Dr. Dhiraj Dhotre

Scientist, MCC, National Centre for Cell Science, Pune

September 09, 2014

Microbial Culture Collection

Overview

• MCC has made significant progress towards achieving a premier culture collection status.

• MCC is offering the largest array of services for systematics of microbial cultures by any culture collection in India.

• In addition, MCC authenticates and preserves microbial cultures for public access and is also accepting cultures for patent deposits as an International Depositary Authority (IDA).

• Recently, the Ministry of Environment and Forests, Govt. of India has designated MCC as a National Repository for microorganisms under the Biological Diversity Act 2002 vide its Order No. 26-15/2007-CSC dated 8th July 2013

MCC- BRC Project

• Since its establishment, MCC received ~135,000 cultures collected from various ecological niches as part of the microbial prospecting project undertaken by DBT in collaboration with nine participating institutes/ Universities.

• All cultures received from the nine participants have been preserved in -80 °C freezers.

Preservation in LN2

• In addition, significant proportion of cultures have also been preserved in liquid Nitrogen (-196°C).

• So far, 10,000 cultures have been sequenced.

• The DNA sequencing was done using three/ four different universal 16S

rRNA gene specific primers.

• Preliminary classification of these three star sequences was done using

Ribosomal Database Project (RDP) using Bayesian naïve classifier.

16S rRNA Sequencing of MCC-BRC Cultures

• All 200,000 culture will be identified by either

16S rRNA gene sequence or by MALDI

MALDI-TOF based identification

• To build a strong meta database of the pure cultures, MCC processed them for FAME analysis by MIDI.

• The common aerobic genera identified were Bacillus, Paenibacillus, Staphylococcus, Pseudomonas, Microbacterium, Klebsiella, Arthrobacter, Virgibacillus, Shigella.

FAME Analysis

• Supply of Cultures • General Deposit • Safe Deposit • IDA/Patent Deposit • Identification Services.

– 16S/18S rRNA gene, ITS region sequencing (~ 700 and ~1200 bp) – Phylogenetic Analysis – MALDI-TOF typing – FAME (fatty acid methyl ester) Analysis – G+C mol% (Tm) – G+C mol% (HPLC) – DNA-DNA Hybridization

Services Offered by MCC

• MCC began supplying its public access general deposit

cultures to researchers in India.

• MCC has supplied many bacterial and fungal cultures in form

of stabs and slants, respectively.

• MCC has started supplying lyophilized culture vials of many

high demand cultures to its customers.

Supply of Cultures

• MCC is continually receiving cultures for general deposit from

several institutions across India and overseas.

• Authentication of the taxonomic identity of the cultures is

done by rRNA gene sequencing (>1200 bp).

• MCC has 1000 authenticated cultures (800 bacterial and 200

fungal) in its collection.

General Deposits

• In addition to the general deposits, MCC is also receiving deposits for IDA from Indian and overseas institutes.

• MCC has accessioned 50 bacterial cultures under IDA and seven are in process.

• On priority, all IDA deposits have been preserved by lyophilization in addition to -80 °C and LN storage.

• For each culture, 14 lyophilized vials are prepared and two of these are sent to the depositor and the remaining are stored at MCC at 4°C.

IDA Deposits

> 16S/18S rRNA gene, ITS region sequencing

• In addition to the sequencing of deposit cultures for authentication, a total of 434 cultures for bacteria and fungi together were sequenced at MCC.

• For bacterial identification 16S rRNA gene sequence was used whereas for fungi ITS region sequence was used.

Identification Services

Identification Services

> Phylogenetic Analysis

• MCC receives requests for bacterial identification and phylogenetic analysis.

• Currently MCC is providing phylogenetic analysis based on two methods; Neighbor joining and Maximum parsimony.

• For phylogenetic analysis, only type strain sequences from databases like RDP and EZTaxon are used.

