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Effect of Streptomyces pylosus (LMA1) cell free extract on the staphylococcal-infected cell lines
*Corresponding author: Smaranika Pattnaik, School of Life Sciences, Sambalpur University, Burla, Odisha, India. smaranika2010@suniv.ac.in
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Received: ,
Accepted: ,
How to cite this article: Rath T, Pattnaik S. Effect of Streptomyces pylosus (LMA1) cell-free extract on the staphylococcal-infected cell lines. Indian J Physiol Pharmacol. doi: 10.25259/IJPP_218_2025
Abstract
Objectives:
The experimentation on cell lines is a prerequisite as well as an optimise method for the screening of natural drugs.
Materials and Methods:
The cells with 70% confluency were selected for the viability in the presence and absence of the test metabolite extracted from Laboratory of Microbiology Actinomycetes 1 (LMA1) (Streptomyces pilosus), which was considered for the screening as an ‘anti-infective agent’ using the cell line culture.
Results:
It reveals that the cells present were observed to be at the stage of stationary as well as death or decline period, indicated by the deformity of the cells and coated with some molecules, inferring the phase of senescence.
Conclusion:
The experiment carried out had made an attempt to decipher the mode of action of the test actinomycetal LMA1, cell free extract on the cell line culture infected with test bacteria, namely Staphylococcus aureus (BMS4).
Keywords
Cell Free Extract
Streptomyces pilosus
Staphylococcus aureus
Cell line infection
INTRODUCTION
Actinomycetes[1] have been defined as the most potent bioactive metabolite-producing organisms for many decades. The bioactive compounds[2] are reported with enormous antimicrobial activity[3-6] because of the broad-spectrum mechanism of action of the candidate bioactive compounds. Drug screening is an important factor in the process of designing and developing drugs, specifically when a drug is extracted from natural resources. The actinomycetal strains diffusing the drug components are to be screened for their bioactivity[7,8] against the bacterial strains in vitro, substantiated with in vivo experimentation. In this regard, the experimentation on cell line[9] is a prerequisite as well as an optimised method for the screening of natural drugs.
The cell line culture [Figure 1] for the induction of bacterial infection in the process of drug screening is a technology proven with high performance, high stability and giving profound results. The cell lines are developed from the animal models in the form of tissue. The tissue is allowed to proliferate under appropriate conditions. As a result, the primary culture occupies the substrate (media) and the process is called confluence. After this stage, the cell lines are subcultured, which is called ‘Passaging’ to new media containing vessels. The media are enriched with optimised nutritional conditions, maintaining enough sterility. Observations should be made on the growth of all the cells, which should maintain a stable as well as continued growth. Enough precautions are made so that the cell lines should not be contaminated with opportunistic microbial strains.
- The fundamental steps adapted in cell line culture (a) The growth media containing cell colonies are (b) transferred to culture vessel and observation under microscope depicts presence of (c) individual cells.
The cell culture provides an excellent model system to study various scientific investigations using cellular growth, metabolism, the effect of drugs, mutation and more so tumour genesis. This is the first line of approach to study the drug screening and development while dealing with natural drugs such as actinomycetes or plant products. This further emphasises that the cell culture-based experimental approach is very much consistent and affords reproducibility and is more so laboratory-friendly in comparison to handling multicellular animal models, which requires extended ethical clearance. Keeping this view in mind, this fragment of research work, being a part of thesis work, was converged with experimental work related to cell culture technique for the screening of actinomycetal-induced presumptive drug candidate in the infected and symptomatic cell lines, following the guidelines of the cell culture maintenance and experimentation.
It is pertinent to mention that there is a big gap in the literature regarding the in vivo studies on the cell-free extracts (CFEs) of bioactive actinomycetes on staphylococcal-infected cell lines to date. This study may be a novel approach in the arena of drug discovery and the design of naturally sourced lead drug molecules.
