Meet Dr. Neetha Joseph- Scientist at NCMR-NCCS Pune

Dr. Neetha Joseph’s research interest is in microbial systematics, ecology and community analysis. She is affiliated with NCMR-NCCS Pune from last 8 years. She is in-charge of FAME analysis service and curator of Firmicutes. It was a great pleasure to interact with Dr. Neetha and to know more about her as a person and her work.

Kranti: Dr. Neetha, you have worked with coastal environment micro-organisms during your PhD. At a personal level, what motivated you to enter into microbiology research?
Dr. Neetha: Kranti, my native place is in Kerala, a beautiful coastal area in India. Kochi is a lovely place with lot of Backwaters and Estuaries. When I finished my post-graduation, I got an opportunity to join at National Institute of Oceanography (NIO) where most of the research work is related to Ocean and Estuaries. Nutrient enrichment due to various anthropogenic activities is the most widespread problem in estuaries around the world. Significant spatial and temporal variability of physico-chemical and geochemical characteristics and productivity patterns are the important characteristics of estuaries. Microbial communities are involved in mineralization of organic matter; therefore, I was interested in understanding the response of these sedimentary microbial communities to these regional and seasonal changes using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems .

Kranti: Everybody has someone in their life who inspires them to achieve something. Who is your inspiration in science?
Dr. Neetha: My PhD guide at NIO, Kochi is my inspiration in Science. She inspired me a lot! She encouraged me in various aspects of science and helped in boosting my confidence.

Kranti: Which methods and tools you use in your research?
Dr. Neetha: Microbial communities are involved in mineralization of organic matter in estuarine sediment. To understand the response of these microbial communities to various physiochemical and geochemical factors using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems. Phospholipids are mainly found in the cell membrane, not in storage lipids and have a rapid turnover in aquatic sediments. So it provides a measure of viable cellular biomass in an ecosystem.  Different physiological and functional groups of microorganisms in sediments were described using PLFA analysis.
The extracted PLFAs were analyzed using gas chromatography (Agilent 7890 Series, USA) with a cross-linked phenyl – methyl siloxane capillary column (25 m, 0.2 mm) and FID. Identification of the FAMEs was carried out by comparison of retention time and equivalent chain length with known standards like Eukary calibration mixture – 1201A (Eukary6 method, Version: 3.7) and MIDI peak identification software (MIDI Inc., Newark, DE).

Kranti: You are contributing to microbiology related services offered at NCMR Pune. What are those services ?
Dr. Neetha: I am in – charge for FAME analysis service and curator of Firmicutes at NCMR. Under FAME analysis, the bacterial (aerobic and anaerobic) or yeast samples are identified based on their cell membrane fatty acids. Also cell membrane fatty acids are analyzed for novel taxa along with their closely related type strains for publication.

Kranti: Are journals necessary in the age of internet? Don’t you think research should be done not just to publish a paper but also to have real life impacts?  
Dr. Neetha: We know that nowadays we can extract all the information we require via internet. But we cannot compare the beauty of reading a book or journal with internet. Yes, I totally agree that we should do research not only to publish a paper but also to have real life impacts.

Kranti: Being a woman in science, what are the challenges that you’ve faced?
Dr. Neetha: Being a woman in science, the major challenge I face is to manage family, children and their education along with my research work. Another challenge is to get time to spend for research along with my routine services and other commitments.

Kranti: How do you maintain the balance of your family and work-life?
Dr. Neetha: For that I should thank my husband and children for their co-operation and moral support throughout my career.

Kranti: What advice would you like to give to young women who want to pursue research?
Dr. Neetha: If you have an actual interest in science along with sincerity, dedication and hardworking nature, you will be able to succeed in your research career. As a woman, you should be able to manage your time and having patience is also equally important to succeed in your life.

Kranti: Would you share with us any memorable incident/moment of your research life?
Dr. Neetha: In the year 2000, I got an opportunity to participate in Cochin – Alleppey – Mangalore Cruise on board CRV Sagar Paschimi, under DOD, COMAPS Programme. It was a rare experience and golden memory in my research life.

Kranti: Most of the scientist’s children opt for career in science. Do you want your child to become a scientist too? 
Dr. Neetha: Yes, if they are showing real interest in science and research, definitely I will encourage him or her to opt for career in Science.

Novel bacterial species isolated from the Rann of Kachchh, India

-By Kranti Karande

The genus Rhizobium is a large group of bacteria, and most species are known for their symbiotic fixation of nitrogen within the root nodules of leguminous plants. Rhizobium is a major genera in the Rhizobiaceae family. Presently, this genus comprises 90 recognized species. Non-symbiotic and free-living Rhizobium members have been found in different types of soils.