• The alignment is done in ClustalX software. Phylogenetic trees are constructed in MEGA software.

> MALDI-TOF typing

• Since its installation in April 2013, the methods for sample preparation and analysis have been standardized for the Bruker MALDI-TOF MS.

• So far, only internal MCC cultures have been run on the instrument with very high congruence to rRNA gene sequence identification.

• MCC is now ready to provide MALDI analysis as a part of bacterial identification service.

Identification Services

> FAME Analysis

• MCC has started providing FAME analysis as a service in February 2013.

• The major anaerobic genera identified were Bacteroides Camylobacter, Tissierella, Treponema , Peptococcus, Clostridium, Coprococcus etc. Among the aerobes, Sphingomonas, Virgibacillus, Bacillus, Paenibacillus, Psuedomonas, Rhodococcus, and Rhizobium were identified as common genera.

Identification Services

> DNA-DNA hybridization and GC (mol %)

• MCC standardized and started DDH and GC content analysis as a

service in June 2013.

• These services are now being utilized by scientists of MCC and

other national institutes for classification and delineation of taxa

at species and subspecies level.

Identification Services

• MCC has implemented ISO 9001 in October 2013 for its general

and IDA deposit services.

• A set of standard operating procedures (SOP) for various

activities performed during processing of cultures for deposit

have been devised.

• As per the ISO 9001 requirement, the first audit review for

certification is due in early 2014.

ISO Certification

• In order to efficiently perform activities, it was necessary to have

a digital catalog of the cultures, and the transactions relating to

these cultures.

• MCC got a web based system developed during the past 1 year.

MCC Database Management System

Nitrincola lonarensis Lonar lake, Maharashtra

Allonitrincola sp. Lonar lake, Maharashtra

New genus Lonar lake, Maharashtra

Rufibacter immobilis Pangong lake, India

New genus Industrial waste, Pune

New Cyanobacterial genus Sugarcane Field, Ahmednagar

Chaetomium jatrophae Endophyte on Jatropa podagrica

Arthrinium jatrophae Endophyte on Jatropa podagrica

Alanobotryoshaeria indica Soil from industrial area

Micrococcus aloeverae Endophyte from Aloe vera

Pelistega indica Human gut

Reclassification of Physicola gilvus

Reclassification of Microterricola viridarii

Gordonia sp. VT40 Coal mine drainage

Domibacillus sp. SD111 Marine sediments, Lakshyadweep islands, India

Baciluus enclensis Mangrove Sediment

Fictibacillus enclensis Marine sediment

New Taxa

Scientist Facility Taxonomic group

Dr. Ashish Polkade Lyophilization, DNA sequencing

Actinomycetes

Dr. Amaraja Joshi - Firmicutes

Dr. Amit Yadav - Beta-Proteobacteria, Tenericutes

Dr. Avinash Sharma IDA

Dr. Kamlesh Jangid Verrucomicrobia, Gamma-Proteobacteria, Acidobacteria

Mahesh Chavadar - Alpha-Proteobacteria

Dr. Neetha Joseph FAME Firmicutes

Dr. Om Prakash Anaerobic Anaerobes, Gamma-Proteobacteria

Dr. Praveen Rahi MALDI-TOF Rhizobia

Dr. Venkata Ramana DDH, G+C content

Anoxygenic Photosynthetic bacteria

Achievements

• Symposia on Molecular Ecology and Systematics of microbes

• Establishment of Microbial Diversity Assessment Club (MDAC)

• Workshop on Molecular Phylogeny and Taxonomy

• 32 publications in last one year in reputed international journals

• BISMIS conference to be help in MCC, Pune in 2016

• Additional services

– MALDI-TOF

– DNA-DNA Hybridization and GC content analysis

– DNA isolation from cultures

– Anaerobic microbes

– Hazard group 3 microbes

• Supply of cultures to Industry

Additional services

http://www.nccs.res.in/mcc/index.html

Thank You!