Extraction and Characterization of Antibiotic Compounds produced by Streptomyces spp. VITGV01 against Selected Human Pathogens


 Streptomyces are well known to produce a large number of diverse antibiotic compounds. New environments are being explored and some new Streptomyces strains have been discovered; the potential of the plant endophytic environments has been highly explored during the last decade. In the present study, an endophytic actinomycete, VITGV01, was isolated from a farm tomato plant collected from an agricultural field. These actinomycetes are cultured in ISP2 medium supplemented with nystatin (15µg/ml) and nalidixic acid (50µg/ml). Based on the 16S rRNA analysis and physiological tests, this isolate was found to be a member of the known genus Streptomyces spp. and suggesting that it might be a new spp. The isolated strain showed morphological and chemical characteristics of the genus Streptomyces. The strain VITGV01 produced different antibiotics on different media such as yeast extract, malt extract, glucose medium, which were active against some Gram-positive and negative bacteria [Bacillus subtilis (MTCC2756), Staphylococcus aureus (MTCC737), Escherichia coli – (MTCC1687), and Klebsiella pneumoniae (MTCC109)]. The primary and secondary screening was performed against these human pathogens. Maximum zone of inhibition was observed against K. pneumoniae (22 mm) and minimal zone of inhibition against B. subtilis (8 mm). Maximum biomass production was recorded in media containing 1% sucrose and 1% yeast extract cultured at pH 7.0 for 7 days at 30◦C. FTIR analysis revealed amine, alkane (C = C) of the aromatic ring, secondary alcohol, and alkyl halide groups. The GC–MS data showed twenty compounds, of which fifteen are antimicrobial compounds.


Introduction
Antibiotics are chemical compounds used to prevent and treat bacterial infections. Antibiotic resistance occurs when bacteria change in response to the use of these drugs. Today, drug-resistant bacteria are a serious threat to people's health. Currently, at least 7,00,000 people die each year due to drugresistant diseases, including 2,30,000 people who die from multidrug-resistant tuberculosis (WHO 2019). The discovery of new antibiotics that reduce bacterial drug resistance is essential for modern medicine. The World Health Organization (WHO) claims that Antimicrobial Resistance (AMR) presents a signi cant challenge to public health and the ecosystem. (Allcock et al. 2017) .
Among the antibiotic-producing microorganisms, the class actinomycetes represent a broad range of valuable and prominent sources of pharmaceutically active metabolites (Subramani and Sipkema 2019). Over 40 genera of lamentous Actinobacteria have been detected as endophytes in plants. Among the studied endophytes, Streptomyces species, which are isolated from a diverse plant species, are one greater group of interest because of their ability to produce antibiotics as well as other bioactive secondary metabolites (Yan et al. 2010). About 23,000 bioactive secondary metabolites were discovered from endophytic microorganisms, approximately 10,000 different substances were produced by these endophytic actinomycetes and 7,600 metabolites were found in the Streptomyces genus (Hong-Thao et al. 2016). Actinomycetes produce approximately 75% of the commercially used antibiotics (Sanghvi et al. 2014). The actinomycetes are one of the most attractive sources of new bioactive metabolites. However, the rate of discovery of new compounds has decreased since the available species have been studied extensively (Badji et al. 2007). Recently, rare actinomycetes are an important source of novel and useful antibiotics (Takahashi and Nakashima 2018). Several studies have been oriented towards the isolation of new uncommon actinomycetes from unexplored environments. Although numerous actinomycetes have been isolated and used as a producer of key drugs and biomedical agents, it is increasingly di cult to nd novel compounds to identify a new organism.
To achieve this objective, we have isolate actinomycetes from tomato plants and the following parameters were studied such as morphological, antimicrobial, biochemical, phylogenetic characters, and optimization of growth for Streptomyces spp. VITGV01. Antimicrobial properties (human pathogens) of the secondary metabolites production and characterized on FTIR and GCMS.

