INTRODUCTION:

The health and wellness of animals, including humans, are influenced by the existence and make-up of endogenous microbial inhabitants known as the microbiome¹. The microbiota is considered a supporting organ because it plays many key roles in influencing host physiology and function. Recently, the human microbiome is receiving more attention as a result of its correlation with a variety of diseases, including cancer, and metabolic disorders such as type 2 diabetes².

Much research has been undertaken on bacteria as the causal agent of cancer as evidenced by the example of gastric cancer caused by Helicobacter pylori. On the other hand, bacteria such as Bifidobacterium and Lactobacilli or their products are considered potential rescuers in the treatment of some of the deadliest cancers. Over the years in cancer therapy, “the microbiomes have gone from ignored to super-popular organisms”³. However, little is revealed as to how the host microbiome and the response to cancer treatment interact. Interpreting the bacterial strains in the microbiome with potential therapeutic activities and the purification of the bio-compounds responsible for the biological activities will aid in the discovery of various lead molecules.

The upper reproductive tract, including the fallopian tubes and ovaries, was traditionally assumed to be sterile, but new evidence casts doubt on that assumption^4,5. Recently, some studies have demonstrated the isolation of Bacillus cereus strain from the ovaries of broiler hens and S. daejeonense strain and Bacillus sp. from the oviducts⁶. Dysbiosis also referred to as oncobiosis in neoplastic disorders, is a feature of ovarian cancer. Oncobiosis can lead to a lower diversity of microbiota and has been found in a variety of compartments in ovarian cancer, such as in the tumour tissue directly, the upper and lower genitalia, the peritoneum, and the intestines⁷. With the introduction of bacteriotherapy, cancer therapy has undergone a paradigm shift, where bacterial dysbiosis and metabolite signalling are exploited. It is also known that microbial natural products are extremely adaptable and interesting due to their distinct properties and functions. Different compounds isolated from various microbiota have shown antioxidant, antibacterial, and anticancer activities and are used in pharmaceutical industries. The best way for conducting microbiome studies as part of preclinical trials is in various animal models. The mouse has been the subject of extensive microbiome study, and it is the animal of choice for preclinical proof-of-concept experiments. In light of this, the present study has attempted to isolate both aerobic and anaerobic culturable bacterial strains from the ovaries of 8-week-old mice and to evaluate the antioxidant, antibacterial, and antiproliferative potential of the isolates.

MATERIALS AND METHODS:

Animals:

Swiss albino female mice aged 8 weeks were purchased from NIN, Hyderabad, and housed under a 12h light/dark regimen at a temperature of 25±2°C and relative humidity of 40–60%. Throughout the experimentation, the animals were fed a regular commercial diet and provided with water ad libitum⁸. The number of animals permitted and approved by the Institutional Animal Ethics Committee (IAEC), for the study was n=2 (Sanction No. SAC/IAEC/02/2018). The experiments were carried out under the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Govt. of India.

Isolation of bacteria from mice ovaries

The 8-week-old female mice were euthanized by cervical dislocation, and then the whole ovaries were excised through the surgical procedure⁹ under sterile conditions. Immediately, the ovaries were aseptically transferred to a 10mL of sterile PBS solution and macerated using a homogenizer (LabGEN®125, Cole-Parmar, USA). This suspension was then spread plated onto four different culture media, including Luria-Bertani agar, nutrient agar, blood agar, and Bifidobacterium agar. The inoculated plates were incubated at 35±2°C under both aerobic and anaerobic conditions. After incubation for 72h, the colonies were randomly selected and streaked on the respective medium to obtain pure cultures. The isolated pure cultures were stored at −20°C in the presence of 30% w/v glycerol for further experimentation.