Rann of Kachchh is reputed to be the world’s largest salt desert. The temperature of this desert goes upto 50 degree celsius during summers and drops below zero degree celsius during winters. Due to the hot and hyper saline environment, there is a vast possibility of identifying novel microbes with high economic and industrial potential in this region. A bacterial strain was isolated during investigations on the bacterial diversity of the saline desert soil obtained from Kachchh Rann, India.

For phylogenetic analysis, the 16 S rRNA based sequencing technique was used. Genomic, physiological, and chemotaxonomic approaches were used to analyze the strain. The API 20E and API ZYM systems and BIOLOG GN III systems were used to understand biochemical characteristics, enzyme activities, and oxidation / reduction of carbon sources.The genotypic and phenotypic data generated for this strain revealed that the strain represents a novel species of the genus Rhizobium, for which Rhizobium desertarenae sp. nov. name is proposed.

The cells of this strain were rod-shaped, gram-negative, and non-motile. At 28 degrees Celsius and pH 7.0 the strain grows well and can tolerate up to 2 percent NaCl. It is oxidase and catalase positive. Based on 16S rRNA gene phylogeny, the strain belongs to the genus Rhizobium, with the highest similarity to Rhizobium wuzhouense and Rhizobium ipmoeae. The average nucleoide identity of this strain was less than 82%, to members of the family Rhizobiaceae. The genomic DNA G+C content is 58.6%. This strain showed differences in physiological, phenotypic and protein profiles estimated by MALDI-TOF MS analysis to its closest relatives.


Comparative genomics of Whooping cough vaccine strains from India

-By Kranti Karande

File:Bordetella pertussis on Charcoal Agar supplemented with ...

Pertussis also known as Whooping cough is a highly contagious respiratory disease, endemic in all countries. It is caused by the bacterium Bordetella pertussis that survives in mouth, nose and throat region. Pertussis is known for uncontrollable, frequent coughing and breathing difficulties, a disease found most dangerous in infants.

The aim of pertussis vaccination is to reduce risk of severe disease in infants and young children. There are two types of pertussis vaccines: whole-cell vaccines based on killed B.pertussis organisms and acellular pertussis dependent on one or more highly purified pertussis antigens. Despite high vaccine coverage, re-emergence of pertussis is observed globally. There are different factors contributing for this disease resurgence. Genetic divergence in the circulating strains of B.pertussis has been reported as one of the important contributing factors for the same.

Our current knowledge of the genetic evolution of B.pertussis in circulating strains is largely focused on studies carried out in countries using ACVs (Acellular Vaccines) targeting only a few antigens used in the production of ACVs. In order to better understand adaptation to vaccine-induced selection pressure, it will be essential to study B.pertussis populations in developing countries where WCVs (Whole-Cell Vaccines) are used. India is a significant user and global supplier of WCVs.

This article briefly describes a study conducted by researchers to compare genomes of B.pertussis vaccine strains and clinical isolates reported from India. Genetic divergence was mostly studied in circulating strains of B. pertussis concerning vaccine antigens such as pertussis toxin, pertactin, fimbriae and filamentous hemaglutinnin. Whole genome sequences obtained from five different vaccine strains were compared with reference strain (Tohama-I) and two recently isolated clinical isolates from India. Core-genome based phylogenetic analysis was also performed using isolates reported from countries using ACV.

Whole-genome analysis of vaccines and clinical isolates reported from India revealed high genetic similarity and conserved genome among strains. Phylognetic analysis showed that clinical and vaccine strains share genetic closeness with reference strain.

This study provides detailed characterization of vaccine and clinical strains reported from India, which will further facilitate epidemiological studies on genetic shifts in countries which are using WCVs in their immunization program.


Genomic analysis of a bacterial strain isolated from the Lonar Lake

-By Kranti Karande

Bacterial isolates of the Rhodococcus genus are widely recognized as being capable of catabolizing a wide variety of aliphatic, aromatic and polyaromatic hydrocarbons. Taxonomically genus Rhodococcus belongs to the Nocardiaceae family under phylum Actinobacteria. The tremendous catabolic diversity and remarkable biotransformation ability of genus Rhodococcus is mainly due to the existence of a diverse array of catabolic genes reported for several enzymatic groups such as monooxygenase, dioxygenase, and hydroxylases.

This study identified the genome sequencing data and the draft genome of the Rhodococcus rhodochrous strain originally isolated from Lonar Lake sediments located in Maharashtra province of India. The strain was identified as Rhodococcus rhodochrous based on the mass spectra of the cell extracts of overnight grown pure cultures and DNA sequencing results.

Specific gene and gene groups responsible for catabolism of various compounds have been identified and assigned to the respective compound. Total 39 genes were assigned to subsystem category ‘Stress response’ and the analysis of the genes for this subsystem category revealed the presence of specific genes. The presence of these specific genes implied that the isolate has the ability to synthesize and uptake ‘Glycine betaine (N,N,N-trimethylglycine)’ which may be the main osmolyte synthesized by this bacterium under osmotic stress conditions usually present in the soda lake environment. 

