The PML5 isolate showed poorly developed white airy mycelium and yellow vegetative mycelium, a relevant taxonomic criterion for the identification of Streptomyces (12, 13) (Fig. 2).
The development of aerial mycelium occurred after seven days of cultivation in ISP2 medium in a greenhouse at 30ºC, something common to actinobacteria, since its growth is slow and can take from 7 to 15 days for complete development at this temperature (14, 15).
Regarding the arrangement of the spores, the presence of flexuous filaments in branched chains, short closed filaments was identified, all these characteristics converging with the typical morphological profile of the genus Streptomyces, cited by Azuma (2012) (16).
The microscopic characteristics observed in the isolates showed organization and shape compatible with the genus Streptomyces, as described in the literature by Alam et.al. (2022). (17).
The biochemical and physiological characteristics of the isolate made it possible to identify the biochemical profile of the isolate with growth in lipase (3,58) and esterase (3,58), biocatalyst enzymes that are used biotechnologically in the synthesis of surfactants, textile, cosmetic and pharmaceutical industries, in addition to participating in the production of biofuels, acting without the need for cofactors, with high stability in organic solvents and wide specificity of substrates (18).
The PML5 isolate was positive for the enzyme Catalase, which in the study by Fang et.al. (2021) was described as a substance directly involved in the biosynthesis of secondary metabolites in 2,634 Streptomyces strains with catalytic properties capable of being exploited in different ways (19).
When exposed to nitrogen sources, PML5 was positive for L-histidine, serine, valine and L-asparigine. Each of the amino acids—L-histidine, serine, valine, and L-asparagine—plays key roles as sources of nitrogen in the metabolic processes of organisms. During the metabolism of L-histidine, the nitrogen present can be released and utilized in the synthesis of other nitrogen-containing molecules, such as nucleotides and neurotransmitters (20).
Similarly, Serine and L-asparagine, also containing amino groups, contribute as sources of nitrogen in metabolic pathways. When metabolized, these amino acids release nitrogen, which is then used in the synthesis of a variety of molecules vital for cellular function (21).
Valine can be incorporated into other nitrogen-containing molecules. These processes are essential for balancing the nitrogenous metabolism of organisms, ensuring adequate nitrogen availability for various biological functions, including protein and nucleic acid synthesis (20, 21).
The growth of PML5 in these nitrogen sources suggests that these microorganisms may be present in the environment where these compounds are found and may be involved in the production of bioactive compounds.
PML5 grew in media with carbon source D-xylose, Sucrose, Maltose, D-mannose, D-lactose and Glucose. D-xylose is a monosaccharide, commonly used in industry as a carbon source in microbiological culture media. On the other hand, sucrose is a disaccharide composed of glucose and fructose, widely found in food and used in the food industry and in microbiological culture media. Maltose, another disaccharide composed of two glucose molecules, is often employed as a carbon source in the food industry and microbiology (22).
D-mannose, a monosaccharide isomer of glucose, is commercially synthesized and used in dietary supplements due to its beneficial health properties. D-lactose, a disaccharide, is an important source of carbon and energy for infants and is used in the food industry in dairy production. Finally, glucose, a monosaccharide, is essential for the metabolism of organisms and is used in various industrial sectors, from food and beverages to biofuels and pharmaceutical production. These carbon sources play crucial roles in the nutrition and metabolism of various organisms and have significant applications in industry and scientific research (23).
The growth of PML5 strains in these media indicates the ability of actinobacteria to use these carbohydrates as a carbon source, which can influence the degradation and cycling of nutrients in the carbonate rock environment.
The Antibiotic Susceptibility Test showed that the PML5 strain is sensitive to Bacitracin (23mm) and Azithromycin (35mm) and resistant to Nalidixic Acid, Cefepime, Tetracycline, Cethazidine, Rhinoampiline, Oxacillin, Penicillin, Ketalhotin, Cephalitriaxone, Amphotericin B and Amoxicillin.
Resistance to drug classes may be related to the production of secondary metabolites of PML5 similar to the antibiotics tested. Quinolone, Penicillin, and 1st-, 3rd-, and 4th-generation cephalosporins are classes of broad-spectrum antibiotics with different modes of action. The simultaneous production of these classes may suggest that PML 5 has the ability to synthesize compounds that act on different bacterial targets and with different mechanisms of action. Resistance to rifamine and antifungal polyene may indicate the ability of the isolate to synthesize compounds with antituberculosis and antifungal properties, respectively(24).
