Screening antibacterial activity ODS fractions of marine sponges against non-pathogenic bacteria tuberculosis Mycobacterium smegmatis

Nine unidentified marine sponges from Bunaken Island waters in Manado Indonesia were observed on antituberculosis (TB) against non-pathogenic Mycobacterium smegmatis NBRC 3207 and it was found that the ethanol crude extracts and ODS fractions of these sponges inhibited the growth of bacteria M. smegmatis. Sponge SP.323 is the most inhibiting and showed Intermediate (I) activity with inhibition zones of 9, 11, 11, 8 mm/disk at 10μg/disk., for fraction 3,4,5,6 respectively, and need further experimental analysis to justify the antimicrobial activity, NMR analysis and structure elucidation.


INTRODUCTION
Tuberculosis (TB) disease is one of the top 10 causes of death and the leading cause from a single infectious agent (above HIV/AIDS). Tuberculosis (TB) caused by Mycobacterium tuberculosis is still one of the main infectious diseases in the the world, including with the human immunodeficiency virus (HIV) and malaria (WHO, 2020). According to World Health Organization (WHO), around 10 million people continue to fall sick with TB each year with the leading cause of M. tuberculosis. In 2019, TB caused an estimated 1.2 million deaths (range, 1.1-1.3 million) among HIV-negative people and there were an additional 208.000 deaths from TB (range, 177.000-242.000) among HIV-positive people (WHO, 2020). Because of lack of treatment or lack of adapting treatment, many researchers are interested to find antituberculotic drugs. Urgent action is required to improve the coverage and quality of diagnosis, treatment and care for people with drug-resistant TB.
Marine organisms such as invertebrate are a much sought-after sources of drugs candidate. Sponges are exclusively aquatic animals that dominate in many benthic habitats, are rich source of biologically active natural products with unique structures (Caroll et al., 2020). Sponges are thought to be used for pharmaceutical and biotechnological industries and have shown the highest potential for natural product discovery (Mehbub et al., 2014). A number of metabolites have been isolated and they were found to have various biological activities, such as antibacterial, antifungal, anti-HIV, antiinflammatory, anticancer, and as enzyme inhibitory activities (Mehbub et al., 2014). Some sponge derived natural and synthetic compounds have been approved for clinical use (Newman and Cragg, 2014;, such as Eribulin (an anticancer agent). Therefore, marine metabolites might be developed as a new recommended source of promising drugs.
For several years our research has focused on antimicrobials with purpose to find new antimycobacterial substances from marine sponges and microorganisms collected in North Sulawesi, Indonesia. We found microbe M. smegmatis which has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis.
Mycobacterium smegmatis is used as an alternative microorganism to detect antibacterial activity against tuberculous bacteria. During our group research on sponges, we found antimycobacterial activity of a bisfunctionalized sphingolipid from marine sponge Agelas sp. collected in Manado , that was the first study to show that Agelas sp. inhibited the growth of M. smegmatis. Three new dimeric 3-alkyl pyridinium alkaloids, called haliclocyclamines A-C, were found from Indonesian marine sponge Haliclona sp. and showed inhibited antimicrobial activities against M. smegmatis (Maarisit et al., 2017). On the screening of antimicrobial, it was found that the ethanol extract ODS fractions of an Indonesian marine sponge Auletta sp. inhibited the growth of nonpathogenic M. smegmatis (Sumilat, 2019).
In the course to continue studies on anti-TB metabolites from Indonesian marine sponges we have tested nine unidentified species of marine sponges against M. smegmatis NBRC 3207 and found that the crude extracts and ODS fractions of these nine sponges exhibited prominent activity.

Sample Collection and Extraction
Marine sponges were collected by scuba diving at Bunaken Island in Manado Indonesia, in 2014. The nine-voucher specimens were deposited at the Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University.
Marine sponge was cut into small parts and put in a bottle containing ethanol 500 ml, kept at room temperature for 1x24 h. The ethanol extracted was filtered and subjected to the rotary evaporator for drying to obtain the final weight (Ebada et al., 2008).

