilled and dehydrated according to the standard procedures 38 . The high-resolution mass spectrometry (HRMS) was performed using a micromass Q-TOF MicroTM instrument by using methanol as a solvent. 1 H-, 13 C-NMR spectra were collected at 400 and 100 MHz, respectively, on a Bruker DRX spectrometer. For NMR spectra, CDCl 3 was used as solvent using TMS as an internal standard. Chemical shifts are expressed in δ ppm units and 1 H-1 H and 1 H-13 C coupling constants in Hz. The following abbreviations are used to describe spin multiplicities in 1 H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet. KBr pellets have been used to record the FTIR spectra using a spectrophotometer. Fluorescence spectra of the compound SM02 were recorded on a Perkin Elmer Model LS 55 spectrophotometer whereas the UV-Vis spectra were recorded on a SHIMADZU UV-3101PC spectrophotometer. Elemental analysis of the SM02 compound was carried out on CHNS/O analyzer. Specific rotation of the compound SM02 were recorded on Bellinham Stanley Ltd. polarimeter.

Estimation of MIC and MBC.
Ethical approval. The research is not associated with any prior ethical approval.

Consent of publication.
The data used for the manuscript is original and does not require any consent from third party for publication.

Results
Isolation and identification of PSAA01. A Streptomyces sp. PSAA01 (= MTCC 13157) was isolated from soil collected from Manas National Park, Assam, India. Streptomyces is the most prevalent actinomycetes present in the soil and have a huge contribution towards the development of pharmaceuticals. The strain was grown on the ISP-2 medium. The colony of the isolated strain has been found to be irregular, blackish and rough (Fig. 1a).
Scanning electron micrograph of PSAA01 shows that the filamentous mycelia and the spore surfaces are rough (Fig. 1b). The 16S rDNA sequence (MT829328) of the strain PSAA01 (= MTCC 13157) is having a 99.72% similarity with Streptomyces melanosporofaciens DSM 40138, as analysed with EZBioCloud. Phylogenetic analysis www.nature.com/scientificreports/ with Neighbour Joining (NJ) algorithm shows that the isolate originated from the same ancestor as S. melanosporofaciens DSM 40138, but based on the branch length and other molecular and biochemical observation (data not shown), it can be assumed that the isolate might be quite different from its closest neighbour (Fig. S1). It has been found to secrete a bioactive metabolite SM02, which is having antimicrobial property.
Culture conditions for SM02 production. ISP-2 medium was found to be the best-tested medium as the cell-free extract that contains the desired antimicrobials showed the highest inhibitory effect against S. aureus MTCC 96 (Fig. S2). To estimate the culture incubation time-point that has highest antimicrobial production, we prepared the cell-free extract and checked the antimicrobial potency. The maximum antibiosis on the test pathogen was assumed as the point where there is maximum SM02 production has occurred. The incubation has been continued for 15 days to get the interval time points. The production (as determined by the test-pathogen killing assay) was found to be maximum after 7 days of incubation (Fig. S3a). Similarly, it has been found that the optimum pH was 7.0 for SM02 production (Table S1; Fig. S3b). This indicates that the maximum efficacy of the desired compound SM02 can be obtained while the strain PSAA01 were grown in ISP-2 medium (pH 7.0) in shaken condition (180 rpm) for 7 days at 30 °C.