The PML5 strain showed a positive response to PKSII and NRPS, indicating its potential for the production of polyquetides and also non-ribosomal peptides. The presence of PKSII and NRPS is reported by Paulus et.al. (2022) and Alonso et.al. (2021) as part of genome mining for identification of biosynthetic genes from natural products. The discovery of these clusters is necessary for the search for new bioactive compounds, in order to then create the necessary conditions for the clusters to produce them, for which the PML5 strains were provided with carbon and nitrogen sources necessary for their growth and metabolism (23, 25)
The PML5 isolate showed antagonistic activity against gram-positive and gram-negative pathogens, as shown in Chart 1
Table 1
Halos of pathogen growth inhibition against isolates
Pathogens | PML5 |
Streptococcus pneumoniae ATCC700699 | 12 ± 0,58 |
Staphylococcus aureus ATCC14458 | 12 ± 1,0 |
Staphylococcus epidermidis ATCC12228 | 12 ± 0,56 |
Micrococcus lutteus ATCC7468 | 12 ± 1,00 |
Escherichia coli ATCC25922 | 15 ± 0,00 |
Escherichia coli ATCC10536 | 13 ± 0,00 |
Klebsiella pneumoniae OXA48 | 12 ± 0,59 |
Acinetobacter baumannii ATCC19606 | 13 ± 1,15 |
Salmonella enteritidis INCQS00268 | 16 ± 0,00 |
Proteus vulgaris ATCC13315 | 13 ± 0,29 |
Aeromonas hydrophyla INCQS00318 | 10 ± 0,58 |
The antagonistic activity revealed a broad antimicrobial spectrum against Gram-positive and Gram-negative bactéria of medical interest in humans, with emphasis on Salmonella enteritidis INCQS00268, exhibiting na averafe inhibition halo of 16 ± 0,0 mm. The most resistant pathogen was Aeromonas hydrophyla INCQS00318with na inhbition halo of 10 ± 1,15 mm.
In the research by Bhat e Nayaka (2023), the cave isolate YC69 had a greater halo of inhibition against Staphylococcus aureus and Escherichia coli when compared to the other 69 isolates. Similarly, Jaroszewicz et.al. (2021) tested isolates from limestone rocks, where the one with the highest spectrum against Staphylococcus aureus, Salmonella enterica, Enterococcus sp., Escherichia coli, and Pseudomonas aeruginosa for anticâncer testing (26, 27).
The acetatethyl extract of the PML5 isolate showed greater cytotoxic action (96.5%) against the growth of human colorectal cancer cells (HCT-116). The cytotoxic action of extracts of PML11 and PML15 isolates against HCT-116 cells was 33.40% and 58.55%, respectively.
Researchers Bhat and Nayaka (2023) obtained a 41.98% inhibition of cell proliferation in vitro by subjecting the YC69 isolate against human cervical cancer cells (HeLa) to cytotoxic testing. For Jaroszewicz et.al. (2021), the isolates of Streptomyces sp. selected for cytotoxic testing reduced the viability of the breast cancer cell line (T47D). (26)
The use of the cytotoxic MTT assay facilitates the determination of the cytotoxicity of a sample, although it does not clarify its mechanism of action, the analysis of cytotoxicity represents a crucial stage in the process of creating new drugs for clinical use (28).
Researcher Huang et.al. (2023) tested the cytotoxic action of mangrove sediment isolate 4503, with morphological and chemical properties converging to the genus Streptomyces. The isolate showed cytotoxic activity against nasopharyngeal carcinoma (NPC) cell lines with antiproliferation, antimetastasis, induction of cell cycle arrest and apoptosis, in addition to increasing the production of reactive oxygen species, which led to the need to also explore the antioxidant potential of the isolate (29).
The antioxidant action detected by the ABTS method showed an action of 35.60 + 2.01%, while the antioxidant potential by the capture of the free radical DPPH had an inhibition of 10.65 + 1.95%. The antioxidant activity revealed by the ABTS and DPPH method suggests a significant capacity of actinobacteria isolated from carbonate rocks in the Amazon to neutralize free radicals, reflecting a promising biological potential for health and technology applications. This capacity is evidenced by the outstanding performance of the actinobacterium Streptomyces sp., isolated from the Caatinga, whose cultural filtrates demonstrated high antioxidant activity through the DPPH and ABTS methods, in addition to having high phenolic and flavonoid contents (30).