The genomic data reported in this publication will pave the way for further study of the different catabolic genes implicated in hydrocarbon metabolism in this bacterium.


Pea plant shapes its rhizosphere microbiome for nutrient uptake and combating stress

-By Kranti Karande

Legume crops like Pea are used as rotation crops along with rice cultivation in long term conservation agriculture experiments in the acidic soils of the North East region of India. Rhizosphere microbiomes present in the soil have significant influence on plant growth and productivity. The study aims at understanding the bacterial composition of microbiomes present in bulk soil as compared to the rhizosphere. It also aims to understand how the pea plant influences the bacterial communities present in soil and the rhizosphere microbiome in order to improve nutrient uptake and stress improvement. Pea cultivation is a practice used in conservation agriculture which strives to preserve and enrich the environmental resources to sustain and improve crop productivity. The study conducted will help devise future strategies to expand pea cultivation and improve soil health in the region. 

Crop rotation is an important and effective strategy as part of conservation agriculture practices. The North East region of India is a fragile, marginal, inaccessible and diverse ecosystem. Generally a mono-cropping system of rice is followed in this region.  Zero tillage (without disturbing the soil) cultivation of pea (Pisum sativum L.) has been considered beneficial to enhance the cropping intensity in the region. The majority of soils in North-East India are acidic in nature. The pH of soil among many other environmental factors has a significant influence on the type of nutrients and microorganisms present in the soil which in turn have an influence on the productivity of crops. Similarly, nutrient and residue management practices like the application of chemical fertilizers often influence the endogenous microbial communities. 

Sample collection for the study was done from experimental fields of the ICAR Research Complex for NEH Region, Umiam, Meghalaya, located in Eastern Himalayan region. Different tillage and residue management treatments were maintained in these fields for the last eight years by alternatively cultivating rice followed by pea cultivation. For microbial community analysis, bulk soil and pea rhizosphere samples were collected from each treatment plot. All the samples were processed for community DNA extraction. Analysis of the chemical properties of the soil samples was done using available methods. Rhizosphere soils were harvested from roots of pea plants. 

Soil pH (1:2.5) was found to be influenced by tillage and nutrient management practices at depth  0-15 cm. The combined effect of tillage and nutrient management practices on available N, P and K content and SOC,TOC of soil were significant. The rhizosphere showed higher diversity indices in comparison to the bulk soil samples. A total of 71 bacterial phyla were detected in the bulk soil and rhizosphere samples. A higher abundance of Firmicutes was recorded in bulk soil (~41.7%) in comparison to the rhizosphere (~17.8%). On the contrary, Proteobacteria were highly abundant in the rhizosphere (~43.9%) in comparison to bulk soil (~18.6%) samples. Significantly higher abundance of Proteobacteria and Bacteroidetes was observed in pea rhizosphere samples in comparison to bulk soil. 

Impact of residue management practices on abundance of specific microbial communities was observed across both rhizosphere and bulk soil samples. The impact of tillage history was also observed on the enrichment of specific OTUs in the bulk soil and rhizospheric soil. Differences in the abundance of 11 genera were recorded in the rhizosphere sample across the history of different tillage treatment. All these genera showed higher abundance in the conventional tillage fields. The correlation between soil properties and microbial community structure was also studied as part of the study. Significant correlations were observed between relative abundance of few bacterial phyla & genera and soil properties in both bulk soil and rhizospheric soil samples. However, the number of significant correlations was low in rhizosphere samples, in comparison to bulk soil samples. 

The study was designed to investigate the effect of long-term exposure to various tillage and residue management practices on the bacterial community structures of the bulk soils and how pea plant (a rotation crop) shapes the rhizosphere communities. A higher species diversity and evenness was observed in rhizospheric samples. There was no significant difference in bacterial richness and evenness among different tillage and residue management treatments in both rhizospheric and bulk soil samples. This is an indication that the plant rhizosphere effect (a plant’s ability to alter microbial communities in rhizospheric soil) is the key driver of alpha diversity. Plants can alter the microbial communities by secreting a variety of nutrients and bioactive molecules into the rhizosphere. Enrichment of specific OYUs in the Pea rhizosphere were also confirmed which can be attributed to the selection pressure of the Pea root. The results of the Pea rhizosphere and bulk soils were consistent with the fact that the majority of members of microbial communities in the host plant are horizontally acquired from the surrounding environment, and the soil is the main reservoir of a plant rhizosphere microbiome. The genus Nitrobacter was at higher abundance in pea rhizosphere samples than bulk soils, suggesting its enrichment by the host plant as Nitrobacter converts nitrite to nitrate making nitrogen more readily available to the host plant. Higher abundance of genes related to nitrogen fixation, phytohormone and siderophore production, phosphate solubilization in the rhizosphere soil substantiate the conclusion that the selection of bacterial communities is always based on plant growth promoting potential in the rhizosphere. 

