INTRODUCTION:

The actinomycetes being a co habitant in soil environment, able to defend itself by producing self-protecting chemical compounds, which are known as chemical weapons, the output of competetion for common niche. This process is dynamic and continuous. Therefore, mankind was interested for the discovery of antibiotics and allied compounds secreted from actinomycetes, making it one point programme.

Actinomycetes are regarded as ‘Antibiotic factory’ of nature, with immense potentiality of inhibiting the growth of many a number of bacteria, notorious for multi antibiotic resistance, persistence and more over tolerance.

However, with emergence of new strains of bacteria in clinical scenario, of course due to human error, the currently used antibiotics are getting banned by World Health Organisation as well as Food and Drug administration. The antibiotics fail to enter into the system of bacteria or unable to satisfy the ADME (Absorption, Distribution, Metabolism and Excretion) process. Among the bacteria of hot discussion, the ESBLs (Extended Spectrum of Beta lactam producing Bacilli)^1-6, Methicillin resistant Staphylococcus aureus (MRSA)⁷, VRE (Vancomycin Resistant Entereococcus, Glycopeptide)⁸, Enterococci (involved the alteration of the peptidoglycan synthesis pathway, CRE (Carbapenem resistant Enterobacteriaceae)⁹, CPE (Carbapenemase-producing Enterobacteriaceae)¹⁰ are considered to be life threatening agents^11,12.

Worrying with problem of said antibiotic resistance, researches stand in queue to discover promising new chemical compounds. Hence, it is important to carry out experimental as well as assays with sufficient productivity. The technical advancements¹³ have been enough opportunities for the detection of antibiotics in both cellular (CE) or cell free extract (CFE) of actinomycetes, using robust analytical methods such as Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)¹⁵, where-as, UV- Visible spectroscopy¹⁴ in scanning mode is very much helpful to screen for presence of putative antibiotics considering the λmax peaks as markers for detection of known as well as unknown compound. The specific λmax of standard drugs/antibiotics¹⁶are referred and accordingly the compound of interest is identified. Contemporarily, UV- Visible spectrophotometry¹⁸ is an obligatory instrumentation for the detection of unknown compounds of specific interest.

With the advancement of mass spectrometry, the identified compound is further authenticated by the tandem MS along with structural elucidation and m/z (mass determination). Therefore, this communication is defined to carryout analytical studies on the cell free extract of three actinomycetal isolates co- incubated with counterpart bacteria using UV- Visible^17,18and LC-MS. Further with the use of OMICS language¹⁹ the data inferred from the analysis, the putative antibiotics molecules and/or its derivatives could be identified²⁰.

MATERIALS AND METHODS:

Preparation of Cell Free Extract (CFE):

The actinomycetes under study Microbacterium barkeri (LMA4), Corynebacterium argentoratense (LMA5), Gene bank No.OP023130 and Streptomyces shenzhenensis (LMA6), Gene bank No.OQ092768 were grown in Actinomycetes Isolation Agar (AIA)²¹, pocurred from Hi-Media (Mumbai) were subjected to preparation of Cell Free Extract (CFE) for the purpose of detection of putative antibiotics/ bioactive compounds³⁰, using a “Co-culture method”²²method. Purposefully a multidrug resistant (MDR) strain of Staphylococcus aureus (BMS4) and a Gram negative, Escherichia coli (BME4) were taken as co-culture partner in the broth media, maintained in the Laboratory of Medical Microbiology, School of Life Sciences, Sambalpur University. After requisite incubation period, the flask inoculated with both actinomycetes cellular mass (0.05gm/ml) and 10³Cfu/ml of bacterial cells respectively. The broths containing both the organisms were kept in a rotary shaker maintained at 37^oC and keeping the shaking rotation at 120rpm was incubated for a period of total 48hrs. For the analysis of LC-MS, the CCFE (Cell free extract of respective actinomycetes co cultured with BME4 and BMS4)²³.

