Antibacterial and cytotoxicity activities of bioactive compounds from Micrococcus species OUS9 isolated from sea water

1 Introduction

Marine bacteria are documented as an invaluable resource in delivering bioactive compounds and the marine natural products plays a vital role in biomedical research and pharmaceutical industry (Karbalaei-Heidari et al., 2020). For thousands of years, nature was the origin of medicinal products. There are an imposing number of contemporary drugs, primarily based on traditional medicine use, isolate from microorganisms. Microorganisms have played a growing part in antibiotic manufacturing and in another drug manufacturing in the previous century. As sources of precious bioactive metabolites, terrestrial fungi and bacteria have been extremely important for more than half a century. There has been a continuing search for fresh drug products from the ocean for over two centuries. Prior researchers have revealed as marine invertebrates are significant source for fresh biochemical compounds and well-known fact as established through the large number of compounds present in the clinical trials, proper and reliable supplies of these compounds have proved notoriously hard to acquire from the nature. Due to these issues, (Faulkner, 2000) studies have well-documented about the avenue centered on marine microorganisms for considerable attention. Therefore, the anticipated huge biodiversity of sea organisms could have been at first sight for one of reasons (Burch-Brown and Archer, 2017); though marine derived microorganisms were not taxonomically sounded. The groundwork studies showed the wealth of microbial diversity in the oceans of the world making the capable of frontiers for discovering fresh drugs (Blunt et al., 2004).

Marine derived bacteria are defined through marine water demands in particular with sodium for growth (Fenical, 1997). Among the cases of marine associated with fungi are without ion demands and mandatory marine species are lives only sea water (Jensen and Fenical, 2000). This issue is compounded in samples near shore/estuarine, where the large proportion of resident microbes were modified for variable marine levels of exposure. All microbes may be isolated from the environmental marine for the purpose of microbial drug discovery (Gerwick and Moore, 2012). Maximum of fresh complexes have recorded from marine microorganisms which have been acquired from various species, isolated either from land, sea or both. Coherent investigation for all the microbes has been isolated from extreme sea water environment for the purpose of microbial drug discovery. Based on different studies on species, the majority of new bioactive metabolites from sea associate microorganisms reports were derived from species which can be isolated. While, these optional species of marine constitute obvious sources of new metabolites, their environmental functions and adaptation levels for marine environment are not known (Garcia and Rosenberg, 2010). Survey carried on marine bacteria were separated from marine algae and invertebrates which showed as antimicrobial metabolites are produced in a large proportion (Burgess et al., 1999). The production of non-bacterial bioactive metabolites which inhibit the setting of potential competitors has also been shown to cause by epiphytic bacteria present in marine combines with rich algal surface and invertebrates (Petersen et al., 2020).

The novel cyclical decapeptide antibiotic Loloatin B, which prevent development by M−resistant S. aureus MRSA (Staphylococcus aureus methicillin resistant), and VRE (Vancomycin Enterococcus resistant) was made by species Bacillus in Papoa-Nova Guinea (Calfee, 2012). Thiomarinol, eminent antibiotics has discovered to produce marine bacterium Alteromonasrava. Marine microorganisms, including Bacillus loloatins, have been recorded as antibiotics. Ascomycin through ascomyceticus var. Streptomyces hygroscopicus, Rhamnose from Saccharopolyspora spinosa (Madduri et al., 2001), pyrones from Pseudomonas (Singh et al., 2003), Kanglemycin from Nocardia mediterranei var. kanglensis, Zorbamycin from Streptomyces flavovirdis. Therefore, current study aims to explore to isolate bioactive potential bacteria with seawater for an antibacterial activity.

2 Materials and methods

3 Results and discussion

The composition of the marine ecosystem and biogeochemistry of the oceans play a crucial role. The great variety of microbes in our waters continues therefore vital to be understood. In order to capture rare, less abundantly organisms, efforts are required to investigate regions containing the biggest biodiversity, such as aquatic settings (Salihoglu et al., 2013). The notable variety of marine micro-organisms is due to their initial development, speed of generations and micro-environmental heterogeneity (Staley, 2006). The ocean is heterogenous owing to its nutrient patches and microscale incline, which can help into various inches (Kassen and Rainey, 2004).