Biochemical and molecular characterization of bacterial isolates:

Presumptive identification of isolates was carried out based on their morphological and biochemical characteristics. The characteristics of pure cultures, such as colony morphology and response to Gram staining were analysed. The biochemical characterization of isolates was carried out by using a standard HiBacillus^TM identifications kit (Himedia, India). Genetic identification at the species level was done by PCR analysis and 16S rRNA sequencing. The genomic DNA was isolated according to the manufacturer's instructions using the Himedia bacterial genomic DNA purification kit. The 16S rRNA gene was then amplified using the universal PCR primers, 27F (AGA GTT TGA TCC TGG CTC AG) and 1492R (CGG TTA CCT TGT TAC GAC TT). Following that, the amplified fragments were used for sequencing¹⁰. The obtained 16S rRNA sequence was subjected to BLAST analysis at NCBI.

Phylogenetic analysis:

The MUSCLE multiple sequence alignment software of MEGA X was used to align the 16S rRNA sequences of all bacterial isolates with reference sequences from the NCBI database that showed sequence homology. The phylogenetic tree was built with the UPGMA algorithm with bootstrap test results from 1000 replications¹¹.

Extraction of bioactive compounds:

The bacterial isolates were cultured in an Erlenmeyer flask containing 100mL of production medium (pH -7), which was inoculated with an exponential-phase culture and incubated on an orbital shaker at 150rpm and 37°C for 48 h. Centrifugation at 5000rpm for 20min was used to separate the bacterial cells from the supernatant at 4°C. Further, the cell-free supernatant was subjected to solvent extraction using ethyl acetate (EtAC). The organic phase was collected using a separating funnel and concentrated in a vacuum evaporator at 45°C under reduced pressure¹².

Antibacterial assay by the disc diffusion method:

The screening of the antibacterial activity of the isolates on the test bacteria was performed using the disc diffusion method. Briefly, 20μL 200µg/mL of the ethyl acetate extract (EtAE) in 1% DMSO from each isolate was impregnated on sterile blank disks of 6mm diameter. Then the discs were fully dried at 40°C and sterilized under UV light for 2h. The Mueller-Hinton agar media plates were swabbed with 100μL of appropriately diluted (turbidity was adjusted to a 0.5 McFarland standard) freshly grown test microorganisms. The discs were then carefully placed, and the plates were incubated at 37°C. The antibiotic zone scale (HiMedia, India) was used to measure the zone of inhibition accurately. Discs soaked with 1% DMSO served as solvent control while Tetracycline (30 mcg) served as the standard drug¹³.

Determination of Minimum Inhibitory Concentration (MIC):

The MIC of EtAE from the selected strain was determined by the microtitre broth dilution method¹⁴. Each test bacterial suspension was adjusted to a cell density of 1x10⁵ CFU/mL in Mueller-Hinton Broth (MHB). The various concentrations of EtAE, ranging from 6.25 to 800µg/mL were prepared in 1% DMSO. For all investigations, a sterility inspection (MHB + 1% DMSO), negative control (MHB + 1% DMSO + test organism), and positive control (MHB+ test organism + tetracycline in 1% DMSO with a concentration range of 6.25 to 800µg/mL) were also included. In general, 80μL of MHB, 10μL of test chemicals, and 20μL of the test organism were dispensed into each well of the 96 well plates. After incubation at 37°C for 20h the microbial growth in each well, as well as the lowest concentration of EtAE that prevented visible growth of microorganisms, were measured.

Antioxidant activity by DPPH scavenging method:

The free radical scavenging activity was determined by the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay¹⁵. 100 µL of each concentration of EtAE (100- 1000µg/mL) was mixed with 100µL of 0.2mM DPPH in ethanol and incubated in the dark at room temperature for 1h. The absorbance of the mixture was determined at 517nm using a MULTISCAN GO plate reader (Thermo Scientific, USA). The ascorbic acid was maintained as a positive control. The scavenging ability was calculated by the formula:

SA_DPPH(%) = {(Absorbance of control-Absorbance of sample)/(Absorbance of control)} x 100

H₂O₂ scavenging activity:

In summary, a 43 mM solution of hydrogen peroxide was prepared in a 0.2 M phosphate buffer with a pH of 7.4.Subsequently, 0.6 mL of the H₂O₂ solution was mixed with 4 mL of ethyl acetate extract from bacterial isolates, which were tested at various doses ranging from 100 to 1000 µg/mL, or with standard ascorbic acid. The reaction mixture was then incubated in the dark for 10 minutes, and the absorbance was measured at 230 nm using a UV-visible spectrometer and computed the percentage of H₂O₂scavenging activity¹⁶.

