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T-4-126 - Identification and taxonomic classification of microorganism(s) represented for use as supplements under the Fertilizers Act

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May 2017

1. Introduction

1.1 Purpose

The objective of this document is to provide guidance to applicants/product proponents on acceptable scientific methodologies and techniques for the identification of microorganisms intended for use as active ingredients in microbial supplementsFootnote 1.

This document focuses on the taxonomic identification of pure cultures of microorganisms belonging to the kingdoms of:

  1. bacteria,
  2. archaea,
  3. fungi.

It also includes guidance on identification methods for microbial consortia which contain multiple genera of microorganisms that function as a community. The document lists standard resources and reference materials on taxonomic classification and nomenclature for the above groups of microorganisms.

1.2 Microbial Identification

Knowledge of the phenotypic, genotypic and biological characteristics of a microorganism is imperative in differentiating it from its pathogenic and/or toxigenic relatives or other microorganisms that are detrimental to the health of plants, animals, humans and the environment. As such, accurate identification of the active microorganism(s) is a fundamental component of all safety assessments of microbial supplements regulated under the Fertilizers Act. Furthermore, the conclusions pertaining to product safety or its impacts on human health or the environment are valid only if the active microorganism(s) are correctly identified.

General guidance regarding hazardous properties of microorganisms known to be human pathogens may be sourced from the Public Health Agency of Canada's Centre for Biosecurity websiteFootnote 2.

2. Polyphasic approach to microbial identification

The selection of method(s) used for microbial identification depends on the type and nature of the microorganism. The method(s) chosen should be well-described in scientific literature and consistent with those currently used in the field of microbial identification and taxonomic classification and they must enable identification of the organisms to the genus and species and, if possible, strain level. The robustness, precision and validity of the methodologies used to identify the microorganism are critical elements in the assessment of the safety of the product.

The choice of methods for microbial identification is at the discretion of the product proponent. However, the Fertilizer Safety Section recommends that applicants adopt an integrated polyphasic approach that includes classical microbiological and phenotypic analysis along with molecular tools, to accurately identify the active microorganism(s). The strengths and weaknesses of the various identification methods should be taken into consideration, such that the methods chosen complement each other to result in a conclusive and definitive identification of the microorganism, and allow for clear differentiation of the organism from any closely related pathogenic and/or toxigenic species and strains. The methods commonly used in identification and substantiation of taxonomic classification of microorganisms are summarized below.

2.1 Phenotypic analysis

Preliminary analysis in microbial identification often involves one or more phenotypic methods. Phenotypic methods are suitable for microorganisms that are culturable (i.e., can grow as pure culture on artificial media), have well-established growth parameters, and physiological and biochemical profiles.

The expression of microbial phenotypes is highly dependent on environmental variables (e.g., culture pH, temperature, selective vs non-selective media, depletion of nutrients, presence of stressors, etc.), and thus, may introduce inconsistencies in the identification process. The phenotypic methods are only acceptable if the response criteria are sufficient to identify the microorganism with a high level of confidence and distinguish it from phylogenetically close relatives that potentially pose safety concerns. Furthermore, the applicability of the method is based on the robustness of information in reference databases. In consequence, results from phenotypic methods require supporting data from other methods to accurately identify a microorganism.

2.1.a Analysis of morphological traits

Colony and cell morphology are used to obtain an initial identification of a microorganism. This is accomplished through simple isolation and culturing of the microorganism and subsequent visual observation using microscopy. The morphological properties include:

  1. shape,
  2. size,
  3. surface characteristics and pigmentation,
  4. cell wall characteristics (Gram-staining),
  5. sporulation characteristics,
  6. mechanisms of motility, and
  7. other cellular inclusions and ultrastructural characteristics.

2.1.b Analysis of biochemical, physiological and metabolic characteristics

The study of the biochemical profile and metabolic properties of a microorganism by testing its growth requirements, enzymatic activities and cellular fatty acid composition are part of a phenotypic evaluation.