The study concluded that pea plant is the most dominating selection factor shaping the microbial communities under diverse residue management and tillage treatments. The rhizospheric soil was found to be enriched with bacterial taxa known for plant growth promotion which indicates that the plant plays a role in selecting the rhizospheric communities to meet its requirement of nutrient uptake and combating stress.


Identification of pathogenic yeasts from different clinical samples

-By Kranti Karande

Yeasts have always been a part of human microbiota. Some of the species belonging to the yeast family are opportunistic pathogens leading to infections of cutaneous, mucosal, bloodstream or deep-seated organs known as Candidiasis. These infections have become a major threat to humans. Different species of the Candida genus lead to different medical conditions and demonstrate varied sensitivity towards anti fungal agents used in practice. Failure to commence accurate anti-Candida therapy at an appropriate time has led to an increase in fatal cases of Candidiasis. Hence, it is important to correctly identify the Candida species to start appropriate and timely treatment. There are very few studies of yeast infections in Indian context. Thus this study is important as it is a study of a large number of Indian clinical samples for yeast infection reporting opportunistic and emerging pathogens. The study conducted on 176 clinical samples collected from Bharati Hospitals, Pune, Maharashtra was aimed at identifying the pathogenic species of yeast, understanding their anti fungal susceptibility and cell invasion capabilities.

Existing techniques for species identification of Candida species may be time consuming and even resulting in non-authentic identification if carried out by classical usage of blood culture technique. Recently developed molecular tests provide real time PCR assays and rapid results. Although these methods are specific, they do not improve the diagnostic sensitivity of Candidaemia and Candidiasis. Nonetheless DNA sequencing of amplified PCR products continues to be the most reliable method for authentic fungal species identification. A comparison of three different techniques for yeasts species identification was carried out along with characterization of these species for antifungal susceptibility and cellular invasion capabilities.

The clinical samples obtained were chemically treated as per standard protocol before being microscopically examined to find out the presence of unicellular yeast cells and other yeast organs. Samples with probable yeast were further treated to obtain isolated colonies and consequently obtain pure cultures which were deposited at NCMR- NCCS Pune, India. The colonies were further characterized biochemically using different identification tests. 

PCR amplification, DNA sequencing and phylogenetic analysis:
Genomic DNA from each isolate was extracted using lithium acetate. The precipitated DNA was used for PCR amplification (increasing the copies of DNA) using suitable primers. After confirmation of amplification, the PCR product was sequenced to identify different species present in the samples. A phylogenetic tree was constructed individually for each strain to confirm its species identification using known sequences of different Candida species available in GenBank at NCBI. 

MALDI-TOF/MS biotyper and chromogenic media method:
Actively growing pure cultures were used for protein extraction for MALDI-TOF MS analysis. The extracted protein samples were analyzed using a 60 Hz Nitrogen Laser and Flex Control software. Chromogenic media screening of yeasts was also performed and identification of colonies was done using colony color and appearance. Chromogenic media is used commonly in clinical laboratories for rapid identification of Candida strains. It is a fast method but not entirely reliable. Test results showed that chromogenic media successfully identified only 64 out of the 75 strains identified as C. albicans by sequencing. Seven strains were misidentified and four remained unidentified. Hence, it is safe to say that chromogenic agar technique is not a reliable method for yeast species identification. 

A total of 176 isolates were analyzed using MALDI-TOF/MS and the results were compared with results of DNA sequencing. MALDI-TOF MS could identify 157 of yeast isolates correctly. Overall, the correct identification rates of the 176 yeast isolates to species levels by the Bruker MALDI Biotyper systems was 89.2. This study confirms that MALDI-TOF MS presents an effective alternative to the sequencing for the correct identification of the emerging yeast pathogens however, there is a need to improve the database by regularly adding newer yeasts species from clinical and environmental scenarios.

Invasive fungal infections are becoming common and its rapidity of invasiveness by pathogens demands early arrest of infection by antifungal agents. There are immense changes in the host factors, infecting fungi, and antifungal agents and hence there is a need to accomplish antifungal susceptibility tests. The study conducted involved characterization of yeast species in terms of their antifungal susceptibility. A total of 157 isolates were tested for their susceptibility to antifungal drugs. Clinical yeasts showed increased resistance to fluconazole (55%) which is the most common antifungal used for treatment of candidiasis. Some emerging pathogens were found to be sensitive to most of the tested antifungal agents. It has been observed that strains of C. glabrata have highest resistance among various species of Candida. Many researchers have also isolated multi-drug resistant strains of C. glabrata

In vitro cell invasion assay involved investigation of the ability of various yeasts isolates to invade epithelial cells (HeLa Cells were used for this study). Since the invasion of host cells by pathogenic yeast cells is one of the important virulence factors in invasive candidiasis, this study is significant for the purpose of understanding potential threats from each Candida species. A total of 88 yeast isolates tested were isolated from invasive Candidiasis patients and only few could show invasion in vitro. Although the results of invasion capabilities study for different species like C. albicans, C. glabrata, C. parapsilosis showed varied results, it was observed that yeast cells invade host cells with the help of pseudo-hyphae or hyphal structures. Some studies have also shown that the transformation of C. albicans into hyphal form increases its interaction with the tissue cells, thus increasing its ability to adhere to human cells and potent invasion.