Spectrophotometric analysis of Co-cultured CFE (CCFE):

The Cell free extracts²⁴of the actinomycetes isolates (LMA4, LMA5 and LMA6) of 0hr, 2hrs, 4hrs, 6hrs, 12 hrs, 24hrs and more so 48hrs incubated CCFEs were subjected to spectrophotometric analysis. The CCFEs were subjected to centrifugation at 10,000rpm for 10 minutes using refrigerated, microprocessor based (Brush Less) centrifuge. Then the supernatant was decanted into sterile vials. Further, the CCFE was diluted into eight times and was analyzed for its absorbance in the spectrophotometer using “Scanning Mode”. The selected wavelengths were in a range of 300nm (λ) -700nm (λ) for the detection of antibiotic molecules in the respective CCFE. The λ^max of putative compounds were noted and was correlated with published reports found in contemporary literature^25,26.

Liquid Chromatographymass spectrometry (LC-MS/MS) of co-cultured Cell free extracts (CCFE):

The Liquid chromatography (LC) conjugated with mass spectrometry (MS) of CFE of co-cultured broth was carried out at Sophisticated Analytical Instrument Facility (SAIF) at CSIR- Central Drug Research Institute (CDRI), Lucknow, Uttar Pradesh, India using the instrument “Alliance e2695/HPLC-TQD Mass spectrometer”²⁷. The column for the analysis was ACCUCORE “C18 250× 4.6 5um”. The chosen wavelength was 280nm (λ), while the flow rate was maintained 1.5ml/ mint. The Solvent System (A) was “Acetonitrile”, while the Solvent System (B) was “5mM AcNH4 buffer, PH 6.5”. For solubility water (H₂O) was taken as solvent.The name of the instrument was “ACQ-TQD/QBB1152”. The instrument was calibrated using the path file C-Mass linux/ intellistart/ results/ unit mass resolution. While, tandem mass spectrometry was carried out with MS1 static considering the mass range 20 Da to 1974 Da with resolution 8.9/15.1. The ion energy used was 0.3 and the name of the equation file was SCN-MS1. The scan speed was compensated with 100-2500 amu/ sec. resolution was 8.9/15.1, while the MS2 scanning mode acquition file name was Fast MS2. The polarity was ES+, Calibration dynamic was 1 and capillary in (kV) was 3.50, cone (V) was 30.00, 15.87, which RF values was 0.10. The source temperature was 120 to 119^oC. Desolvation temperature was 350^oC, cone gas flow rate (L/hr) was 30, and Desolvation gas flow was 0.17-to- 0.01 (ml/mint.). Ion energy was 1, MS mode entrance was 50.00, MS- mode collision energy was 2.00, and MS-mode exit was 50.00. While, the tandem MS/MS mode entrance was 2.0, MS/MS mode collision energy was 2.0, MS/MS mode exit was 2.0, Ion energy was 20.50, gain was 1.00, multiplier was -484.45, active reservoir was B. While the engineers setting in the instrument was MS1 Low mass position 519, MS1 High mass position 379, MS1 Low mass resolution 518, MS1 High mass resolution 299, MS1 resolution linearity 822, MS1 High mass DC balance-0 and MS1 DC polarity was Negative.While, MS2 low mass position was 521, MS2 High mass position 419, MS2 Low mass resolution 513, MS2 High mass resolution 179, MS2 resolution linearity 850, MS2 High mass DC balance -0, MS2 DC polarity Positive, HM RF lens correction +0 and HM RF lens correction -0.

The run method parameters are as follows:

a (A %) final flow setting being held at the end of run at volume of 130, which is (Normal stroke volume) was 0.0 H₂O, b (B %) was 5.0 ACN, c (C %) 0.0 MEOH, d (D %) was 95.0 0.1% FA. The Flow rate was (ml/ min) 1.000. The Flow ramp was 0.30 stroke time was in minutes 40. The column temperature was 35 ^oC; the column temperature limit was 5^oC, Minimum pressure (Bar) 0.0, Maximum pressure (Bar) 300.0.

The auto sampler Needle depth was 0.00mm. The Sample temperature was 20^oC, while the Sample temperature limit was 3^oC. The Purge loop volume was 0.00 and the Injection volume was 5 µL. The end of the experimental results was recorded with the following parameters categorized in to Function 1 and Function 2 given in the Table 1.