The biochemical tests use specific growth media, nutrients, chemicals or growth conditions to elicit an observable or measurable biochemical response from the microorganism, thereby enabling its identification and characterization. These tests include: utilization of carbon and nitrogen sources, growth requirements (anaerobic or aerobic; temperature-optimum and range, pH optimum and range), preferred osmotic conditions, generation of fermentation products, production of enzymes, production of antimicrobial compounds, as well as sensitivity to metabolic inhibitors and antibiotics. Examples of recognized tests include: phenol red carbohydrate, catalase and oxidase tests, oxidation-fermentation tests, methyl red tests, Voges-Proskauer tests, nitrate reduction, starch hydrolysis, tryptophan hydrolysis, hydrogen sulfide production, citrate utilization, litmus milk reactions, etc. Several miniaturized and automated commercial systems are currently available with well-defined quality control procedures that allow for rapid identification of microorganisms.

2.1.c Analysis of Fatty Acid Methyl Ester composition (FAME analysis)

Microorganisms can be identified by analysing the fatty acid profiles of whole cells or cell membranes using gas-liquid chromatography or mass spectrometry. The data on the type, content, proportion and variation in the fatty acid profile are used to identify and characterize the genus and species by comparing it against the fatty acid profiles of known organisms.

2.2 Molecular Methods

Molecular biology contributes a set of powerful new tools: molecular methods that have helped to detect the smallest variations within microbial species and even within individual strains. These methods have greatly improved the ability to rapidly detect, identify and classify microorganisms and also establish the taxonomic relationship among closely related genera and species. Identification, using molecular methods, relies on the comparison of the nucleic acid sequences (DNA, RNA) or protein profiles of a microorganism with documented data on known organisms. The molecular methods are considered sensitive enough to allow detection of low concentrations of viable or non-viable microorganisms in both pure cultures and complex samples (e.g., soil, peat, water etc.).

2.2.a Genotypic methods

Microbial identification using genotypic methods is an alternative or complement to established phenotypic methods. Broadly, genotypic microbial identification methods can be divided into following two categories:

  1. Pattern- or fingerprint-based techniques
  2. Sequence-based techniques

In pattern-based techniques, a series of fragments from an organism's chromosomal DNA are generated using a systematic method. The generated fragments are then separated based on their sizes to generate a profile, or a fingerprint that is unique to that organism and its very close relatives. With enough of this information, a library or database can be created. The profile of test micro-organism can then be compared to the library or the database. Based on the similarity between the profiles of two organisms, they can be considered either very closely related or not related. Examples of some of the pattern or fingerprint based techniques are:

  1. Nucleic acid hybridization (Southern blot analysis or Solution-phase hybridization),
  2. Amplified Fragment Length Polymorphism (AFLP),
  3. Pulsed Field Gel Electrophoresis (PFGE),
  4. Random amplification of polymorphic DNA,
  5. Multiplex PCR, etc.

In sequence-based techniques, the sequence of a specific stretch of DNA is determined. Similar to genotyping, a database of specific DNA sequences is generated, and then a test sequence is compared with it. The degree of similarity, or match, between the two sequences is a measurement of how closely related the two organisms are. To date, a number of computer algorithms have been created that can compare multiple sequences to one another and build a phylogenetic tree based on the results. Some examples of sequence- based techniques are:

  1. 16s rRNA gene sequence analysis,
  2. Multilocus Sequence Typing (MLST),
  3. Multilocus Sequence Analysis (MLSA), etc.

Both fingerprinting techniques and sequence-based methods have strengths and weaknesses. Traditionally, sequence-based methods, such as analysis of the 16S rRNA gene, have proved effective in establishing broader phylogenetic relationships among bacteria at the genus, family, order, and phylum levels, whereas fingerprinting-based methods are good at distinguishing strain or species-level relationships but are less reliable for establishing relatedness above the species or genus level. Other limitations associated with genotypic methods include:

  1. difficulties in differentiating between species that share identical and/or similar conserved region sequences
  2. limited information on the quality of sequence data available in public databases, and
  3. the complexity of taxonomic nomenclature overall.

Considering the above, it is important to validate the results of genotypic microbial identification methods with data from other sources (e.g., morphological and/or phenotypic analysis).

Reliable genotypic identification requires databases with accurate and complete sequence information from a large number of taxa. The commonly used gene sequence databases include:

  1. GenBank®Footnote 3;
  2. Ribosomal Database Project (RDP)Footnote 4
  3. Europe's collection of nucleotide sequence data (EMBL)Footnote 5; and
  4. Universal Protein Resource (UniProt)Footnote 6.

Product proponents are not limited to using the reference materials listed above; these are intended as guidance only.

