Unexplored deep sea habitats in active volcanic Barren Island, Andaman and Nicobar Islands are sources of novel halophilic eubacteria
Authors: Meena Balakrishnan
Anburajan Lawrance
Kirubagaran Ramalingam
Dharani Gopal
Rajaguru Srinivasan
Rajaprabhu Gunasekaran
Vinithkumar Nambali Valsalan
Journal: Infection Genetics and Evolution (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Antibacterial activity; Barren Island; Deep Sea; Halophilic eubacteria; Osmoprotectant.
Affiliations: Andaman and Nicobar Centre for Ocean Science and Technology, Earth System Sciences Organization-National Institute of Ocean Technology (ESSO-NIOT), Port Blair 744103, Andaman and Nicobar Islands, India. Electronic address: ., Andaman and Nicobar Centre for Ocean Science and Technology, Earth System Sciences Organization-National Institute of Ocean Technology (ESSO-NIOT), Port Blair 744103, Andaman and Nicobar Islands, India., Marine Biotechnology Division, Ocean Science and Technology for Islands Group, ESSO-NIOT, Ministry of Earth Sciences, Government of India, Chennai 600100, Tamil Nadu, India.

Abstract:  Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman &amp; Nicobar Islands. A total of 24 halophilic eubacteria were isolated and identified based on their biochemical and 16S rDNA sequences. Three major classes (Gamma-Proteobacteria, Alpha-Proteobacteria and Bacilli) of bacteria were detected in the deep sea sediments of active volcanic Barren Island. Among those, 37% of isolates exhibited antimicrobial activity against all tested Gram positive and Gram negative clinical pathogens. 60% of isolates revealed the presence of either PKS or NRPS genes and 65% isolates disclosed medium to higher level of cytotoxicity in MB-231 breast cancer cell line. Majority of the isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance. 

Retracted: No

PubPeer comments:
 **I request authors to provide detailed clarification for the following queries to better understand the paper and appreciate its scientific output.** **Query 1:** For isolation of deep sea halophilic eubacteria the method followed was as per the reference Wei et al., 2013 (section 2.2). Going through this reference, one finds **only uncultured based genomic study**. Authors kindly explain how to use uncultured based information for isolating deep sea halophilic eubacteria? **Query 2:** For the isolation of **halophilic eubacteria simply** Zobell marine agar was used (section 2.2). This media is used for the enumeration of **heterotrophic marine bacteria**. Authors kindly provide the base for reporting the isolates as halophilic? ![file] ![file] **Query 3:** Concerns regarding the percentage values reported in abstract and result section. In many instances the **percentage values reported donu2019t match** with the percentage calculated using given numeric values. For example, **@ 1:** The author reports 60% of the isolates revealed the presence of either PKS or NRPS genes. If it is 14 bacteria out of 24, the percentage will be 58.0, and if it is 15 bacteria out of 24, it comes to 62.5%. What is the rationale behind reporting 60% value? **@ 2:** Similarly the reported values 47% NRPS genes, 10% PKS and NRPS genes, 65% cytotoxicity are not matching based on simple mathematical calculations as expressed above. **Query 4:** Authors reported just the percentage values of different activity but the important information that which of the bacterial species showed these properties is missing. Without names of bacterial species, citing just the percentage values do not provide any scientific information. Authors, kindly provide all the 24 bacterial species names, their NCBI accession numbers, to support claims and mention for each bacterium, the presence or absence of reported activities. **Query 5:** Paper title, abstractu2019s first line and the last sentence of section 2.1 point towards multiple sediment cores used in this study. Going through the article, Lat Long details of only one box core sediment sample collected using research vessel Sagar Manjusha could be found. Sampling plurality used in the title, abstract and in the body misleads. If more than one sediment box core samples were collected around the Barren Island, then authors please provide their Lat Long details? ![file] **Query 6:** Concerns on the method used to determine antibacterial activity (section 2.8). **@ 1** The authors used the solvent extract in **well diffusion assay**. **@ 2** The authors maintained the **negative controls** with **solvents in well diffusion assay**. Using this method, all the wells will get clear zones, whether negative or positive, due to solvent toxicity against the test microbes. Activity in such cases is determined by disc diffusion and not by well diffusion. Do authors kindly explain how the reported % value was obtained when the method itself is not valid? ![file] **Query 7:** Concerns related to the amplification of PKS and NRPS genes (section 2.9). **@ 1** For PKS and NRPS genes amplification the isolation of genomic DNA was performed using HiPurA kit. However, in section 2.3, the authors already reported genomic DNA isolation to amplify 16S rDNA. In both sections, different methods were reported to isolate the genomic DNA from the same group of bacteria. To amplify different sets of genes from the same bacteria, one does not need to isolate the genomic DNA again and again. The authors kindly justify re-isolation of genomic DNA. **@ 2** Amplification of PKS and NRPS genes were carried out using the primers described by Ayuso-Sacido and Genilloud (2005). The primers reported in this reference were targeted to specifically amplify NRPS and PKS-I gene sequences from only actinobacteria. In this paper, *no actinobacteria were reported*, but still, 60 % of the isolates showed the presence of NRPS and PKS-I genes, which is unique and intriguing. It raises the question that how the primers specific for actinobacteria worked for non-actinobacteria. The authors please provide the name of bacterial species that formed 60% under this category. ![file] ![file] **Query 8:** The isolate NIOT-BARREN 35 grouped under **alphaproteobacteria** in the Fig.1 was submitted in the NCBI database as Streptomyces sp. NIOT-BARREN 35 (KX250315) by the same authors. Kindly explain how the grouping of an altogether different phylum actinobacteria is possible under the phylum alphaproteobacteria? ![file] ![file] ,