Table 1: The Parameters used Tandem MS-MS for ES+ and ES-

Parameters	Function 1	Function 2
Scans in function	1173	1173
Cycle time (Sec)	Automatic	Automatic
Scan duration (Sec)	1.000	1.000
Inter Scan Delay (Sec)	Automatic	Automatic
Start and End Time(min)	0.000 to 40.000	0.000 to 40.000
Ionization mode	ES+	ES-
Data type	Enhanced Mass	Enhanced Mass
Function type	Scan	Scan
Mass range	150 to 1500	150 to 1500

NB: ES+, Function 1 and ES-, Function 2

RESULTS AND DISCUSSION:

The flasks (Figure 1 and Figure 2 respectively) containing the biomass of actinomycetes, bacteria and antibiotic were observed for growth of both bacteria and actinomycetes, at the time interval of 0hr, 3hrs, 6hrs, 9hrs, 12 hrs and 24 hrs, were found to be turbid except the control flask containing only Nutrient Broth³¹.

Figure 1: The Flasks containing the Actinomycetes biomass, Test Bacteria and Test Antibiotic.

a:T1, NB; T2,Actinomycetes (LMA4) biomass (1mg/ml), Actinomycetes biomass (1mg/ml) and test bacteria, BMS4 (10³ Cfu/ml), Test bacteria BMS4 (10³ Cfu/ml) and test antibiotic, Vancomycin; b: T1, NB; T2, Actinomycetes (LMA5) biomass (1mg/ml), Actinomycetes biomass (1mg/ml) and test bacteria, BMS4 (10³ Cfu/ml), Test bacteria BMS4 (10³ Cfu/ml) and test antibiotic, Vancomycin; T1, NB; T2, Actinomycetes (LMA6) biomass (1mg/ml), Actinomycetes biomass (1mg/ml) and test bacteria, BMS4 (10³ Cfu/ml), Test bacteria BMS4 (10³ Cfu/ml) and test antibiotic, Vancomycin.

Figure 2: The Flasks containing the Actinomycetes biomass, Test Bacteria and Test Antibiotic.

a:T1, NB; T2,Actinomycetes (LMA4) biomass (1mg/ml), T3, Actinomycetes biomass (1mg/ml) and test bacteria, BME4 (10³ Cfu/ml), Test bacteria BME4 (10³ Cfu/ml) and test antibiotic, Azithromycin; b: T1, NB; T2, Actinomycetes (LMA5) biomass (1mg/ml), Actinomycetes biomass (1mg/ml) and test bacteria, BME4 (10³ Cfu/ml), T4,Test bacteria BME4 (10³ Cfu/ml) and test antibiotic, Azithromycin; T1, NB; T2, Actinomycetes (LMA6) biomass (1mg/ml), T3, Actinomycetes biomass (1mg/ml) and test bacteria, BME4 (10³ Cfu/ml), T4, Test bacteria BME4 (10³ Cfu/ml) and test antibiotic, Azithromycin.

UV-Spectral Analysis of CCFE:

The cell free supernatant (with putative spores) were analysed in UV-Visible spectrophotometer at 600nm (λ) and the spectral absorbance values were recorded. Figure 3 and Figure 4 are the graphical representations of the absorbance values noted from the UV-Visible spectral analysis of samples (T1-T5). It was observed that the T1 samples (only NB) were with absorbance value zero, which was obvious for blank samples. The T2 samples were found to be with growth of respective actinomycetes namely LMA4, LMA5 and LMA6.

In Figure 3, it was observed that there was drop of absorbance value recorded for sample T3, containing only BMS4, but rise in absorbance value for the sample T4, containing both LMA4 and BMS4. However, there was also a steady fall of the absorbance value in T5, containing bacteria with the antibiotic, Vancomycin (50 µg/ml). The recorded absorbance values shown in b, the similar trend was observed with LMA5 and also LMA6. In Figure 4, the graphs plotted, also indicate the similar pattern of absorbance values taken for T1, T2, T3, T4 and T5 respectively. From this spectral absorbance values, taken for (a) actinomycetes CFE (b) bacteria, (c) actinomycetes with bacteria and more over (d) bacteria in presence of conventional antibiotic, it was inferred that the actinomycetes cell free extract had minimal growth inhibitory activity²⁸ against the test bacterial strains. The conventional antibiotics, Vancomycin and Azithromycin had substantial growth inhibitory activity against the bacteria, BMS4 and BME4 respectively.