Anti-proliferative assay on human ovarian cancer cell lines:

Cell culture:

The NCCS, Pune provided the ovarian cancer cell lines PA-1 and SKOV-3, which were purchased and grown in Dulbecco's Modified Eagle Medium (DMEM) augmented with 10% fetal bovine serum and 2.5mL of an antibiotic and antimycotic solution (Product code: A002, Himedia) at 37°C in a humid environment in a 5% CO₂ incubator (NuAire, USA).

MTT assay:

Using a yeast model, the EtAE from all five isolates were screened for cytotoxic potential using MTT assay¹⁷. The isolate that displayed the highest level of cytotoxicity was subjected to a test against ovarian cancer cell lines. Briefly, SKOV-3 and PA-1 cells were cultivated in 96-well tissue culture plates and exposed to EtAE at varying concentrations (6.25 to 1800µg/mL) for 24 and 48h, respectively. Metabolically active cells converted the supplied MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) into insoluble purple formazan dye crystals. The absorbance of the crystals was measured using a microplate spectrophotometer (Multiskan Go, Thermo Fisher Scientific) at 570nm after they had been solubilized.

High-Resolution Liquid Chromatograph Mass Spectrometer (HR-LCMS) analysis:

The EtAE of bacterial isolates that exhibited high pharmacological activities were characterized by HR-LCMS¹⁸ (Agilent Technologies, USA). The separation of EtAE of the bacterial isolate was performed using UHPLC equipped with Hypersil GOLD C18 column (100 mm in length x 2.1mm diameter x 3 µm particle size). The gradient solvent system used for elution contained 0.1% formic acid in channel-A and acetonitrile in channel-B, with a high-pressure limit of 1200 bar and a rate of flow of 0.3mL/min. The gradient elution started with 95% A and 5% B, reaching 5% A & 95% B within 30 min, which was maintained at 40°C. The metabolites eluted from the column were ionised with a dual ajs esi ionisation source. MS scan was commenced at 100 mass and ended at 1100 mass, with a scan rate of 1 spectra/second under +ESI source and MS-MS collision energy was 2.0. The following MS source parameters were used: capillary voltage of 3500 V, gas temperature of 250°C, drying gas flow of 13 L/min, sheath gas temperature of 300°C, sheath gas flow of 11, the nebulizing gas pressure of 35 psi, fragmentor of 175V, the skimmer of 65V, octopole RF peak voltage of 750V, and mass range of m/z 50-1000. There was a 40,000 FWHM resolution. Agilent Mass Profiler Professional (MPP) software is used for metabolite identification. Open chemistry database search engines, such as Pub-Chem, NIST Chemistry, and Chem-spider, were utilised to evaluate the biological activity of identified compounds.

Statistical Analysis:

The results are reported as the standard error of the mean (SEM). Using the IBM SPSS Statistics 23 software suite, one-way ANOVA and Tukey's honestly significant difference (HSD) test were used to analyse statistical differences between the data, p<0.05 was deemed statistically significant.

RESULT:

Isolation and identification of the bacterial isolates:

Five distinct bacterial strains were isolated from the homogenate of the ovaries of mice under aseptic conditions. This was accomplished by culturing under aerobic and anaerobic conditions and making use of a range of different culture media, including Luria-Bertani agar, nutrient agar, blood agar, and Bifidobacterium agar. These strains were designated OM01 through OM05. Among the two mice used in the study, the samples taken from the ovaries of mouse-A resulted in the isolation of three different bacterial strains, such as OM01, OM02, and OM03. When ovarian tissue homogenates from mouse-A were spread-plated on different culture media, only 56.25% of those plates yielded bacterial isolates under aerobic conditions. On each of the four different kinds of media that were employed, the strains OM01 and OM02 were shown to grow, although the rate of growth was most rapid on the nutrient agar plates. The samples that were collected from the ovaries of mouse -B revealed the existence of a total of three different bacterial isolates, two of which were given the designations OM04 and OM05. However, the isolate that was found to be present in common in both of the mouse ovaries was OM03.