MALDI-TOF MS is an important tool in clinical set up for microbial identification as it is fast, low-cost, simple to use and wide spectrum applications in the identification of bacteria, archaea, and fungi. However, there is an urgent need to identify the species directly from the sample since it will greatly save on time of treatment which is crucial in serious cases like invasive Candidiasis. At present, both the methods which are considered reliable require pure culture, i.e. DNA sequencing and MALDI-TOF MS. The antifungal susceptibility tests showed that a large number of isolates showed resistance to all antifungals tested. A molecular analysis of all these strains is essential to substantiate the cause and mechanism of resistance. In the present study, nystatin was found to be the most effective antifungal agent. Many new types of yeast were reported in the study as probable human pathogens which should be included in diagnostic protocols.


Description of a novel species isolated from the surface of tomato

-By Kranti Karande

A new bacterial strain designated as TOUT106T was isolated from the tomato surface. Though there were many bacterial species isolated from various fruits and vegetables like sweet potato, banana, tomato, lettuce and cucumber, TOUT106T was found to represent a novel species. This study was aimed at providing a detailed taxonomic description of this novel strain TOUT106T isolated from tomato surface. A novel strain is a micro organism which hasn’t been identified earlier.

The strain was isolated by washing the outer surface of a tomato collected from a local vegetable market in Pune, India. Colonies grown on a trypticase soy agar turned out to be 1-3 mm in diameter, circular, raised with an entire margin, and translucent opacity. The identification was done using MALDI-TOF MS technique. After extracting high quality genomic DNA from the strain, the 16s rRNA sequence was amplified. The similarity search for the 16S rRNA gene sequence of strain TOUT106T was performed against the type strains of prokaryotic species in the EzBioCloud’s database. 

Genome sequencing was performed and from the concatenated sequences of 92 core genes extracted, a possible phylogenetic tree was inferred. The Average Nucleotide Identity (ANI) was determined between strain TOUT106T and closely related strains of the Enterobacteriaceae family. Analysis of chemotaxonomic features (based on fatty acids and cell proteins)was done after harvesting cell biomass from culture grown on TSA at 28°C.  Antibiotic susceptibility was determined using the disc diffusion method. The susceptibility to antibiotics was interpreted based on the Clinical and Laboratory Standards Institute (CLSI) guidelines determined for members of the family Enterobacteriaceae

A search of the 16S rRNA gene sequence of strain TOUT106T showed the highest similarity to Salmonella enterica subsp. arizonae strain NCTC 8297T (98.4 %) . The phylogenetic tree constructed based on the 16S rRNA gene sequence placed the strain TOUT106T within the Salmonella clade. The genomic DNA content of strain TOUT106T was well within the specifications of genus Klebsiella. Comparative analysis of ANI value and dDDH relatedness of Enterobacteriaceae strains suggested that the strain TOUT106T is a novel species.  A comparison of MALDI-TOF MS spectra based dendrogram showed that the strain TOUT106T separated from the type strains of Salmonella and was placed along with Klebsiella variicola DSM 15968T and Raoultella terrigena DSM 2687T, corroborating well with the results of genome-based analysis. Colony morphology, as examined on blood agar medium, were mucoid and translucent.

Based on different methods of identification, scientists reported that the strain TOUT106T is a member of genus Klebsiella. However, it differs from closely related species of the genus Klebsiella in several aspects, such as biochemical features, physiological features, protein profile, and overall genome relatedness indices. Thus, it represents a novel species in the genus Klebsiella, for which the name Klebsiella indica sp. nov. is proposed.

Klebsiella indica (in’ L. fem. adj. indica, of or belonging to India, where the type strain was isolated from the outer wash of a tomato collected from the vegetable market in India). Cells are Gram-negative, straight rods with round ends (0.7-0.9×2-3 μm), and non-motile. Colonies grown on trypticase soy agar are 1-3 mm in diameter, circular, and with translucent opacity. The optimal temperature for growth is 28 °C, and the optimal pH is 7.0. Growth occurs in the absence of NaCl with up to 2% tolerance in trypticase soy broth. It is weakly positive for catalase and negative for oxidase activity. It is susceptible to the majority of antibiotics. 