Figure 3: The Graphical representation of the Spectral absorbance values of respective test samples. a: LMA4 CFE + BMS4+ Vancomycin; b:LMA5 CFE+ BMS4+ Vancomycin; c:LMA6 CFE+ BMS4+ Vancomycin at 600nm (λ).

Figure 4: The Graphical representation of the Spectral absorbance values of respective test samples. a: LMA4 CFE + BME4+ Azithromycin; b:LMA5 CFE+ BME4+ Azithromycin; c:LMA6 CFE+ BME4+ Azithromycin at 600nm (λ).

Therefore, this result may be analysed in a different angle. It is important to bring back the concept of cell free extract of test actinomycetes, say LMA4, LMA5 and LMA6, which were studied for the respective bio efficacy against the test bacteria belonging strains of Staphylococcus aureus (BMS4) and Escherichia coli (BME4). Both the absorbance values as well as the subculture plates were in agreement with each other with slightly differential result. It was very keenly observed that there were indication of growth of each of actinomycetes, when absorbance values were analysed at 600nm (λ) as well as viability in subculture plates. The plausible explanation for this result could be due to presence of spores, which were very small in size (<0.5µm), which could have not been separated with the vegetative mycelia of Actinomycetes. As the broths were allowed to incubate for a period of 24 hours, with rotational shaking, there would have establishment of favourable condition for germination of spores, due to activation of putative growth favourable enzymes. This assumption was substantiated with the growth pattern result observed in subculture plates. In addition to this, it was also found that there was a steady increase of bacterial growth in presence of the ACFE of LMA4, LMA5 and more over with LMA6 and also in presence of test antibiotics, namely Vancomycin/ Azithromycin, used as reference drugs for Staphylococcus aureus and Escherichia coli strains (BMS4 and BME4). Hence, this communictaon could infer about the resistance of test bacteria towards both the ACFEs and antibiotics, at the test concentrations. The bacterial strains were growing in presence of both ACFEs and also test antibiotics.

LC-MS analysis of Cell Free Extract (CCFE) of co-culture broth:

The Liquid Chromatography Mass spectrometry (LC-MS)²⁹ generated from co-culture CFE of LMA4, LMA5 and LMA6 are given in the Figures. The mass spectra of the cell free LMA4 was preliminarily generated with a set of peaks are depicted in Figure 5 and Figure 6 using “Full- Scan- only” of putative metabolites present in the said extract; which is been plotted intensity (mV) vs. Retention time in minutes. From these scan peaks the specific peaks were considered which are eluted with respective mass spectra. The peaks under consideration are enlisted in Table 2.

Figure 5: LC-MS Graph of ES+ reading of LMA4

Figure 6: LC-MS Graph of ES- reading of LMA4+BMS4

Azithromycin with chemical formula C₃₈H₇₂N₂O₁₂ with low resolution considering minimum abundance 0.1% the mass intensity is given in the below Table 2.

The mass spectra generated in the first scan mode, the Peak No- 53 with intensity 2% having m/z 751.3 can be declared as mass spectra of Azithromycin. Besides the full scan mode, that is ‘b’ with Peak id-45 the spectra with base peak at 749.8 with intensity of 8%, mass spectra with m/z 751.7 with intensity 8% are referred as mass spectra of Azithromycin.The mass spectra of the cell free LMA5 was preliminarily generated with a set of peaks are depicted in Figure 7 and Figure 8 using “Full- Scan- only” of putative metabolites present in the said extract; which is been plotted intensity (mV) vs Retention time in minutes. From these scan peaks the specific peaks were considered which are eluted with respective mass spectra.

Figure 7: LC-MS Graph of ES+ reading of LMA5 and BMS4

Figure 8: LC-MS Graph of ES- reading of LMA5

The mass spectra of the cell free LMA6 was preliminarily generated with a set of peaks are depicted in Figure 9 and Figure 10 using “Full- Scan- only” of putative metabolites present in the said extract; which is been plotted intensity (mV) vs Retention time in minutes. From these scan peaks the specific peaks were considered which are eluted with respective mass spectra.

