The Antibacterial Efficacy of a Compound Extracted from Marine Sediment Bacterium Enterococcus Lactis (S-2): A Comparative Analysis Through In-Vitro and In-Silico Assessments.

BACKGROUND: Marine sediment bacteria have been generating considerable attention lately due to their potential as valuable reservoirs of novel antimicrobial agents. AIM: In vitro and in silico antibacterial activities of antibacterial compounds isolated from the marine sediment bacterium Enterococcus Lactis (S-2). METHODS: Coastal sediment samples were collected from Rameswaram, Ramnathapuram District, Tamil Nadu, India. Bacteria were isolated using the crowded plate method, and their phenotypic and genotypic characteristics were studied. Purified bacteria were cultured in large volumes, secondary metabolites were extracted, and novel antibacterial agents were isolated from the aqueous extract. Novel compound antibacterial activity was studied through in-silico and invitro. The mechanism activity of antibacterial activity was confirmed by a high-resolution transmission electron microscope. RESULTS: Genotypic analysis confirmed that the isolated S-2 bacteria were Enterococcus lactis, and the aqueous extract showed antibacterial activity against Staphylococcus aureus (17 mm zone of inhibition) and Proteus mirabilis (12 mm zone of inhibition). A bioactive molecule, 13- hydroxy-9-(1-hydroxyethyl)-11-methoxy-2,4dioxapentacyclo[10.7.1.0³,4.05,²¹.0¹³,¹6]icosa- 1(20),5,7,12,14(19), 16-hexane-18-one, was isolated from aqueous extracts of the S-2 bacterium. Chromatography and spectroscopic analysis confirmed the identity of the isolated compound. Novel compound potential antibacterial activity showing against S. aureus (18 mm zone of inhibition) and MIC 250 µg/mL, which was confirmed by tetrazolium staining. The antibacterial activity mechanism was confirmed by transmission electron microscopy. Molecular docking studies show good binding (-9.9 kcal/mol) of the compound with 3U2D, while molecular dynamic simulation studies confirm the conformationally stable structure of the complex between 3U2D and 13-hydroxy-9-(1-hydroxyethyl)-11-methoxy-2,4-dioxapentacyclo [10.7.1.0³,4.05,²¹.0¹³,¹6]icosa-1(20),5,7,12,14(19), 16-hexane-18-one. It has been observed from the docking study of 3U2D with standard drug ciprofloxacin that the lower affinity is compared to the test ligand, which has a docking score of 7.3 kcal/mol. Out of interacting residues of protein 3U2D residue, Thr173 and Ile86 formed conventional hydrogen bonds. CONCLUSION: Marine bacterium E. lactis produces a novel antibacterial compound (13-hydroxy- 9-(1-hydroxyethyl)-11-methoxy-2,4-dioxapentacyclo[10.7.1.0³,4.05,²¹.0¹³,¹6]icosa- 1(20),5,7,12,14(19),16-hexane-18-one), which shows antibacterial activity against clinical S. aureus, confirmed by in vitro and in silico analysis. This molecule can used as a lead molecule for antibacterial activity.

Isolation and Identification of Bioactive Compounds with Antimicrobial Activity from Marine Facultative Anaerobe, Bacillus subtilis.

INTRODUCTION: The marine ecosystem contains many microbial species that produce unique, biologically active secondary metabolites with complex chemical structures. We aimed to isolate and identify bioactive compounds with antimicrobial properties produced by a facultative anaerobic strain of Bacillus subtilis (AU-RM-1), isolated from marine sediment. METHODOLOGY: We optimized the AU-RM-1 growth conditions, analyzed its growth kinetics and its phenotypic and genotypic characteristics. Extracts of the isolate were studied for antimicrobial activity against three clinically important microorganisms and the structure of the active compound was identified by spectroscopy. RESULTS: Antimicrobial activity of the AU-RM-1 DMSO extract was evaluated by disc diffusion assay and by serial dilution. The AU-RM-1 DMSO extract showed antimicrobial activity against Candida albicans, Escherichia coli, and Klebsiella pneumoniae. The bioactive fraction of the AURM- 1 DMSO extract was separated by TLC-bioautography at Rf = 0.49. We then used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to study the morphological changes in the bacterial cells treated with the isolated compound. It was observed that cells seemed to shrink, and the cell walls appeared to be damaged. A bioactive compound was identified, and its structure was examined by spectroscopic analysis: a LC-MS molecular ion peak (ESI) m/z (% of relative abundance) was calculated for C19H22O3: 298.38, and found to be C19H22O3 +1: 299.51 [M+1]. The chemical structure of the compound (2-(2-{8-methoxy-5aH,6H,7H,8H,9H, 9aH-naphtho[2,1-b]furan-7-yl}ethyl)furan) was determined using 1HNMR and 13CNMR, and its purity was confirmed by HPLC. Fifteen known and previously reported compounds were also identified, in addition to the novel compound; these were lipopeptides, antibiotics and chemical moieties. CONCLUSION: The facultative anaerobic marine organism Bacillus subtilis (AU-RM-1) produces a novel bioactive secondary metabolite with antimicrobial and antifungal activity.

Resistance of pearlspot larvae, Etroplus suratensis, to redspotted grouper nervous necrosis virus by immersion challenge.

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty-day-old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post-infection, and the infected larvae were subjected to nested RT-PCR and histology. The virus was isolated from infected larvae using SSN-1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT-PCR and the virus was isolated using SSN-1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT-PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.

Silencing and augmentation of IAG hormone transcripts in adult Macrobrachiumrosenbergii males affects morphotype transformation.

Morphotypic differentiation is the external manifestation of dominance hierarchy in Macrobrachium rosenbergii The intermediate morphotype orange claw (OC) male exhibits the highest growth rate and is subordinate in hierarchy to blue claw (BC) male while dominant on small male (SM). The present study was undertaken to examine the specific role of insulin-like androgenic gland (iag) hormone in morphotype differentiation of M. rosenbergii To achieve this, RNAi mediated knockdown as well as augmentation of iag transcripts were effected in ∼60 g OC males using plasmid-based constructs pcD-IAG-lh and pcD-IAGorf, respectively. The treatments were administered to animals maintained in isolation as well as in community. The knockdown plasmid construct that expresses iag-specific long hairpin RNA caused 16-fold reduction of iag transcripts in the SSN1 cell line in vitro When injected into OC males living in a community, 2.3-fold iag knockdown was recorded, while in isolated OC males it was 4.2-fold initially, but returned to normal subsequently. Compared with the respective controls, OC to BC transformations in the iag silenced animals were significantly lower in the community-reared group, while no difference was observed in the isolated animals. It is reported here for the first time that iag augmentation in OC males resulted in significantly higher OC to BC transformations, when animals were reared in community. This plasmid-based IAG knockdown approach could be developed into a low stress, feed or immersion treatment for controlling heterogeneous individual growth of M. rosenbergii males in aquaculture.

Protection of shrimp Penaeus monodon from WSSV infection using antisense constructs.

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3-VP24, pPn-VP24), VP28 (pH3-VP28, pPn-VP28), TS (pH3-TS, pPn-TS), and RR2 (pH3-RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.