microbial culture collection
TRANSCRIPT
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!