MATERIALS AND METHODS
In vitro-based assay
Culturing actinomycetes of interest
The post-dried soil samples were mixed with sterilised distilled water at a ratio of 1 g of soil samples in 100 mL of sterilised water and further diluted as 1:1, 1: 10, 1: 100 and 1:10,000 serially using the ‘Serial dilution technique’. The petri-plates poured with inoculant were incubated in an incubator, maintaining a temperature of 37°C ± 2 for 24 h. Further, the presumptive actinomycetal growth was identified and was sub-cultured onto actinomycetal agar medium using the ‘Streak plate technique ’.
Bacterial strains
Bacterial strain of Staphylococcus aureus (BMS4) was given by the Department of Microbiology, Veer Surendra Sai Institute of Medical Sciences and Research, BURLA, Odisha, with permission. The ethical guidelines were strictly followed.
Screening for antibacterial constituent’s synthesis by isolated actinobacteria
The actinomycetal isolate (LMA1) was screened for its bacterial growth inhibitory activity following the ‘Patch inoculum diffusion’ method. The bacterial lawns were made using the ‘Pour plate’ method. The actinomycetes (5 days old) with spores were put on the surface of poured plates in triplicate at equidistance from each other. These ‘co culture’[10] plates were incubated at 37°C. Periodically (24th, 48th and 72nd h), observations were made for inhibition of bacterial growth around the patch inoculum (if any). The appearance of zones of inhibition (ZOI) around the patch inoculum was considered as the inhibition of bacterial growth due to the activity of the test actinomycetes. The respective ZOIs were measured, and the degree of growth inhibitory activity[11] was inferred.
Preparation of actinomycetes CFE (ACFE)
The actinomycetes under study (LMA1) were grown in actinomycetes isolation agar were subjected to preparation of CFE for the detection of putative antibiotics/bioactive compounds. Purposefully a multidrug-resistant bacterial strain of the Gram-positive category, S. aureus (BMS4), was taken taken as co-cultured partners in the broth media. Hence, the bacterial strain was revived from the stock culture, maintained in the Laboratory of Medical Microbiology, Department of Biotechnology and Bioinformatics, Sambalpur University. The bacteria were grown in the subculture media for 12 h in the broth medium. After the requisite incubation period, bacteria broths containing 105Cfu/mL were considered as the inoculants for the co-culture technique. The flask was inoculated with broth actinomycetes cellular mass (1.5 g) per 0.05 g/mL and 103Cfu/mL of bacterial cells, respectively. The broths containing both the organisms were kept in a rotatory shaker maintained at 37°C, and the shaking rotation was kept at 120 rpm. The incubation period was incubated for a total of 48 h. The CFE decanted from culture broths (supernatant) was kept at +4°C for further use.
Optimisation of concentration of ACFE against test bacteria
The nutrient agar plates inoculated with test bacteria (BMS4) using the ‘Pour Plate’ method were used to make bacterial lawns. Further, agar cups were made in inoculated agar plates using an agar borer of 3 mm in diameter, after the poured plates were solidified. The ACFEs were put in each of the agar cups in a range of concentrations 10 µL/well, 20 µL/well and 30 µL/well, respectively. A nutrient Agar plate without wells, but inoculated with test bacteria, was taken as a control. The plates were incubated at 37°C ± 2 for 24 h. The plates were observed for zones of inhibition, if any, after the scheduled incubation period. The zones of inhibition (diameter) in mm were measured, and the results were inferred.
Culture of Streptomyces pylosus (LMA1) was assigned with Gene Bank ID: ON015754, onto Nutrient agar plates, which were marked with specific slots using markers. The co-incubated broth was sub-cultured onto the marked slots using an inoculation loop following the streaking method, starting from 0 h to 72 h with a gap of 2 h. The streak subculture plates had been kept in incubators maintained at 37°C, but not in a rotary shaker cum incubator. The observations were made after 24 h of incubation on the appearance of growth or no growth in the specific slots; accordingly, the presence of live cells or dead cells was inferred.