Tomato plant sample collection:
Healthy tomato plants (Solanum lycopersicum) were collected from ve different locations (Tirunelveli − 8.7815° N, 77.3942° E;Tuticorin 9.1727° N, 77.8715° E;Madurai − 9.9420° N, 77.9724° E;Dindigul − 10.4489° N, 77.9360° E and Theni − 10.0015° N, 77.6164° E) from the southern part of Tamil Nadu in India during August and September 2017. Plants were collected from the eld at the fruiting stage. Three tomato plants were randomly collected from the corners and center of the eld. Thus, a total of fteen plants were collected in sterile polythene bags and passed directly to the laboratory for microbiological processing 2.2 Surface sterilization 2.3 Screening for effective strain The preliminary antimicrobial screening was done by spot inoculation method (Shomura et al. 1979) using four test microorganisms. Gram-positive (B. subtilis (MTCC2756), S. aureus (MTCC737), and two Gram-negative strains (E. coli -(MTCC1687) and K. pneumoniae (MTCC109). The diameter of inhibition zones was determined after 24 hrs. The experiment was performed in triplicate. The actinobacteria that showed promising results in the preliminary antimicrobial screening were subjected to secondary screening by the agar well diffusion method (Bauer et al. 1966). The above-speci ed microorganisms were used. Actinomycete antimicrobial extracts were recovered from the culture ltrate by solvent extraction using ethyl acetate (1:1, v/v). The diameters of the zone of inhibition were determined after 24 hours. Samples were assayed in triplicate.
Morphology of the strain was observed in a phase-contrast microscope (MT5210/MEIJI) and scanning electron microscopy was performed using Carl Zeiss -Evo 18).
Ampli ed PCR product was sequenced and the nucleotide sequence was matched using the BLAST program. The phylogenetic tree was constructed using the neighbor-joining method (Fatima et al. 2019). The sequence of the isolate was submitted to GenBank (Accession ID: MN641011).

Metabolite production
Pure and active cultures of microbial strains selected from the secondary screening experiments were grown in ISP2 broth and incubated at 30°C for 7 days and cellular growth was con rmed by visible pellets, clumps, and aggregates in the ask. The culture broth was centrifuged and the supernatant was used to evaluate the antimicrobial activity against the above-mentioned test microorganisms. Antibiotic activities of the strains were compared with that of known commercially available erythromycin standard.

Extraction of the bioactive metabolites
The culture was grown in 1000 ml Erlenmeyer asks containing 200 ml of ISP2 broth for 7 days as speci ed in metabolite production. The culture was centrifuged at 10,000 rpm for 20 min to separate the biomass. The secondary metabolite was recovered from the fermented ISP2 broth using a twophase solvent extraction system with organic solvent (ethyl acetate). (Shetty et al. 2014). The mixtures of separating ask crude culture ltrate: solvent (1:1 ratio) were vigorously shaken for 10 min and in kept shaker from 18 to 24 hrs at 200 rpm. After 24 hrs this was left to settle for 30 minutes for separating ask kept stationary from 15 to 30 min. until the separation of aqueous and organic phases. Organic phases were collected and concentrated in a rotary evaporator (model RE100-Pro; SCILOGEX, LLC, CT, USA) at 54°C and 80 rpm. The dried crude extracts were then weighed, dissolved in 200µL of methanol, and nally stored at -20°C.

Screening of antibacterial activity
Concentrated crude ethyl acetate extract obtained from Streptomyces spp. VITGV01 was screened for their inhibitory activity against 4 known human pathogenic bacterial strains by agar Well Diffusion Assay (WDA) (Magaldi et al. 2004). The wells were prepared in the plate (already spread with the test organism) by using a sterile cork borer (6 mm in diameter) at 0.5 McFarland turbidity (1x10 8 cells/mL). A volume of 25, 50, 75, and 100 µL of 1 mg/mL of crude extracts was carefully dispensed into each well and allowed to diffuse for 2 hrs and incubated at 37°C for 24 hrs. To validate the experiment, negative control (ethyl acetate) and positive control (1 mg/ml erythromycin) were also maintained, and each experiment was performed in triplicate. Diameters of inhibition zones were measured after 24 hrs. Tested pathogens such as B. subtilis (MTCC2756), S. aureus (MTCC737), E. coli -(MTCC1687), and K. pneumoniae (MTCC109).

FTIR and GCMS analysis of secondary metabolites
FTIR spectra were recorded on SHIMADZU FT-IR spectrophotometer, Model: IR A nity in the range of 4000-400 cm − 1 (Sanghvi et al. 2014). GC-MS (GC trace ultra-version) was used to nd out the chemical compounds. The peaks were identi ed with the library.

Result
The present study focuses on the isolation of potent antibiotic-producing endophytic actinomycetes. From two hundred and forty actinomycetes strains were isolated in ve districts of Tamilnadu ( Fig. 1), India. Among the isolated pure strains, VITGV01 was found to produce a wide spectrum of antimicrobial activities against selected two Gram-positive and two Gram-negative bacteria. The isolated VITGV01 showed a clear zone of inhibition ( Fig. 2) due to antimicrobial activity.

Biochemical characterization
The selected strains were subjected to various biochemical tests and the results are tabulated in Table 1, which supports that it belongs to actinomycetes.