Pyrimidomycin inhibits growth and biofilm formation of Gram-positive pathogen. The MIC
and MBC values of the pyrimidomycin against various sensitive test organisms were determined (Table S3, Fig. S4). The MIC against the Gram-positive organisms like S. aureus MTCC 96, S. pyogenes MTCC 1928, B. cereus MTCC 1272, Methicillin-resistant S. aureus (MRSA), M. smegmatis mc 2 155 was found to be 12 μg/mL or 16.08 μM. However, we found that the MBC values are relatively higher and are more than 50 µg/mL. The cellular alteration was observed after the pyrimidomycin treatment to the test organisms. The cellular morphology of the untreated B. cereus MTCC 1272 and S. aureus MTCC 96 was intact (Fig. 4a,c) whereas the treated cells exhibited altered morphology (Fig. 4b,d), particularly the cellular membrane anticipated to be damaged severely. This data help to reveal the mode of action of the isolated pyrimidomycin. Further, the antibiofilm property of the pyrimidomycin was determined in sub-MIC value (1.56, 3 and 6 µg/mL) by crystal violet assay. In the case of positive control (untreated cells) the optical density was shown to be highest. The optical density was found to be decreased with the increasing concentration of pyrimidomycin indicating that the compound inhibits the biofilm formation by the test pathogen and thus could inhibit the colonization of the pathogen if applied (Fig. 5).
The inhibitory effect of pyrimidomycin with different concentrations (12,24,48,96 and 192 µg/ml) was also checked on human cell line (HuH-7) through MTT assay. The compound did not show any significant toxic effect on the cell line and can be predicted as non-toxic to humans (Fig. 6). However, the standard experiments on the mammalian models and the human subjects will confirm on its suitability for future use.
Fractional inhibitory concentration index of pyrimidomycin. As we determine the MIC of pyrimidomycin is 12 µg/mL but when combined with other individual antibiotics, it shows its MIC much lower than its actual individual MIC value. The individual MIC value of ampicillin, chloramphenicol, vancomycin is 0.4 µg/ mL, 4 µg/mL, 0.5 µg/mL, respectively. When 1/4 MIC of pyrimidomycin was combine with 1/4 MIC of ampicillin, chloramphenicol and vancomycin it showed complete growth inhibition of the test organism. FICI value of both shows equal to 0.5 which indicates pyrimidomycin has a synergistic effect (Table. 3).

Discussions
The studied Streptomyces strain was isolated from the soil sample collected from Manas National Park, Assam, India. The isolated strain PSAA01 has been identified as Streptomyces sp. which is capable of producing an antimicrobial compound named pyrimidomycin. The 16S rDNA sequence of the strain PSAA01 has been showing 99.72% similarity with Streptomyces melanosporofaciens DSM 40138. Small scale fermentation of the strain has www.nature.com/scientificreports/ been performed in different media with different temperatures, pH and incubation time to optimize the production of the pyrimidomycin. The used media contains different major nutritional sources such as carbon, nitrogen. ISP-2 contains yeast extract, malt extract, dextrose; oat meal is present in ISP-3; casein is contained by starch casein medium; TSB contains peptic digest of soyabean meal, dextrose; sodium propionate, sodium caseinate, L-asparagine and glycerol is present in AIA medium. We have found that ISP-2 medium is the optimum medium for the production of the antimicrobial by the strain PSAA01 at pH 7.0 upon 7 days of incubation. The structural elucidation has also been done which reveals that the molecule contains two substructures, substructure A and B. The molecular weight of this compound is 746 dalton. The molecule contains pyrimidine moiety and is yielded by Streptomyces sp., thus we named it pyrimidomycin.  www.nature.com/scientificreports/ Nowadays, bacterial multidrug resistance (MDR) is a challenging issue to the clinician as the control of the MDR pathogens becomes unsuccessful with most of the available antibiotics. Most of such pathogens are the causal agents for many life-threatening diseases in humans 9,39 . The isolated compound pyrimidomycin is specifically effective against Gram-positive organisms including the methicillin resistant S. aureus (MRSA) which shows their resistance property against typical beta-lactam drugs like methicillin, amoxicillin, penicillin, nafcillin, oxacillin and cephalosporin 40 (Table S3). The antibiofilm activity of the pyrimidomycin has been recorded at the sub-MIC values. The SEM assay has been showing the cell wall and cell membrane rupture after the treatment of B. subtilis MTCC 441 or S. aureus MTCC 96 with the MIC value (12 µg/mL). The pyrimidomycin does not affect tested human HuH-7 cell lines up to the concentration of 48 µg/ml concentration. From these experiments it can be concluded that the identified strain is a novel antibiotic producer. The pyrimidomycin has been successfully characterized and the presumable mode of action is also indicated in this study. The inhibitory effect of the compound on drug-resistance strain is a promising outcome. Furthermore, as pyrimidomycin shows a synergistic relationship with very common existing antibiotics like ampicillin, vancomycin and chloramphenicol, it might be a choice of antimicrobials in the future for its combinatorial use.

Data availability
The bacterial culture has been submitted to MTCC, India with the accession number MTCC 13157. The 16S rDNA sequence of the strain was deposited in NCBI and the accession number is MT829328. All the data associated with the research are available as supplementary files.