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Microbiome of indian patrilineal families reveal association with age

-By Kranti Karande

The human microbiome plays an important role in maintaining stable health conditions. It is influenced by age, geography, diet and other factors. This study was aimed at understanding the association of composition of the human microbiome with age in Indian joint families formed through paternal descendants. Oral, skin and stool microbiome of a total of 54 healthy individuals from 6 joint families with three generations were studied and characterized using 16S rRNA gene based methodology. The study population had matching dietary, social habits, hygiene and sanitation habits, economic status and geographic position.  This study highlights that precise and perceptible association of age with microbiome can be drawn when other causal factors are kept constant. 

Human microbiome has evolved with the host and its ancestors for millions of years and it plays an important role in maintaining a good health by performing various functions such as digestion, protection against pathogen colonization to host immunity and regulation of central nervous system. The human microbiome is affected by various factors such as ethnicity, age, diet etc. Hence, it is important to study the same population for the exploration of a precise association of age and microbiome. This study of genetically linked individuals of different generations having similar diet, ethnicity and location will help to understand the ability of microbiome to persevere with increasing age and how they progress with the age. 

Approximately 99% of the gut microbiome was constituted of 5 bacterial phyla. Total of 174 bacterial genera were noted to be present out of which 5 contributed to 77% of the gut microbiome. The oral microbiome showed comparatively higher abundance of some specific bacterial phyla.  Bacterial genera prevalent in 95% of the study population with more than 0.1% abundance were considered as a part of the core microbiome. Gut, Oral and skin microbiome had 3, 13 and 2 core microbiome genera present in the samples respectively. 

Microbiome community structure of gut, oral and skin samples was investigated across three generations (age groups). Gut microbiome of each generation had a unique set of bacterial genera present in abundance out of the prevalent genera for the specific age group. High abundance of few bacterial taxa was recorded in particular age groups in the skin microbiome samples also. Comparative microbiome analysis in three age groups did not show significant difference in abundance of bacterial genera in the gut and skin microbiome. However, the oral microbiome showed significant variations in the abundance of genera Dialister, Fusobacterium, Streptococcus, Selenomonas, Filifactor and Treponema. 

Age-associated changes in the microbiome were further analyzed based on differentially abundant OTUs (a methodology). After performing a correlation analysis it was revealed that phylum Proteobacteria in gut microbiome and phylum Fusobacteria in oral microbiome showed higher abundance with increasing age. However, in the skin microbiome, no such statistically significant correlations were noted. Amongst the total 171 bacterial genera in the gut microbiome, only genus Bacteroides showed age-associated changes. Decreased abundance of Bacteroides was noted with increasing age.

Dietary information of the study population was collected using the food frequency questionnaire (FFQ) and this information is subsequently translated into the daily intake of carbohydrates, proteins, fats, lipids, fibers and calories with the help of a nutritionist. Detailed analysis showed that carbohydrates provide about 74%, 81% and 80% calories in the first, second and third generation members, respectively. Further analysis showed no significant correlation across generations suggesting similar microbiome structure and dietary pattern. This emphasizes the fact that overall homogeneity in the diet helps in maintaining the microbial state.

Bacteria with high fiber degrading potential were found highly abundant in first generation members while the second generation members showed an abundance of metabolism boosting gut microbiome. Early gut colonizers and Bacteroides were higher in the third generation members. The skin microbiome also showed age related changes in abundance of bacterial taxa present. With the increasing age, physiological changes occur in the skin structure which explains the association of key bacterial taxa in the members of the respective age groups. Similarly, in the oral microbiome, Fusobacteria was found to increase with increasing age. It was observed that a negative correlation in the abundance of Bacteroides with age; this is in contrast to previous studies demonstrating the higher abundance of genus Bacteroides with increasing age. Age related changes in oral microbiome could be associated with physiological changes occurring with increasing age in the oral cavity. 

In conclusion, this study particularly highlights the precise and perceptible association of age with the microbiome. The findings suggest that core taxa constitute more than 75% of the gut and oral microbiome, while only 67% of the skin microbiome, indicating a larger variability of the microbiome present on the skin. The baseline data presented from a healthy Indian sub-population can be used as reference for further studies including diabetes, obesity and inflammatory diseases. 


Environmental and public health perspective of WasteWater Treatment Plants (WWTP)- safe or unsafe?

-By Kranti Karande

The population of Pune city located in the state of Maharashtra, India is around 31 lakhs. Imagine how much waste water such a huge number of people produces!  The amount of wastewater produced at Pune City is approximately 744 MLD (millions litres per day). Out of this, the WasteWater Treatment Plants (WWTP) treats about 527 MLD of water. The waste water treatment plants aim to minimize the adverse effect of untreated wastewater on ecology, the environment and human health through its adequate treatment before release into the ecosystem. But the question is, is this treated water indeed safe for environmental and public health? Whether or not the microorganisms present in the untreated water are eradicated following treatment? How WWTP contributing in spread of antimicrobial resistance (AMR) in nature?