Microbes
The strain of M. smegmatis NBRC 3207 was obtained from the Biological Resource Center (NBRC), NITE (Chiba, Japan) and was maintained in 20% glycerol a t -80 °C.
ODS column chromatography was performed to separation in 7 fractions and screening to examine the inhibitory activities on the growth of M. smegmatis. The concentration of samples was arranged from 10, 20, 30 ug/disk. M. smegmatis was cultured in Middlebrook 7H9 broth at 37 °C for 6−12 days and adjusted to 1.0 ×106 CFU/mL. The disk was placed on an agar plate and incubated for 2 days at 37 °C. Streptomycin sulfate and CH3OH were used as positive and negative controls, respectively. A zone of inhibition test in antibacterial assay was carried out using M. smegmatis NBRC 3207 by the paper disk method (CLSI, 2013). The interpretive diffusion disk consists of Susceptible (S) ≥ 20 mm, Intermediate (I) 15-19 mm, Resistant (R) ≤ 14 mm, where the sample concentration is 30 µg (CLSI, 2013).

RESULTS AND DISCUSSION
In this research, we found nine marine sponges collected by scuba diving at Bunaken Island in Manado Indonesia, in 2014. We presented our sample in code number due to their molecular identification is still in progress. The dried crude extract was put into a glass vials, weighed and tested as a crude extract ethanol for screening the antimicrobial activity (Table 1).

Screening Bioassays Antimicrobial
Antimicrobial screening of EtOH crude The ODS fraction for all specimens were screened against M. smegmatis (based on their activities in ethanol crude extracted) and the result shown in Table 3  The results showed that SP.323 revealed the highest inhibiting activity against the growth of M. smegmatis. It is therefore have a high prospect to continue for the next other purification and NMR analysis.
Based on interpretive diffusion disk (CLSI, 2013), which shown in Table 3 and Figure 1, it can be concluded that marine sponges SP.205, SP.206 and SP.356 were categorized as Resistant (R) with diameter of the inhibition zone range ≤ 14 mm, while SP.323 showed Intermediate (I) 15-19 mm on fractions 3-6 at sample concentration 10 µg/disk. Sample concentration 20 and 30 µg of SP.323 were categorized Susceptible (S) ≥ 20 mm. Maarisit et al. (2017) obtained haliclocyclamines A-C and cyclostellettamines A-C, E-F from Haliclona sp. collected in Manado and found that these compounds have antimicrobial activity against    Abdjul et al. (2017) and obtained two compunds 5bromophakelline and leucettamol A, leucettamol A exhibited moderate anti mycobacterial activity, with inhibition zones of 7, 9, 12 mm/disk at 10, 20, 50/µg/disk. Similar to the results observed by Abdjul et al. (2016), for antimycobacterial activities of Okinawan sponge Halichondria panicea against M. smegmatis, the results showed inhibition zones range from 7-16 mm at 10/µg/disk. The difference in the inhibition of each extract against bacteria can be influenced by the structure and composition of bacteria cell, the density/concentration of bacterial cells and the composition of the active compound contained in the extract. The standard concentration according to CLSI (2013) is 30 µg per disk, and when compared in this study, which the concentrations range between 10, 20, 30 µg, it is necessary to do more research to produce accurate data with the aim of obtaining standard categories Resistant (R), Intermediate (I) and Susceptible (S).
In general, the best concentration required for antimicrobial activity is the lowest concentration while A larger zone of inhibition usually means that the antimicrobial is more potent.
Based on the interpretive diffusion disk, marine sponges SP.323 as an inhibitor and more potent to inhibit the growth of non-pathogen bacteria M. smegmatis. This sponge SP.323, might be recommended for detailed research for structure elucidation and other synthesis experiments, to develop as a new promising antibacterial drug.

CONCLUSION
Marine sponges collected in Bunaken Island in this research were found to have inhibiting activities against the growth of nonpathogenic M. smegmatis. Among nine marine sponges, it can be concluded that, marine sponges SP.323 can be used for further experimental analysis as an inhibitor. Its antimicrobial activity needs to be justified and purified with NMR analysis for structure elucidation.