Figure 9: LC-MS Graph of ES+ reading of LMA6

Figure 10: LC-MS Graph of ES- reading of LMA6

The results (Table 2) in the form of eluted peaks structured with spectra generated using X- axis as percentage of intensity vs Y- axis m/z (mass of specific compounds present at different retention time). As intentioned in materials and methods the BLANK containing only the culture broth was also consider in the mass spectral analysis for the purpose of comparison and authentification. Both the mass spectrometry (MS) / electro spray positive mode and negative mode scanning at created the spectrum, which are given in Figures. The control (BLANK) was in the positive ES mode, there was elution of peaks with a range of 15.02 m/z to 34.08 m/z starting from 15 minutes retention time (RT). While the ES negative (ES^-) mode was illustrated with 15.07 m/z to 32.63 m/z range with the same RT as observed with positive mode ES.

Table 2: Presence of Azithromycin in the Cell free extract of LMA4 and LMA6

Table-1. ES+ (LMA4)
Peak ID	Retention Time (Minutes)	m/z
53	26.99	751.3
Table-1. ES-
Peak ID	Retention Time (Minutes)	m/z
45	24.65	749.8
54	27.11	751.7
Table-1. ES+ (LMA6)
Peak ID	Retention Time (Minutes)	m/z
12	5.71	752.4
42	20.55	749.8

CONCLUSION:

Precisely, the strains of actinomycetes, namely Microbacterium barkeri (LMA4), Corynebacterium argentoratense (LMA5) and Streptomyces shenzhenensis (LMA6), when co- cultured with conventional antibiotic resistant bacterials strains, detected in local health care institute (a Gram positive, Staphylococcus aureus, BMS4 and Gram negative, Escherichia coli, BME4), it was observed that there was no indication of absolute inhibition of growth of bacteria, evidenced from subculture plates, at the test concentration of Actinomcetes500µg/ml (W/V). But, the cell free extract (CFE) of co culture broth when was subjected to UV-Visible scanning (300nm, λ-700nm, λ) and LC-MS for detection of presumptive novel antibiotics or derivatives of prescribed drugs/antibiotics. The LC-MS (m/z) data was correlated with published reports and it was inferred that there was similar peak pattern with Streptomycin, Doxorubicin, Pyrazine, Pyrrolizidines, Oxacillin, Ciprofloxacin, Allistatin, Gentamycin, Chlorellin, Penicillin, Penicillin G, Kanamycin, Levofloxacin, Amikacin, Ofloxacin, Imipenem and more over Ampicillin. However, from the LC-MS (m/z) spectrum analysis, it was noted that the peak with ID-53, eluted from the CFE, of Co-culture broth of LMA4 with BMS4, with retention time (RT) 26.99 (min), of m/z 751.3, carried out with Electrospray Ionisation (ES) in +ve mode (ES+), Peaks with IDs, 45, 54 (24.65 and 27.11respective RTs), having resultant m/z, 749.8and 751.7 in ES-, and the CFE containing LMA6 and BMS4, with eluted peak Ids 12, 42 (RTs, 5.71, 20.55), having m/z 752.4 and 749.8 and 749.8 in ES+ mode, which could be assigned with structure of Azithromycin. Hence, it is concluded that LMA4 and LMA6 would have produced Azithromycin or allied class of antibiotic, which may be considered as excellent bioactive compound from the Actiniomycetal strains under study. Further, the LC-MS data had also substantiated the observation, about detection of many antibiotics, the spectra eluted in LC chromatogram, with respective m/z, in which all the strains of actinomycetes including LMA5 was also under the category of source of bioactive metabolite production. But this is to mention that the growth inhibitory activity of LMA5 and LMA6 were superior to the LMA4. This metabolite analytical tool results would substantiate the Genomics based results and interpretations, which will be future prospective of this communication.

REFERENCES:

1. Dhillon RH, Clark J. ESBLs: a clear and present danger? Critical Care Research and Practice. 2012; Oct; 2012. doi:10.1155/2012/625170.