Dr. Om Prakash Sharma’s group from NCMR, NCCS Pune started with an interesting question as to whether there is a difference between the number of bacteria in the sample of untreated and treated waste water. Different types of antibiotics are used in human therapy, veterinary and animal farming, and huge number of antibiotics are released into municipal wastewater which ultimately finds its way into the environment. Dr. Sharma’s group also tried to answer the question of whether or not the wastewater-isolated bacteria develop antimicrobial resistance.

They collected samples from untreated and treated waste water and stored it at appropriate storage conditions for further processing. The bacterial number was estimated by total viable count and most probable number methods and efficacy of current wastewater treatment plant in reduction of bacterial load and spread of antibiotic resistant bacteria in the environment was also studied.

A total of 30 bacterial species belonging to 18 different genera were isolated from the untreated water sample. Whereas in the treated wastewater only 9 species from 6 different genera were present, all 9 species of bacteria are non-pathogenic. They also performed antimicrobial susceptibility  testing using  these bacteria to know the role of WWTP in spread of AMR. Twenty one antibiotics have been used for testing. All of these antibiotics are common in testing antimicrobial resistance in bacteria. The bacteria isolated from untreated water samples indicated high levels of antibiotic resistance.

The research group concluded that, untreated wastewater sample contained wide range of organisms with high levels of antibiotic resistance while bacterial load reduced drastically and pathogens were absent in the treated wastewater. Results indicated that the wastewater treatment plant was working effectively and efficiently by reducing the bacterial load in treated wastewater. Several organisms of clinical significance like Acinetobacter septicus, Citrobacter farmeri, Klebsiella oxytoca, Raoultellao rnithinolytica etc. were also reported in untreated wastewater sample.

This study demonstrated the comparison between culturable bacterial population present in influent and effluent of municipal waste water treatment plant of Pune. The researchers conclude that waste water treatment not only reduces BOD (Biochemical Oxygen Demand), COD (Chemical Oxygen Demand) and TSS (Total Suspended solids) but also bacterial load of the waste water.


Meet Dr. Rohit Sharma: Mycologist at NCMR, NCCS Pune

Dr. Rohit Sharma is a fungal taxonomist working at NCMR, NCCS Pune. He has been working in this field from last 15 years. He has identified 2 novel genera and 11 novel species of fungi. It was a great pleasure to interact with Dr. Sharma and find out more about him and his work.

Kranti: What motivated you to enter into the research field of fungal taxonomy?
Dr. Sharma:
I think it all started quite early when I used to go for sampling with my father, a Plant Pathologist at J.N. Agriculture University, Jabalpur. I used to go on collection trips with him during my school and college days to collect fungi infecting crops and wild plants and initial interest developed from there. During this period, I also got an opportunity to meet Dr. Kalman Vanky, expert in smut fungi and got opportunity to see his dedication towards the field of smut taxonomy. However, the actual involvement started when I began my doctoral work under Dr. Akhilesh K . Pandey and Dr. Ram C. Rajak whose lab was known to work on diversity and taxonomy of fungi. Here, I could get experience on entomopathogenic fungi, pathogenic and saprophytic micro fungi, mushrooms and yeasts. I developed a lot of insight in the lab under their guidance. Regular interactions helped to understand basics and the lab was rich in literature (monographs, journals and manuals) which usually is a bottle neck in the morpho-taxonomy of fungi. In the present organization as a curator of fungi, I got an opportunity to handle many fungi (yeasts, mycelial fungi and mushrooms) that helped in the development of the expertise in fungal taxonomy and systematics including polyphasic taxonomy.

Kranti : Why it is important to study fungal taxonomy?
Dr. Sharma: Fungi are hyper-diverse group of organisms. So far, many have been discovered but many are still to be discovered. Till now, about 1,20,000 fungi are known and researchers are discovering many more with an annual rate of approx. 1000 fungi. Based on the environmental sequence/ metagnomic data, it is now estimated that 22-38 lakhs fungi are yet to be discovered which makes it important to study the fungal diversity and taxonomy. Moreover, since they play an important role in environment and industrial biotechnology, it is important to explore, identify and subsequently study for their bio prospecting. Moreover, human and plant pathogens are increasing day by day and many environmental opportunistic pathogens are causing infections. Hence, it becomes important to have their authentic identification and proper classification for better management. The fungal taxonomy is complex and includes species specific morphological and physiological characters and intraspecific variation. The DNA-based taxonomic studies have helped to resolve many taxonomic problems and describe several cryptic species and are considered more stable than morphological characters.