Optimisation of concentration of ACFE against test bacteria
The nutrient agar plates inoculated with test bacteria (BMS4) using the ‘Pour Plate’ method were used to make bacterial lawns. Further, agar cups were made in inoculated agar plates using an agar borer of 3 mm in diameter, after the poured plates were solidified. The ACFEs were put in each of the agar cups in a range of concentrations 10 µL/well, 20 µL/well and 30 µL/well, respectively. A nutrient Agar plate without wells, but inoculated with test bacteria, was taken as a control. The plates were incubated at 37°C ± 2 for 24 h. The plates were observed for zones of inhibition, if any, after the scheduled incubation period. The zones of inhibition (diameter) in mm were measured.
Cell lines are used for cell culture technique
The cell line, HEK 293, was purchased from the National Centre for Cell Sciences, Pune, India. The cell line exhibiting epithelial morphology had been isolated from the kidney of a human embryo. In drug screening and toxicological studies, this cell line is used in medical science. Further, the testing for the drugs, as well as virucide activity against bacterial infections, is carried out using this cell line.
Cell line proliferation and assay for viability
Selection of the CFE of actinomycetes
The cell lines taken at their exponential growth period (log period) were seeded onto tissue culture (TC) plates (96 wells) at a size of 9000 cells. The culture plates were kept in a humidified CO2 incubator with 70% confluency and were allowed to grow. The cells with 70% confluency were selected for the viability in the presence and absence of the test metabolite extracted from LMA1 (S. pylosus), which was considered for the screening as an ‘anti-infective agent’ using the cell line culture. The CFE stored in a cold environment maintained at 4°C was thawed down to room temperature and used for its efficacy study.
RESULTS
The subculture Congo Red agar plates [Figure 2] is showing the isolated colonies of test bacterial strain, namely S. aureus (BMS4). The colonies were pure and contamination-free, and were carried forward for further experimentation. Further, to state that the bacteria were not biofilm producers, as there was no indication of Black colour colonies on said plates.
- The subculture Congo red agar plates showing the growth of Staphylococcus aureus (BMS4).
Figure 3 demonstrates the microscopic image of LMA1. It was observed that the actinomycetes were aerial with branched mycelia having round spores. More so, the actinomycetes were Garm-positive, which are the basic identifying characteristic features.
- Microscopic structure of Laboratory of Microbiology Actinomycetes 1, the test actinomycetes showing the branched mycelia with round spores.
Screening for biopotent actinomycetal isolates
The experiments related to screening for the potentiality of chemo type production by the test isolates, namely, LMA1, showed that there was remarkable growth inhibitory activity of the test isolate [Figure 4]. The patch inoculum method, which opted for ACFE of LMA1 against BMS4, had inferred the inhibition of growth of bacteria by the activity of diffused drug from ACFE of LMA1, with a zone size of 30.6 mm (µ, diameter). Hence, it may be inferred that there were some inhibitory compounds that could have been diffused from the actinomycetes, which were luxuriantly growing in concentric layers around the bacterial inoculum.
- Demonstrating the ‘Patch inoculum diffusion’ method for screening of growth inhibitory activity of test Actinomycetes. The patch inoculum of LMA1, in triplicates respectively. There was higher degree of inhibition was observed with LMA1 against BMS4, in triplicates respectively, A control plate showing bacterial mat without patch inoculum.
Experimental on cell lines
The observations made for ACFE of LMA1 on the Staphylococcal-infected cell lines, the results captured in images are given in Figures 5-9 for 0 h, 3rd h, 12th h, 24th h and 48th h, respectively.
- The condition of cell lines at 0 hour. (a) Unexposed cell lines; (b) Cell line infected with BMS4; (c) Infected with BMS4 and exposed with azithromycin; (d) Infected with BMS4 and exposed with 12 months old actinomycetal cell free extract (ACFE); (e) Infected with BMS4 and exposed with 10 months old actinomycetal cell free extract (ACFE). There was no substantial differentiation among the inoculated cells present in tissue culture (TC) plate. The possible reason behind may be the application of drugs instantly might not have started its action.