In the present investigation the total 29 different bacterial cultures were isolated from different locations of Nellore district regions the bacterial colonies present on agar plates with morphologically different pigment producing has been identified. The selected colonies were sub-cultured in individual testtubes and observed for morphological characterization shown in the (Table 1). The chosen colonies were screened by well diffusion for antagonistic activity. Process against two pathogenic bacteria out of 29 selected bacterial cultures OUS9 have shown efficient (Table 2) antagonistic activity, the results showed that the selected isolate have good growth and ability of high pigment producing capacity as shown in the (Fig. 1). In one of studies of two human-pathogenic bacteria have discovered that marine isolates are more efficacious than bacteria which live freely living bacteria (Nair and Simidu, 1987). Studies investigating the frequencies of marine bacterial isolates; antagonistic interactions discovered between 5 and 8 percent of isolates expressing activity level (Krasilnikova, 1961). Although these trials concentrated on marine isolates as prospective antibiotic producers, the target species were non-marine bacteria, often human pathogens. Similarly, more than our understanding of antibiotics producing and understanding of environmental antibiotic sensitivity is restricted. Kanamycin and penicillin antibiotics have diverse form of concentration for inhibition of marine bacteria.

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Fig. 1

Pigmentation of isolated bacteria from marine intertidal regions.

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Carlos et al (2013) has evaluated the 16S rDNA for the bacterial diversity for the similar samples of coral settings and for large abundance of proteobacteria. For different weavy softs, stony corals, sponges and sea water of this phylum was recorded earlier in an elevated abundance (Porporato et al., 2013). These strains were further reconfirming through ribotyping molecular technique using 16S rRNA gene sequence and this partial sequence of 16S rRNA gene was obtained with the blast which was documented in Micrococcus species OUS9 (Fig. 2) and further the sequence was deposited in GenBank (Accession number MN108086). Microorganisms living in the rhizospheres of diversity of crops are probable due to the abundance of substrate shown from the roots when compared with non-rhizosphere soils to synthesize and release auxin as secondary metabolites (Ahmad et al., 2005). Nair and Simidu (1987) studies have documented this antibacterial compound are not only inhibits terrestrial bacteria also, for ecological importance to native bacterial strains. Yellow pigment extracted from isolated bacteria for high content of pigments was investigated. High contents of pigment were obtained from methanol solvent when compared with ethanol, hexane and petroleum benzene solvents.

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Fig. 2

Phylogenetic tree of Micrococcus OUS9 bacterial isolate from marine intertidal regions.

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Many new species are acknowledged as promising source for fresh bio-active compounds in taxonomic terms (Huang et al., 2012). Wealthy cause for secondary metabolism is with the novel structures at outstanding biological activities has been found in marine bacteria extracted from deep sea sediments. A strain AB 18–032 termed as actinomycete Verrucosispora have been extracted from the sediment sample obtained at 289 m of depth from Japanese Sea (Bister et al., 2004). Abyssomicin C (8) has shown methicillin-resistant antibiotic activity and S Aureus (MRSA) and S-resistant to vancomycin had 4 and 13 µg/mL of MIC values. A representative class-I spirotetron is regarded as Abyssomicin C. This biosynthesis is initially recognized and obtained over bacterial source which inhibits para aminobenzoic acid (PABA) biosynthesis. Abyssomicin C and its atropoisomeric inhibits PABA respectively. Further, discovering the marine environment is to be promising strategy for active compounds and for development of resistant therapies for many microorganisms, which indicates the requirement of new drugs are required. Precisely, for anti-fungal, anti-bacterial, anti-viral and anti-protozoal activities.

The present study was carried out with crude pigments were verified for antimicrobial activity with pathogenic bacteria has also been observed that out of Chloroform, Ethanol, Methanol and Acetone extracts, with standard drug reference and Methanol crude pigment showed zone of inhibition greater against Bacillus subtilis 15 mm, followed by S. aureus with 12 mm, Salmonella 11 mm, Escherichia coli 09 mm, P. aeruginosa 06 mm and lowest zone was observed to K. pneumoniae 02 mm (Fig. 3, Table 3). This confirms as crude Yellow pigment of Micrococcus species isolates were well and active against both gram positive and negative bacteria. For their natural product contents such as algae, ascidians, corals, microorganisms and sponges which is known to be total forms of marines were investigated (Arif et al., 2004). Throughout thousands of years, ecological pressures like space competition, symbiosis, predation and tide variations have led to the biosynthesis of complicated of secondary metabolite species, which in turn enable them for adopting the competitive and hostile environment (Da Silva et al., 2006). Different marine organisms such as algae and invertebrates in sea surface or cavum has been found to be more nutritive than inanimate and marine water and may have large number of bacteria among them (Bonugli-Santos et al., 2015). These few species of bacteria are usually non-symbiotic of host which can be allied bacteria (Bultel-Ponce et al., 1999) and are compelled to create antibiotic resistance secreted by their host.