Each isolate was further identified based on the colony and microscopic characteristics, as well as biochemical characteristics. All the isolated strains were firmicutes, Gram-positive bacteria, according to microscopic inspection. SEM micrographs validated the rod shape of the isolates and confirmed that they were bacillus strains. Furthermore, 16S rRNA sequencing was used for the identification of the isolates. The 16S rRNA sequences of all five bacteria were edited using the BioEdit software 7.2.5, and the consensus sequences were subjected to BLASTn analysis. The BLAST analysis results suggest that OM01, OM02, and OM04 are 100% identical to Bacillus aerius, Bacillus cereus, and Bacillus paramycoides, respectively, with an E-value of 0.0. OM03 exhibited 97.28% similarity to Bacillus velezensis while OM05 showed 99.93% similarity to Bacillus subtilis, both having an E-value of 0.0. The phylogenetic tree was constructed in MEGA X and the evolutionary history was inferred using the UPGMA method (Figure.1). According to the findings, the isolate OM03 and two Bacillus velezensis bacterial strains belong to the same clade, implying that the OM03 bacterium was Bacillus velezensis. Also, OM01, OM02, OM04, and OM05 created clusters with the Bacillus aerius strain, Bacillus cereus, Bacillus paramycoides, and Bacillus subtilis strain correspondingly. The 16S rRNA sequences of OM01, OM02, OM04, and OM05 were deposited in GenBank under the accession numbers MW888451, MW888398, MW882947, OM746763, and OM736144, respectively.

Table 1. Antimicrobial activity and MIC of EtAE of isolated strains on test bacteria.

The values reported are mean ± SEM (n = 3), NZ = No Zone of inhibition, and TE = Tetracycline 30 mcg (Himedia).

Test Organism	Inhibition zone (diameter, mm) against tested bacteria						MIC(µg/ml) of EtAC-OM03
Test Organism	OM01	OM02	OM03	OM04	OM05	TE	MIC(µg/ml) of EtAC-OM03
Gram-negative bacteria
E. coli	7.7 ± 0.3	NZ	18 ± 0.6	NZ	NZ	29 ± 0.6	200
Klebsiella	NZ	NZ	20.3 ± 0.3	NZ	NZ	25 ± 0.6	150
Pseudomonas aeruginosa	NZ	7.7 ± 0.3	24 ± 0.6	NZ	NZ	27.7 ± 0.9	100
Gram-positive bacteria
S. aureus	NZ	9.3 ± 0.3	25.3 ± 0.3	8 ± 0.6	8.3 ± 0.3	29.3 ± 0.9	50
Bacillus cereus	NZ	NZ	22 ± 1.2	10.7 ± 0.9	NZ	23 ± 1.2	100

Figure 2. Antioxidant activity of EtAE-OM03.

(A) H₂O₂ scavenged activity of EtAE-OM03 at different concentrations.

(B) DPPH radicals scavenging activity of EtAE-OM03.

Results are expressed ± SEM (n = 4). Different letter labels denote significant differences (p<0.001) while means with the same letters are not significantly different, according to the two-way ANOVA and Tukey’s HSD. Uppercase letters compare the concentration of ascorbic acid x percentage of scavenging; lowercase letters compare the concentration of EtAE-OM03 x percentage of scavenging.