Physiological Characters
Optimization parameters for growth of this Strain VITGV01 require pH 7, temperature 30 0 C and 1% yeast extract, 1% sucrose incubation duration 7 days were best suited (Fig. 3).

Morphological observation of VITGV63
The cultural characteristics and colony morphology were summarized in Table 2 and Fig. 4. These morphological observations revealed that VITGV01 was similar to Streptomyces. It showed well developed aerial and substrate mycelium and mycelial cluster showed continuous fragmented long and short chain-like structure (Fig. 5B). The scanning electron microscope image of ( Fig. 5C) sample exhibited continuous lamentous spiral chains in a tubular shape. The continuous aerial hyphae differentiated into a small and smooth-surfaced spore. The center of the spore showed a cavity on the upper side of each spore (Fig. 5D).

Genetic analysis of VITGV63
The 16S rRNA sequence of the strain VITGV01 (1040 nucleotide), to identify the 16S rRNA gene sequence with high similarity strain VITGV01 shared with 16S rRNA high degree of similarity with other types of genus Streptomyces. The phylogenetic tree of the strain VITGV01 forms a single separate branch from other Streptomyces neighbors (Fig. 6). It shares 16S rRNA sequence to nearest neighbor is Streptomyces atacamensis (99.42 %), Phylogenetic tree builder uses sequences aligned with method System Software aligner.

Extraction of bioactive compounds
The crude extract exhibited antimicrobial properties proved by the Well diffusion method (Table 3) for secondary screening. Effective zone of inhibition was also recorded against B. subtilis S. aureus, K. pneumoniae, and E. coli. (Fig. 7). The zone of inhibition was observed between the range from 8 to 22 mm. No zone of inhibition was observed in negative control with ethyl acetate. A maximum inhibition zone of 22 mm was observed against K. pneumoniae, which was also to positive control as erythromycin (22 mm). Negative control (Ethyl Acetate)