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Fig. 3

Antibacterial activity of Methanol crude extracts from Micrococcus species OUS9.

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In the present investigational study, carried out the free radical scavenging activity of Micrococcus species OUS9 extract with concentration range from 25, 75 to 100 mg/ml and ascorbic acid standard used as a standard. The result of DPPH, ABTS and NO scavenging activity indicates Micrococcus species OUS9 extract are potentially active. As shown clearly in the Table 4 that the extract shows 48.44 ± 6.2% inhibition as compared to standard 56.21 ± 4.9 in DPPH activity followed by 44.14 ± 2.9 in ABTS and 42.13 ± 3.4% inhibition in NO with 100 (mg/ml) (Table 5). With promising antioxidant activity, the inhibition effect increased in higher levels. It has a maximum of 176 of 95 per cent. Species of reactive oxygen plays important role to protect human body from damage (Govindarajan et al., 2005). In addition, past epidemiological surveys have shown a reduction in consumption of natural antioxidants in cancer and other oxidatively damaged illnesses (Rietveld and Wiseman, 2003).

Extraction of crude through solvent extraction is a significant phenomenon for opting the appropriate solvent for extracting high yields of bioactive compounds. Previous studies have confirmed the strong antimicrobial potential for ethyl acetate extraction against the pathogens of fungi and bacteria. Here from the eluted fractions with column chromatography is tested for antimicrobial study and the best fraction was further identified. KLUF-10 and KLUF-13 with molecular weight 568.9 and KLUF-13 the molecular weight with 263.38. On the basis of these observations the purified compound is proposed to be KLUF-10 as 3-Hydroxy-β, ε-caroten-3′-one, KLUF-13 1-(1-(4-methoxphyenyl)-2-(methyl amino) ethyl) cyclohexan-1-ol and which was isolated from Micrococcus species OUS9 for the first time and is to appear that novel compound with good antimicrobial activity as shown in the Figs. 4 and 5 supplementary table 1). The most promising results for anticancer activity were obtained with KLUF-10 and KLUF-13 fractions of the Micrococcus sp. OUS9 strain. The inhibitory effect of pure compounds treated cells was observed after 24 h of incubation. The results of cytotoxicity study performed by MTT assay suggest that the given test compound, KLUF-10 and KLUF-13 are anticancer in nature against the both HeLa and MCF-7 Cell lines and the IC50 concentrations are 34.5ug/ml and 34.95ug/ml respectively IC50 concentrations are HeLa 28.9 ug/ml and MCF-7 26.6ug/ml (Supplementary Table 2; Supplementary Figs. 1-2). In multiple microbial metabolites, the role of pigment is to act as coloring agent in food processing and cosmetic industries and also own anti-oxidant, anti-inflammatory, anti-cancer, and anti-microbial activities. The current results of this study suggest that the coastal region's ability as an origin of marine bacteria to generate antibacterial compounds which can and must be explored for many pharmaceutical applications.

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Fig. 4

Antibacterial activity (MIC) of pure compound (13th Yellow fraction) isolated from Micrococcus species OUS9.

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Fig. 5

Antibacterial activity (MIC) of pure compound (10th Yellow fraction) isolated from Micrococcus species OUS9.

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4 Conclusion

In the marine environment, bioactive compounds that produce microorganisms are widely distributed. Bioactive metabolites from marine bacteria which acts as an anchor for survival between pathogenic bacteria and their environment. This research is a step towards understanding and testing bioactive metabolites generating marine bacteria in the region around the shore of Nellore, Andhra Pradesh, India. Screening of these bacteria by screening techniques for the manufacturing of bioactive metabolites. The strain of Micrococcus species OUS9 demonstrated potential. To characterize such activity and recognize the promising isolates, further research is required. This new natural biological product is also used in various sectors such as the pharmaceutical industry, the food industry and the preservation of the natural environment