DISCUSSION:

In the present study, a total of five novel bacterial strains were isolated from the ovary of eight-week-old mice. The isolates were identified as Bacillus species namely, Bacillus aerius (OM01), Bacillus cereus (OM02), Bacillus velezensis (OM03), Bacillus paramycoides (OM04), and Bacillus subtilis (OM05) using 16S rRNA sequencing. Previous studies have isolated various Bacillus species from animal sources, including Bacillus aerius strain from the intestine of healthy catfish¹⁹, probiotic Bacillus paramycoides from the gastrointestinal tract of Nypha worm²⁰, Bacillus subtilis from a housefly’s body²¹, B. velezensis from the intestinal tract of healthy mandarin fish²². However, this is the first study of its kind to report isolating bacillus species from mouse ovaries. The EtAE-OM03 has exhibited a broad spectrum of inhibition against the different bacterial species investigated. In another study, the EtAC from the endophytic Bacillus velezensis strain EB 39 exhibited MIC values of 46.8 to 93.7μg/mL against Streptomycin-resistant Xanthomonas citri²³. The antibacterial property could be attributed to the ability of the strain OM03 to produce diverse types of antimicrobial compounds such as myriocin, benzocaine, borrerine, and dihydrodeoxy streptomycin, which was later identified by LCMS analysis from the ethyl acetate extracts.

The antioxidant activity was examined, and EtAE-OM03 was shown to have strong antioxidant characteristics, which could be valuable for further research development. The EtAE-OM03 showed a substantial dose-dependent suppression of DPPH activity and H₂O₂ scavenging. The ability of biomolecules present in the extract of OM03 to donate electrons or hydrogen to inhibit the DPPH chain reaction is responsible for its potent antioxidant properties. Additionally, numerous earlier investigations have reported the antioxidant capacity of Bacillus velezensis²⁴. Previous in vitro research demonstrated that these bacillus isolates have probiotic properties²⁵. Interestingly, our HR-LCMS results revealed the presence of many antioxidant chemicals in EtAE-OM03, including melongoside K, pinacidil, 3-hydroxyneogrifolin, 1,2,3,4-tetrahydro-2-methyl-b-carboline, 3-isopropyl-2-methoxy-5-methylpyrazine, and 2,3-diethylpyrazine. The combinatorial activity of various secondary metabolites and complexity in signalling pathways leading to apoptosis are thought to be the causes of the cell line-specific cytotoxicity observed in the current investigation. The EtAE-OM03 exhibited cytotoxicity against the ovarian cancer cell lines SKOV and PA-1. Similarly, in another study metabolites from B. velezensis strain, RP137 had shown a cytotoxic effect against the A549 and HepG2 cells with IC₅₀ values of 171.6 and 167.2 µg/mL, respectively²⁶. The cytotoxic action of the EtAE-OM03 is further supported by the HR-LCMS findings, which show the presence of many cytotoxic, anticancer, and antiangiogenic substances such as rilmenidine, 2,3,-dimethylpyrazine, 2-(3-, 5-, or 6-methoxy)-isopropylpyrazine, 3-isopropyl-2-methoxy Lanceotoxin B, chinenoside II, curacin A, cyclo(L-Phe-L-Pro), ammothamnine, camelledionol, butenafine (17alpha, 23S), myriocin, ceanothic acid, epoxy-28,29-dihydroxy-27-norlanost-8-ene-3,24-dione, and benzocaine.

CONCLUSION:

The study showcased the relevance of commensal ovarian microbiota for its potential pharmacological properties. The HR-LCMS analysis of EtAE of Bacillus velezensis OM03 showed its ability to produce diverse types of antimicrobial, antioxidant, and anticancer compounds such as myriocin, benzocaine, borrerine, and dihydrodeoxy streptomycin. Considering the strain OM03's ability to produce varied classes of compounds, it would be a potential candidate for industrial applications and therapeutic purposes. Further studies will give more insight into the clandestine applications of ovarian microbiota.

CONFLICT OF INTEREST:

The study's authors affirm that there were no financial or commercial relationships that might be viewed as having a potential conflict of interest.

ACKNOWLEDGMENTS:

The Mangalore Jesuit Educational Society, St. Aloysius College (Autonomous), Mangalore has provided funding for this research project. The authors wish to express their gratitude to SAIF, IIT Bombay for the HRLC-MS analysis.

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Received on 20.08.2022 Modified on 17.11.2022

Research J. Pharm. and Tech 2023; 16(10):4847-4854.

DOI: 10.52711/0974-360X.2023.00786