Kranti: How many fungi you have collected and identified till now?
Dr. Sharma: So far we have collected and identified more than one thousand fungi from Lonar lake-Maharashtra, Famlong Lho-Sikkim, Achanakmar Sanctuary- Chhattisgarh, and other sites of India. They have been isolated from soil, litter, insect gut, sediments, water and as plant endophytes. We at NCMR-NCCS have described 2 novel genera (Matsushimamyces and Alanomyces) and 12 novel species, viz., Naganishia indica, Coniochaeta dendrobiicola, Leucosporidium himalayensis, Aureobasidium tremulum, Matsushimamyces bohaniensis, Alanomyces indica, Nothophoma raii, Curvularia lonarensis, Arthrinium gutiae, Pyrenochaeta telephoniae, Chaetomium jatrophae and Arthrinium jatrophae.

Kranti: What is your contributing role in services at NCMR?
Dr. Sharma: At present, I have been working as Curator-Fungi and looking after more than 15,000 fungal cultures preserved at our collection. My earlier work mostly was related to development of fungal culture collection at NCCS-NCMR, development of protocols, training human resource and undertaking basic research. So far, we have processed more than 2500 fungal cultures deposited by researchers from academia and from industry and accessioned approximately one thousand of them. We also provide identification services by conventional (morphology and API kit) as well molecular method, customized services and contractual research to industries. I along with my technician and project staff have been able to deposit more than 500 cultures to the NCMR-NCCS culture collection and screened them for various potential industrial applications viz., enzymes, bio active metabolites, waste degradation, etc. During the period, I have handled 4 research projects focusing on fungal diversity and their bio prospecting which in turn contributed to enrich the culture collection and preserve fungal wealth of our country.

Kranti: How has your journey been from being a PhD student to a Scientist at prestigious national institute?
Dr. Sharma: It has been a nice learning process, from working on ectomycorrhizal mushrooms during doctoral thesis to working with micro-fungi and yeasts at NCMR-NCCS. It has state of the art facilities and I can now use advanced techniques for my work. Work culture is very nice in our institute and  freedom to work and pursue my research interest in the area of diversity and taxonomy.

Kranti: What are the challenges you face during a field trip?
Dr. Sharma: During my doctoral thesis, I have went for sampling in the forests of Madhya Pradesh and Chhattisgarh for collection of ectomycorrhizal mushrooms. There are several challenges faced due to lack of communication, remoteness of the forests, etc. Moreover, we used to collect mushrooms from 8 am in the morning to 5 pm in the evening with a small packed lunch. In the evening after arriving at the forest guest house, we used to complete the mushroom data sheets and then around 7-pm we used to start culturing the mushrooms before putting them for drying. Sometimes it used to get 2-3 am in the morning because the mushrooms might decompose as the time passes after their harvest from the field.

Kranti: Which is your favorite fungus?
Dr. Sharma: The whole group of fungi are fascinating and one cannot pinpoint a single fungus. These are morphologically diverse and each one is unique in their features viz., colony color, shape, conidial shape, size, etc. The sexual and asexual stage found in these groups of organism makes them more interesting. From microscopic mycelial fungi to multi-cellular mushrooms to single celled yeasts, all have uniqueness of their own. From exogenous spores to endogenous spores, from pycnidia to cleistothecia, all have different morphological structures.

Kranti: What are the storage methods used at NCMR?
Dr. Sharma: We at NCMR-NCCS preserve the culture by five methods viz., cryopreservation at -80oC and at -196oC (liquid nitrogen) and by freeze drying. Apart from this, we also preserve the fungal cultures in distilled water and mineral oil (at 4oC). 

Kranti: What is your support system?
Dr. Sharma: My family and friends are my support system at individual level and colleagues and my team working with me at the professional level.

Kranti: What would you have liked to become if not a scientist?
Dr. Sharma: I would have been involved in doing agriculture, preferably doing scientific and organic agriculture with little bit of teaching at some academic organization so that I can interact and dissipate knowledge to students.

Kranti: What are the current and future projects in your group?
Dr. Sharma: Currently, we are working on collecting fungi from various sites and bio prospecting them for various applications like antimicrobial activity and treatment of industrial effluent. In future, we would like to focus more on the industrial applications of the fungal resource preserved in our collection and develop biotechnological usage of them.

Kranti: What are you hobbies?
Dr. Sharma: I like to read general books. Gardening is one my favorite hobbies and like to grow flowering as well as vegetable plants.

Kranti: Does research sometimes becomes stressful? What do you do for relaxation?
Dr. Sharma: Yes, research sometimes is stressful as sometimes multiple things gets lined up at the same time. To relax, I listen to classical music or go for trekking at nearby Pashan Tekri.

Kranti: What are your thoughts on Science communication in India? How can scientist contribute for better Science communication?
Dr. Sharma: I remember during my childhood I used to read a magazine ‘Science Reporter’, it used to explain scientific discoveries in simple language. Science communication in India is developing over the years by means of audio-visual and writing medium. Scientists can contribute for better science communication by conducting science talks for undergraduates explaining their research in a simplified manner. It is better now than it used to be. There are more communication channels available now with online platforms like blogs and online articles as compared to earlier days.