- The condition of cell lines at 3rd hour. (a) Unexposed cell lines; (b) Cell line infected with BMS4, depicts about the bunch of cells causing degradation of some of the cells, marked by whitish granulations, inferring a cytopathic effect; (c) Infected with BMS4 and exposed with azithromycin, infected with BMS4 and exposed with azithromycin are inconspicuous covered with a sheet of antibiotic film; (d) Infected with BMS4 and exposed with 12 months old actinomycetal cell free extract (ACFE), covered with a film; (e) Infected with BMS4 and exposed with 10 months old actinomycetal cell free extract (ACFE), appearance of healthier cells.
- The condition of cell lines at 12th hour. (a) Unexposed cell lines, depicting presence of cells with normal morphology, with an increase of cell numbers; (b) Cell line infected with BMS4, a cell mass infection evidenced by presence of muliated, circular with loss of cell membranes, disorganised with loss of intracellular organelles; (c) Infected with BMS4 and exposed with azithromycin, infected with BMS4 and exposed with azithromycin are t defined and healthy, baring presence of a small number of rounded integrities inferring about both infected cells as well as healthy cells demonstrated with dark stained bodies of chromosome; (d-e) Infected with BMS4 and exposed with 12 months and 10 months old actinomycetal cell free extract (ACFE), demonstrating presence of aggregates of cells with nominal presence of infected cells. The cells were elongated, rectangle and also polymorphic types.
- The condition of cell lines at 24 hours. (a) Unexposed cell lines, depicting presence of cells with normal healthy morphology; (b) Cell line infected with BMS4, a cell mass infection evidenced by presence of disrupted as well as deformed cells with whitish granular lesions; (c) Infected with BMS4 and exposed with azithromycin, infected with BMS4 and exposed with azithromycin are healthy cells and no indication of presence of a film; (d-e) Infected with BMS4 and exposed with 12 months and 10 months old actinomycetal cell free extract (ACFE), indication of disappearance of cytopathic lesions.
- The condition of cell lines at 48 hours. (a) Unexposed cells present observed to be at the stage of stationary as well as death or decline period indicated by the deformity of the cells and coated with some molecules; (b) Cell line infected with BMS4, presence of disrupted and coalesced/merged cells; (c) Infected with BMS4 and exposed with azithromycin, infected with BMS4 and exposed with azithromycin, inferring conspicuous cells; (d) Infected with BMS4 and exposed with 12 months old ACFE showing presence of inconspicuous merged colonies with vacuoles containing air; (e) 10 months old actinomycetal Cell Free extract (ACFE) showing healthy mass of cells in a greater number without any cytopathic lesion.
0 h observation on the growth and morphology of test cell lines
It is observed that the cell morphology showed no substantial differentiation among the inoculated cells present in the TC plate.
3rd h observation on the growth and morphology of test cell lines
It depicts the images taken for the negative control, containing only the cell line without bacterial inoculation and drug exposure. It was observed that the cells were distinct, elongated and healthy. Some of the cells were found to be isolated, whereas some of the cells were joined with each other, but there was no cell merging effect.
12th h observation on the growth and morphology of test cell lines
It demonstrates the behaviour of cells in response to bacterial infection, exposure to both conventional antibiotics (azithromycin) and test LMA1 CFE (12-month-old) and CFE (10-day-old).
24th h observation on the growth and morphology of test cell lines
The figure depicted in Figure 8a (24 h) indicated that the cells not exposed to either bacterial infection or test drugs were still healthy after 24th h incubation. From the image as shown in (24 h) [Figure 8a], it is noted that there are events inferring about bacterial infection, evidenced by the presence of disrupted as well as deformed cells with whitish granular lesions.
48th h observation on the growth and morphology of test cell lines
In Figure 9 it was found that the cells present were at the stage of stationary as well as death or decline period, indicated by the deformity of the cells and coated with some molecules, inferring the phase of senescence.