Discussion
Actinomycetes play a major role in combating various diseases. Earlier exploration has shown actinomycetes having excellent potential to control the growth of pathogens (Ayoubi et al. 2018). In our study, a total of 240 endophytic actinomycetes were isolated and screened for their antimicrobial activities, out of which 77 actinomycetes showed the antimicrobial property. A similar report of recognizing 619 endophytic actinomycetes was reported by Tan et al., (2006) out of this 8 alone exhibited antagonistic property.
The size of the inhibition zone is the rst criterion for screening antimicrobial activity on pathogens, in the present study, there was a good number of microbes (77/240) which shows a good zone of inhibition (Fig. 3). A total of 44 (18%) isolates inhibited the growth of both Gram-positive and Gramnegative pathogen, while 18 isolates (8%) inhibited Gram-negative and 15 isolates (6%) inhibited Gram-positive bacteria. Thus seventy-seven (32%) isolates were selected to explore their potentials. Strain VITGV01 showed the highest antimicrobial activity against all four pathogens.
Morphological characteristics such as aerobic, powdery, at appearance, aerial and substrate mycelia with different colors in different media and colony size suggested that the isolates are a liated to genus Streptomyces. The above comparison with the description given in Bergey's manual of systematic bacteriology indicates a major resemblance of this strain with Streptomyces species. This study coincides with reports of Al-Ansari et al., (2019) SEM results showed hyphae's were compartmentalized and spores chain-like and continuation (Fig. 5). The mature spore's surface was smooth and unique with a small concave depression; this morphology was the same as that of actinobacteria AFD7 strains (Fatima et al. 2019) The nature and concentration of sugars such as sucrose, maltose, fructose, and glucose are crucial for the growth, nutrient consumption, and production of secondary metabolites. And are important for signal transduction, gene regulation, and development. The utilization rate of carbon and nitrogen sources by VITGV01 seems to be unique. Out of ve different carbon sources, the highest growth was obtained with 1 % sucrose. On the other hand, glucose recorded the lowest growth rate. Similarly, El-Hadi et al., (2019) reported lactose for maximum and mannitol for minimal growth in Streptomyces greseoplanus. This study support that according to carbon sources, growth in Streptomyces spp. is strongly in uenced.
Nitrogen source is required for the synthesis of cells and energy sources. Also, biomass yield and cell morphology are strongly in uenced by the nitrogen source. Similar results have been reported by (Al-Ansari et al. 2020) in their studies associated with Streptomyces greseoplanus. In another study, by Wang et al., (2010) nitrogen sources from yeast extract supported the greatest mycelium biosynthesis in Saccharothrix yanglingensis sp. nov.
Microorganisms are capable of growing over a wide pH range. Moreover, pH is the third important factor that in uences microbial growth. In this way, responses to environmental signals are transmitted through global transcription factors that mediate environmental signals like carbon (CreA) (Janus et al. 2008), nitrogen (AreA) (Tudzynski et al. 1999), and pH (PacC) (Tilburn et al. 1995), translating environmental cues into Secondary Metabolites and concomitant physiological responses. The enhancement or inhibition of the secondary metabolite is in uenced by a change in pH and can also affect the growth of microorganisms (Kamjam et al. 2019). In this experiment, we report the optimum pH for maximum growth is pH 7, and the lowest growth was observed in pH 2. This is very similar to the study by Streptomyces polaris sp by Kamjam et al., (2019).
An important criterion for the potency assessment is the time course for growth plateau formation and continuous growth was observed till the 14th day. A similar report was documented by Yagüe et al., (2012) in Streptomyces coelicolor for growth optimization. Another factor is the incubation temperature, which affects the microbial growth, metabolite production, rate of the chemical reaction, which in turn affects the rate of metabolite production. In the present study, the maximum activity was recorded at 30°C. By increasing the incubation temperature, a gradual decrease in growth was observed. This is a negative impact on this organism; this could be due to the thermolabile nature of extracellular enzymes.
Studies have suggested that to identify actinomycetes details of biochemical, morphological, and phylogenetical characterization are necessary. The biochemical characterization proved that it belongs to actinomycetes ( Table 2). The 16s rRNA study and phylogenetic tree result again proved that this strain clustered within the genus Streptomyces and exhibited close phylogenetic a nity and high sequence similarity to Streptomyces atacamensis (99.42 %), Streptomyces mangrovi (99.33 %), Streptomyces chitinivorans (99.23 %), and Streptomyces atacamensis (99.04%), This is the rst study to identify this Streptomyces spp. (Gen Bank Accession -MN641011). A similar type of conformation for Streptomyces was reported by Duddu & Guntuku, 2016).
Isolation of antibacterial compounds from the endophytic environment is in advance interest in recent times to isolate novel bioactive actinomycetes. Musa et al.,(2020) isolated fty-four endophytic actinomycetes, which showed antagonistic activities. During secondary screening with crude extracts, a wide range of inhibition zone against tested bacterial strains were observed. Similar ndings were reported earlier by Gebreyohannes et al., (2013) in actinomyces spp. In general, the antibacterial activity of crude extracts has uctuated widely. The results demonstrate that a Gram-positive and Gramnegative bacterium both were highly susceptible to the tested crude extracts. This result was in agreement with the report of Sanghvi et al., (2014) who reported it with Streptomyces werraensis.
VITGV01 crude extract showed excellent activity on Gram-positive and Gram-negative bacteria, suggest that it could inhibit DNA synthesis, RNA synthesis, cell wall synthesis, or protein synthesis (Kohanski et al. 2010). There was a marked difference between the crude extracts and pure antibacterial drug (Erythromycin). Some of the crude extracts showed a higher or equivalent zone of inhibition compared to the standard drug erythromycin, which shows excellent activity against Gram-positive organisms and modest activity against Gram-negative bacteria. Rex et al. (2001) reported that a signi cant difference was normally presented in crude (unpuri ed) extracts compared with a pure drug that was already in clinical use..
The MIC varied among the tested isolates against all the tested pathogen. These results were similar to the report of Núñez-Montero et al., (2019).
Therefore, crude extracts could be a potent source for antibiotic production, which leads to the development of novel drugs for the treatment of infectious diseases. The report of antibacterial activities of actinomycetes isolated from Tomato endophytes of agro eld Madurai in Tamilnadu was rst-hand information as per our knowledge.

Conclusion
Plants are a rich source of endophytes. The identi cation of novel Streptomyces strains may lead to new drug discovery. In this study, 240 endophytic actinomycetes were primarily screened and among these 77 isolates showed antimicrobial activity against selected human pathogens. Based on the biochemical characterization, and 16s rRNA studies, it can be a new strain. Our study encourages the exploration of diverse ecosystems for the isolation of new species for novel bioactive compounds. As there is an alarming increase in multi-drug resistance in pathogens, exploration of new bioactive compounds helps to combat the multi-drug resistance.

Declarations
Funding: Vellore Institute of Technology provided 'VIT SEED GRANT' for carrying out this research work, is acknowledged.
Authors' contributions: All the authors contributed equally Informed consent statement: Not applicable Ethical statement: Not applicable Data availability statement: Data supporting the results are given in tables. Figure 1 Map showing the different districts of samples were a collection. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.