2. Rawat D, Nair D. Extended-spectrum β-lactamases in Gram Negative Bacteria. Journal of Global Infectious Diseases. 2010; Sep; 2(3): 263. DOI: 10.4103/0974-777X.68531.

3. Livermore DM. Has the era of untreatable infections arrived?. Journal of Antimicrobial Chemotherapy. 2009; Sep 1; 64(suppl_1):i29-36. doi:10.1093/jac/dkp255.

4. Teklu DS, Negeri AA, Legese MH, Bedada TL, Woldemariam HK, Tullu KD. Extended-spectrum beta-lactamase production and multi-drug resistance among Enterobacteriaceae isolated in Addis Ababa, Ethiopia. Antimicrobial Resistance and Infection Control. 2019; Dec; 8: 1-2. https://doi.org/10.1186/s13756-019-0488-4.

5. Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) in the community. Journal of Antimicrobial Chemotherapy. 2005; Jul 1; 56(1): 52-9. doi:10.1093/jac/dki166.

6. Tooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VH, Takebayashi Y, Spencer J. β-Lactamases and β-Lactamase Inhibitors in the 21st Century. Journal of Molecular Biology. 2019; Aug 23; 431(18): 3472-500. https://doi.org/10.1016/j.jmb.2019.04.002.

7. Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus. Annual Review of Biochemistry. 2015; Jun 2; 84: 577-601. doi: 10.1146/annurev-biochem-060614-034516.

8. Terreni M, Taccani M, Pregnolato M. New antibiotics for multidrug-resistant bacterial strains: latest research developments and future perspectives. Molecules. 2021; May 2; 26(9): 2671. https://doi.org/10.3390/molecules26092671.

9. Durante-Mangoni E, Andini R, Zampino R. Management of carbapenem-resistant Enterobacteriaceae infections. Clinical Microbiology and Infection. 2019; Aug 1; 25(8): 943-50.https://doi.org/10.1016/j.cmi.2019.04.013.

10. Nordmann P. Carbapenemase-producing Enterobacteriaceae: overview of a major public health challenge. Medecine et Maladies Infectieuses. 2014; Feb 1; 44(2): 51-6.

11. Bhakyashree K, Kannabiran K. Anti-MRSA activity of actinomycetes isolated from marine soil sample of ariyaman beach, Tamil Nadu, India. Research Journal of Pharmacy and Technology. 2018; 11(5): 2036-9. DOI: 10.5958/0974-360X.2018.00377.3.

12. Ravi L, Kannabiran K. A review on bioactive secondary metabolites reported from actinomycetes isolated from marine soil samples of indian peninsula. Research Journal of Pharmacy and Technology. 2018; 11(6): 2634-40. DOI: 10.5958/0974-360X.2018.00489.4.

13. Dawadi S, Shrestha S, Giri RA. Mixed-methods research: A discussion on its types, challenges, and criticisms. Journal of Practical Studies in Education. 2021; Mar 1; 2(2): 25-36. DOI: https://doi.org/10.46809/jpse.v2i2.20.

14. Elshafie HS, De Martino L, Formisano C, Caputo L, De Feo V, Camele I. Chemical Identification of Secondary Metabolites from Rhizospheric Actinomycetes Using LC-MS Analysis: In Silico Antifungal Evaluation and Growth-Promoting Effects. Plants. 2023; May 2; 12(9): 1869. https://doi.org/10.3390/plants12091869.

15. Maleki H, Mashinchian O. Characterization of Streptomyces isolates with UV, FTIR spectroscopy and HPLC analyses. BioImpacts: BI. 2011; 1(1): 47. doi: 10.5681/bi.2011.007.

16. Muralidharan V, Deecaraman M. A source of novel therapeutic drugs-marine actinomycetes. Research Journal of Pharmacy and Technology. 2017; 10(10): 3598-600. DOI: 10.5958/0974-360X.2017.00652.7.

17. Chandrashekar A, Muralidharan A, Koteshwara A, Alex AT, Subrahmanyam VM. Isolation and Characterization of an actinomycete strain producing an antifungal metabolite effective against Candida albicans. Research Journal of Pharmacy and Technology. 2019; 12(10): 4601-6. DOI: 10.5958/0974-360X.2019.00791.1.