- Graphical abstract prohibition of conventional drugs by World Health Organization may affect the health of society. The actinomycetes being the treasure trove of metabolites can be mined out for metabolites using high throughput metabolomics approaches with the detection of compounds of interest, which may act as lead drug molecules or functional moieties to replace banned drugs for the health benefit of society.
DISCUSSION
From the test regarding the screening for bacterial growth inhibitory activity of actinomycetal isolate, it was observed that LMA1 (S. pylosus) had the remarkable bacterial growth inhibitory activity against bacterial strains of BME4 and BMS4.
The experiment carried out had made an attempt to decipher the mode of action of test actinomycetal LMA1, CFE on the cell line culture infected with test BMS4. There were five sets of experiments considering negative control comprising a cell line as a normal cell not infected by the test bacteria and not exposed to any test drug. The second set of experiments included the positive control, where the cell lines were exposed to the said bacterial inoculation. Further, the third set included cell lines inoculated with bacteria and exposed to the reference drug azithromycin. The fourth and fifth sets considered the cell lines inoculated with bacteria and exposed to 12-month CFE and 10-day CFE, respectively. There was an observation for each of the set cell lines starting from 0 h to 48th h (0 h, 3 h, 12th h, 24th h and 48th h) while incubating in a humidified CO2 incubator (95% air and 5% CO2).
CONCLUSION
Therefore, the LMA1, CFE 10-day-old was considered as a source of metabolites that had substantial bacterial growth inhibitory activity along with substantial cellular protectivity activity.
Ethical approval:
Institutional Review Board approval is not required as the bacterial strain had been taken from the Department of Microbiology, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, Odisha with prior permission for purpose of research.
Declaration of patient consent:
Patient’s consent not required as there are no patients in this study.
Conflict of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
Financial support and sponsorship: Nil.
References
- Current approaches in metabolomics study of actinomycetes: An analysis. Zeichen J. 2024;10:1-9.
- [CrossRef] [Google Scholar]
- Screening of antimetabolites of interest present in a streptomycetal isolate using analytical tools. Res J Biotechnol. 2025;20:195-201.
- [CrossRef] [Google Scholar]
- Biodiversity of antimicrobial-producing actinomycetes strains isolated from dry dipterocarp forest soil in northeast Thailand. Braz Arch Biol Technol. 2016;59:1-11.
- [CrossRef] [Google Scholar]
- Antimicrobial activity of actinomycetes and characterization of actinomycin-producing strain KRG-1 isolated from Karoo, South Africa. Braz J Pharm Sci. 2019;55:e17249.
- [CrossRef] [Google Scholar]
- Antibacterial activity of actinomycetes isolated from different soil samples of Sheopur (A city of central India) J Adv Pharm Technol Res. 2013;4:118-23.
- [CrossRef] [PubMed] [Google Scholar]
- Isolation, characterization, and screening of antimicrobial-producing actinomycetes from soil samples. Int J Microbiol. 2020;2020:2716584.
- [CrossRef] [PubMed] [Google Scholar]
- Isolation and screening of actinomycetes from Sundarbans soil for antibacterial activity. Afr J Biotechnol. 2010;9:4615-9.
- [Google Scholar]
- Screening of actinomycetes from soil for antibacterial activity. Nepal J Biotechnol. 2020;8:102-10.
- [CrossRef] [Google Scholar]
- Endocrine and targeted therapy for hormone-receptor-positive, HER2-negative advanced breast cancer: Insights to sequencing treatment and overcoming resistance based on clinical trials. Front Oncol. 2019;9:510.
- [CrossRef] [PubMed] [Google Scholar]
- Induction of bioactive compounds in a co-cultured strain of Micromonosperma echinospora (DST-4) isolated from secluded Hirakud dyke soil. Indian Journal of Biotechnology (NSCAIR). 2018;17:586-92.
- [Google Scholar]
- Bioprospection of actinobacteria derived from freshwater sediments for their potential to produce antimicrobial compounds. Microb Cell Fact. 2018;17:68.
- [CrossRef] [PubMed] [Google Scholar]