For Rammali et.al. (2022) a response percentage greater than 30% demonstrates the high capacity of secondary metabolites of actinobacteria to capture the ABTS free radical. For Weslati et.al. (2023) A DPPH eliminator response above 1.3 µg is highly active. By relating the research with the result of 35.60% of activity in the capture of ABTS and 138.56 µg of PML5, a greater potential of the carbonate rock isolate is evidenced (31).
In addition, previous studies highlight the importance of ABTS and DPPH methods as reliable tools for evaluating the antioxidant capacity of plant extracts and isolated compounds. Weslati, Simões, Texeira, Parpot et.al. (2023) detailed the ability of several antioxidants to react with the DPPH radical, providing a solid basis for future comparisons of antioxidant activity. These methods are widely recognized for their efficacy in quantifying antioxidant capacity, which is fundamental for studies such as the present one, which seeks to explore the potential of new sources of antioxidants (32).
The metabolic pathway of PML5 was developed to synthesize four different substances, as shown in Fig. 5, where well-defined peaks were identified.
In the mass spectrum, it was identified that the absorbance of substances 5,791 and 13,556 were similar to 9,921 and 17,077, respectively. This indicates that they have the same chromophores with conjugated double bonds, i.e., the same skeleton, suggesting the possibility that they are of the same class (Fig. 4).
GC-MS analysis of the extract showed strong antimicrobial and antitumor activity (Table 6). We identified 10 different volatile compounds from the active metabolite of the isolate (between 14.81min and 37.53 min) and were recognized for their antimicrobial and antitumor activities. Cyclododecane (83.75%) was the main compound identified in the extract.
Table 2
Identification of chemical compounds of the PML5 isolate using GC-MS analysis.
Time (min) | Area (%) | M.W (g/mol) | Molecular Formula | Compound Name | Bioactivity |
14.816 | 0.76 | 206.32 | C14H22O | 2,4-Di-tert-butylphenol | Antitumor activity (33) |
19.029 | 83.75 | 168,32 | C12H24 | Cyclododecane | Antimicrobial and antitumor activity (34) |
21.255 | 1.11 | 316.5 | C18H36O4 | Tetradecanoic acid, 12-methyl-, methyl ester, (S)- | Antioxidant and antitumor activity (35) |
23.144 | 2.76 | 270.5 | C17H34O2 | Pentadecanoic acid, 14-methyl-, methyl ester | Antioxidant and antitumor activity (36) |
23.551 | 1.32 | 196,2 | C10H16N2O2 | Cyclo(L-prolyl-L-valine) | Antimicrobial activity (37) |
23.902 | 2.96 | 210.27 | C11H18N2O2 | Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- | Antimicrobial activity (38) |
25.262 | 0.62 | 316.5 | C18H36O4 | Tetradecanoic acid, 12-methyl-, methyl ester, (S)- | Antioxidant and anti-rumoral activity (35) |
25.537 | 1.08 | 168.32 | C12H24 | Cyclododecane | Antimicrobial and antitumor activity (34) |
32.289 | 1.36 | 172.18 | C8H12O4 | 4-Octene, (Z) | Antimicrobial Activity (39) |
37.536 | 4.27 | 166.13 | C8H6O4 | 1,4-Benzenedicarboxylic acid | Antimicrobial Activity (40) |
The components determined by GC-MS showed antimicrobial, antioxidant and antitumor activities. 2,4-Di-tert-butylphenol has anticancer properties against Mycobacterium tuberculosis, antioxidant activity of 2,4-DTB, and response of 88% and 89% against colon cancer and uterine cancer, respectively, in Kaari's research; Joseph; Manikkam; Kalyanasundaram et al., (2023). The compound Cyclododecane, which appeared in two moments (19.02min and 25.53min) during chromatography. In the literature, it is described as a substance present in medications for bronchitis, hemomatirua, epilepsy, asthma, leprosy, eczema, debre and jaundice, in addition to having antitumor and antioxidant capacity.
The Tetradecanoic acid, 12-methyl-, methyl ester, (S)- also appeared twice during the analysis (21.25min and 25.26min) and its action has antioxidant and antitumor activity as well as the Pentadecanoic acid, 14-methyl-, methyl ester (KEMUNG; TAN; CHAN; SER et al., 2020). Other antimicrobial substances were Cyclo(L-prolyl-L-valine), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 4-Octene, (Z), 1,4-Benzenedicarboxylic acid, which have antimicrobial potential against gram-positive and negative bacteria, some even resistant to antimethicillin resistant Staphylococcus aureus (MRSA)(40).