18. Haag H, Gremlich HU, Bergmann R, Sanglier JJ. Characterization and identification of actinomycetes by FT-IR spectroscopy. Journal of Microbiological Methods. 1996; Nov 1; 27(2-3): 157-63. https://doi.org/10.1016/S0167-7012(96)00943-8.

19. Martorell-Marugán J, Tabik S, Benhammou Y, del Val C, Zwir I, Herrera F, Carmona-Sáez P. Deep learning in omics data analysis and precision medicine. Exon Publications. 2019; Oct 31: 37-53. Doi: http://dx.doi.org/10.15586/computationalbiology.2019.ch3.

20. Thomas PD. Actinomycetes synthesized nanoparticles and their antibacterial activity. Research Journal of Science and Technology. 2017; 9(2): 219-23. DOI: 10.5958/2349-2988.2017.00038.9.

21. Nabila MI, Kannabiran K. Antibacterial activity of soil actinomycetes against fish and shellfish pathogens. Research Journal of Pharmacy and Technology. 2018; 11(5): 1923-6. DOI: 10.5958/0974-360X.2018.00356.6.

22. Zimmerman N, Presto AA, Kumar SP, Gu J, Hauryliuk A, Robinson ES, Robinson AL, Subramanian R. A machine learning calibration model using random forests to improve sensor performance for lower-cost air quality monitoring. Atmospheric Measurement Techniques. 2018; Jan 15; 11(1): 291-313. https://doi.org/10.5194/amt-11-291-2018.

23. Gayathri A, Madhanraj P, Panneerselvam A. Diversity, Antibacterial Activity And Molecular Characterization of Actinomycetes Isolated From Salt Pan Region of Kodiakarai, Nagapattinam DT. Magnesium. 2011; Sep 28;7:15.

24. Paul S, Kavitha S, Vimala R. Isolation of actinomycetes and optimization of process parameters for antimicrobial activity. Research Journal of Pharmacy and Technology. 2016; 9(11): 1913-21. DOI: 10.5958/0974-360X.2016.00392.9.

25. Kamalakannan K, Kumar U, Kandpal KC, Minz S, Pradhan M, Saravanakumar A, Sivakumar T. Isolation and screening of endophytic actinomycetes producing antibacterial compound from different parts of Citrus aurantifolia. Research Journal of Pharmacy and Technology. 2008; 1(2): 112-5.

26. Sumathi R, Rajeswari R, Pavani S. Isolation, purification, partial characterization and antibacterial activities of compound produced by some actinomycetes from sedimented waters. Research Journal of Pharmacy and Technology. 2009; Sep 28; 2(3): 521-6.

27. Ahlawat J, Sharma M, Pundir CS. An Amperometric Acetylcholine Biosensor Based on Co-Immobilization of Enzyme Nanoparticles onto Nanocomposite. Biosensors. 2023; Mar 15; 13(3): 386. https://doi.org/10.3390/bios13030386.

28. Vaijayanthi G, Cholarajan A, Vijayakumar R. Isolation, characterization and antibacterial activity of terrestrial actinobacteria in the soils of Thanjavur district, Tamil Nadu, India. Research Journal of Science and Technology. 2012 May 1;4(3):IV.

29. Suárez M, Macià A, Romero MP, Motilva MJ. Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil. Journal of Chromatography A. 2008; Dec 19; 1214(1-2): 90-9. https://doi.org/10.1016/j.chroma.2008.10.098.

30. Pattnaik S. Induction of bioactive compounds in a co-cultured strain of Micromonosperma echinospora (DST-4) isolated from secluded Hirakud dyke soil.

31. Devi PN, Warma SS, Srinivasan M, Keziah SM, Devi CS. Antimicrobial Activity of Actinomycetes Isolated from Different soil samples of Vellore Region. Research Journal of Pharmacy and Technology. 2018; 11(7): 3123-7. DOI: 10.5958/0974-360X.2018.00573.5.

Received on 20.11.2023 Modified on 02.04.2024

Research J. Pharm. and Tech 2024; 17(10):4922-4928.

DOI: 10.52711/0974-360X.2024.00757