Services at NCMR Pune

PART-II Microbial identification and characterization services

NCMR Pune offers multiple services for microbial identification and characterization. All of these services are listed below:

A. rRNA gene/ ITS sequencing for microbial identification: rRNA gene sequence of bacteria/ Internal Transcribed Sequence (ITS) of fungi is one of the most reliable molecular methods for microbial identification. We offer this sequencing service for identifying bacteria, archaea and fungi using universal primers for each type of organism.
Once the organism is received at NCMR, it goes through the following procedure: DNA isolation, PCR using universal primers for the type of organism, purification of the PCR amplicons, cycle sequencing reactions, purification and run them on an automated capillary-based Sanger DNA Sequencing system. At every step, there is in-house quality check to ensure success of the sequencing reactions. Post sequencing, fragments are manually checked and only good quality sequences are used to form contigs, which are then matched in well-curated databases for assigning closest neighbor as the tentative identification of your submitted organism.

B. Phylogenetic analysis: Phylogenetic analysis based on 16S rRNA gene is a powerful tool to study the evolutionary relationships among micro-organisms. It provides insights of genes or sequences which include relationships, origins, and closest taxonomic identification. These relationships are described by a branching of the tree.

C. Determination of Polar lipids: Lipids are one of the most verdiagram, or tree, with branches joined by nodes and leading to the terminal at the tips of satile compounds present in living system. Bacterial cell membrane is mostly composed of polar lipids like phospholipids, amino lipids and glycolipids etc. Lipids profile of a bacterial species is well suited to be used as a taxonomic character and is required to be included for description of novel taxa.

D. Determination of G+C mol% in DNA: G+C content in DNA of a species of bacteria is a characteristic feature and in bacterial world G+C mol% varies from 24% to 76%. This value is essential for description of a novel species.
There is a linear correlation between the melting temperature of DNA and G+C mol %. NCMR uses fluorimeteric method using double strand specific fluorescent dye and real-time PCR thermocyclers for determination of melting temperature (Tm) and G+C mol % is calculated from the Tm. This service includes isolation of high quality DNA from microbial culture(s), melting temperature experiment (in triplicate) and calculation of the G+C mol% content.

E. DNA-DNA hybridization: DNA-DNA hybridization (DDH) values determine relatedness between bacteria and is one of the minimum criteria for the description of novel taxon when 16S rRNA gene sequence similarity is more than 97%. Since 1960s DDH values have been used for species delineation with 70% as a recommended cut-off. At high temperature DNA gets denatured and when it cooled down gradually, it starts reassociating with the complementary sequences and results into the formation of double stranded DNA again. But when the DNA of two organisms mixed together and denatured, while reassociation of these hybrid DNA the degree of binding is directly proportional to the sequence similarity between the two DNAs. This degree of binding is converted into the percentage hybridisation. NCMR uses fluorimeteric method for the calculation of DDH.
This service includes isolation of high quality DNA from related species, hybridisation experiment (in triplicate) and calculation of the DDH value.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterisation includes: Colony morphology (such as size, shape, colour, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterization includes: Colony morphology (such as size, shape, color, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.
G. Phenotypic characterization by API: The Analytical Profile Index (API) is a miniaturized panel of biochemical tests compiled for identification of groups of closely related bacteria. Different test panels are available in dehydrated forms which are then reconstituted at the time of use by addition of bacterial suspensions of desired cell density. After proper incubation, test results are scored (after 24 and some time 48 hr) as +/- to generate a seven-digit code (profile). Identity of the bacterium is then derived from the database with the relevant cumulative profile code book or software (apiweb™).
NCMR offers phenotypic characterization by four type of API methods using either one or a combination of strips depending on the users’ requirements.
API 20E: API 20 E is a standardized characterization/identification system for Enterobacteriaceae and other non-fastidious, Gram-negative rods. It consists of 20 microtubes containing dehydrated substrates.
API 20NE: A standardized system characterization/identification of non-fastidious, non-enteric Gram-negative rods (e.g. Pseudomonas, Acinetobacter, Flavobacterium, Moraxella, Vibrio, Aeromonas, etc.), combining 8 conventional and 12 assimilation tests.
API 50 CH: Used for characterization/identification Bacillus and related genera as well as gram-negative rods belonging to Enterobacteriaceae and Vibrionaceae families based on fermentation of 49 carbohydrates.
API ZYM is a semi-quantitative micro-method designed for detection of enzymatic activities.

For more details on these services visit: http://210.212.161.138/services

Services at NCMR Pune

PART-I

National Centre for Microbial Resource, Pune is India’s microbial culture collection centre recognized by World Federation of Culture Collections (WFCC). The main objective of NCMR is to provide authentic microbial cultures to industries as well as academic and research institutes.

Culture collections play an important role in the area of biotechnology and are valuable resources for the sustainable use of microbial diversity and its conservation. But their meaningful exploitation is possible only if the properties of cultures are properly documented and the information is easily accessible. National Centre for Microbial Resource at NCCS Pune established by the Department of Biotechnology, Government of India is one among country’s largest culture collection centres. It is recognized as an International Depository Authority for the deposition of patent cultures under Budapest Treaty and designated national repository under the Biodiversity Act 2002.

NCMR focuses on fundamental research in the fields of microbial diversity, microbial taxonomy, microbial genomics and proteomics etc. One of the main objective of NCMR is providing microbial culture related services. NCMR provides multiple services like culture deposit, culture supply, identification/characterization of microorganisms, genomics/ metagenomics, imaging by scanning electron microscopy, phytoplasma detection, testing for microbial load for food safety certifications etc.

