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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 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.
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.
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.
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.
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.
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.
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