At present, NCMR accepts bacteria, fungi, and plasmids belonging to hazard group 1 and 2. Culture deposition service at NCMR is divided into following categories:

General deposit, IDA deposit and safe deposit.


A. General deposit: Cultures deposited under this category are accessible to public for teaching and /or scientific research and no restriction is imposed. NCMR reserves the right to reject a request for deposit of culture. Unidentified cultures are not accepted for deposit under this category. Once accepted and accessioned by NCMR, a culture cannot be withdrawn from the collection. There is no fee for such deposits.

B. International Depository Authority (IDA): Microorganisms deposited under IDA fulfill the requirement of the deposit for the purposes of patent procedures in all states signatory to the Budapest Treaty. NCMR is the second IDA in India. All IDAs operate as per the regulations of the Budapest treaty. The advantage of depositing microorganisms under IDA is almost similar forms are used and uniform procedures are followed in dealing with such deposits. Confidentiality and security of such deposits are maintained by NCMR. NCMR is obliged to keep the deposits under IDA for the period of 30 years from the date of deposit.

C. Safe deposit: Confidentiality and security is maintained while depositing such cultures. These cultures are not listed in the catalogue. There is an annual fee for such deposits. Depositor needs to sign an agreement with NCMR for making such a deposit.

NCMR supplies cultures listed in NCMR catalog (hard copy/electronic) for teaching and research purposes.

For more information on culture deposit and culture supply services at NCMR, visit following website: http://210.212.161.138/services

In the next article, we will learn about other services offered at NCMR.

MALDI-TOF Mass Spectrophotometry (MS): Emerging technique in Microbial Ecology

-By Kranti Karande

Microorganisms have been identified by their biochemical properties and using techniques like 16S rRNA sequencing for more than a century. Recently, MALDI-TOF Mass Spectrometry (MS) has become a key technique for microbial identification due to its rapid and reliable performance.

MALDI-TOF MS facility at National Centre for Microbial Resource, National Centre for Cell Science Pune

Mass Spectrometry is an important tool for the detection of atoms or molecules in a chemical complex. A mass spectrometer works by turning atoms into ions. Ions get separated when they are passed through electric field and magnetic field respectively. A spectrum is generated determining types of atoms present in the sample.

MALDI-TOF MS technique has a variety of applications in biology including intact mass determination, peptide mas fingerprinting, MALDI imaging. This method is widely used for the identification of DNA and proteins, but recent advances in this technique have incorporated use in microbiology for pathogen detection, which is contributing immensely to research in microbiology.

Dr. Praveen Rahi from NCMR-NCCS Pune and Dr. Parag Vaishampayan from the California Institute of Technology, USA, recently published an editorial detailing the recent advancements and challenges in MALDI-TOF MS for Microbial Ecology applications.

This technique is in the early stage of development for the direct identification of microorganisms in positive blood cultures, detection of drug resistance factors and for bacterial function assessment. Recently, researchers identified Brucella infection from positive blood culture using this technique which is a great breakthrough in MALDI-TOF MS application. Researchers are also trying to develop this technique for studying antimicrobial resistance in bacteria and fungi.

In one of the recent publications, data from MALDI-TOF spectra of intact protein and specialized metabolite spectra from bacterial cells grown on agar were combined to study the link between microbial identities and their potential environmental functions.

This technique is becoming a fast and effective method for filtering out same species strains and genetically identical clones. Until now, identification of same species strains was done with the help of techniques like dendrograms. Now MALDI-TOF MS is used for identifying identical strains as it provides accurate results with rapid analysis.

With the help of MALDI-TOF MS technique medical researchers revealed a potential clonal route of transmission of Enterobacter spp. This research is a significant source of information on the spread of Enterobacter, an evolving pathogen for its ability to acquire antimicrobial resistance factors and the role of MALDI-TOF MS in microbiological surveillance of diseases.

Dr. Praveen Rahi
NCMR, NCCS Pune

While discussing about MALDI-TOF MS applications with Dr. Praveen Rahi, he commented that, “MALDI-TOF MS will be ‘new microscope’ for microbiologists in the era of ‘culturomics’, which include large-scale cultivation of microorganisms and require high-throughput identification tools. Right now the commercial MALDI-TOF MS spectral databases primarily include profiles of microorganisms of clinical relevance, and researchers across the globe are making efforts to generate spectral profiles for microbes according to their research interests. However, most of these efforts are being made in isolations, which might lead to erroneous database entries. There is urgent need to develop universal SOPs for MALDI-TOF MS profile generation, and subsequent curation and validation of newly generated database profiles.”

Though this technique has emerged as a key player in microbial identification and microbial ecology research, but there are many fine tuning steps required to improve this technique.

One study noted that only 35% of species level identifications done using MALDI Biotyper correlated with those detected by 16S rRNA gene sequences, suggesting a poor species level detection by this technique. Insufficient coverage of bacterial species in the repositories was proposed as the primary reason for this difference. The lack of a public repository to send new spectral references created by researchers further worsens this issue. The major challenge is to find the solution for same.

During the identification of the collection of microorganisms isolated from cell phone surfaces, researchers observed that the efficiency of MALDI-TOF MS spectrum filtering can be achieved by improving the methods of sample processing and enriching the spectral database leading to better and highly reliable results for MALDI-TOF MS microbial identification.

If we find feasible solutions for the challenges in this technique, this technique has the potential to make an immense contribution to the field of microbial ecology.

NCMR-NCCS Pune provide services for microbial identification using MALDI-TOF MS.

Reference: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02954/full