2.2.b Protein based methods

Although genotypic information is valuable in identifying an organism and determining its relatedness to other organisms, methods based on an organism's phenotypic properties remain critical for understanding the physiological and functional activities of an organism at the protein level. Phenotypic methods that determine the activity of specific enzymes, such as catalase or oxidase, or metabolic functions, such as the ability to degrade lactose, have long been used as a basis for bacterial identification. The arrival of new proteomics tools that are based primarily on mass spectrometry and allow rapid interrogation of biomolecules produced by an organism offers an excellent complement to classical microbiological and genomics-based techniques for bacterial classification, identification, and phenotypic characterization. The predominant proteomic technologies that have been used for bacterial identification and characterization include:

  1. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS),
  2. Electro Spray Ionization Mass Spectrometry (ESI-MS),
  3. Surface-Enhanced Laser Desorption/Ionization (SELDI),
  4. Mass Spectrometry,
  5. One- or two-dimensional Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE),
  6. The combination of mass spectrometry, gel electrophoresis, and bioinformatics, etc.

Furthermore, serological methods such as Western blotting, Immuno-precipitation (IP) and Enzyme Linked Immunosorbant Assay (ELISA) use antibodies to detect specific proteins that is unique and/or characteristic of a microorganism. The applicability of serological methods is dependent on the availability, sensitivity and specificity of the antibodies used. There are commercial kits available for immuno-detection of several microorganisms.

2.3 Genomics

More recently, complete profiling of the transcriptome, genome, proteome or metabolome have been used to identify and characterise organisms. Several modern technologies such as DNA and protein microarray analyses, mass spectral protein profiling, nuclear magnetic resonance (NMR) spectral analysis, in-silico microbial metabolome platforms are increasingly used in identification and characterization of microorganisms.

The knowledge of the sensitivity and specificity of genomic tools and their application in microbial identification is rapidly evolving. However, challenges related to standardization of genomics methodologies (including optimization of protocols and bioinformatics tools for reliable data annotation, interpretation, etc.) continue to hinder their applicability in safety (risk) assessment and regulatory decision making. The Fertilizer Safety Section will consider data generated by genomics-methods on a case-by-case basis.

3. Identification methods for Microbial Consortia

For microbial consortia, the Fertilizer Safety Section recommends that applicants adopt one of the following three approaches:

3.1 Identification of all individual species through a polyphasic approach.

Where possible, this approach should be chosen. A combination of the following molecular, biochemical and microbiological methods can be used:

3.1.a Molecular methods

  1. PCR-based methods: PCR, ribosomal DNA and other taxonomically differentiated genes, amplified ribosomal DNA restriction analysis, denaturing gel electrophoresis, temperature gradient gel electrophoresis, terminal restriction fragment length polymorphism, ribosomal intergenic spacer analysis, single strand conformation polymorphism, length heterogeneity polymerase chain reaction, sequencing of cultured isolates, randomly amplified polymorphic DNA and functional PCR.
  2. Direct cloning and sequencing.
  3. Probe hybridization: DNA microarrays

3.1.b Biochemical methods

DNA composition and kinetic assays, metabolic assays and lipid analyses. Please also refer to 2.1.b and 2.1.c above.

3.1.c Microbiological methods

Cell counting techniques, flow cytometry and cell sorting, selective growth and microscopic examination.

3.2 Identify taxonomic groupings

Where precise taxonomic designation is not possible due to consortium size or low amenability to culturing, identification to the level of genus (and possibly family) can be used to describe the major constituents in a consortium. For example, 16S rRNA gene cloning and sequencing can be used to detect operational taxonomic units (OTUs) assigned to specific genera or families of microorganisms. The limited availability of environmental isolates in public databases and/or low concentrations of constituents within the consortium may be limiting factors to constituent identification. This approach or similar approaches can serve as a means of major genera or family identification for microorganisms within a consortium.

3.3 Screening for hazardous microbial components

Where taxonomic designation of all microorganisms is not feasible, data on the presence of indicator microorganisms can be submitted. The consortium should be screened for hazardous species relevant to the product in question with the level and taxonomic designation of each species clearly stated. Product-type and source-dependent indicators should be chosen by the proponent. For example, a product may be screened for species pathogenic to humans such as Salmonella sp., Listeria monocytogenes, Vibro sp., Campylobacter sp., Clostridia sp., Bacillus anthracis, Pseudomonas aeruginosa, Yersinia sp., Candida albicans, Aspergillus fumigatus, faecal coliforms, Enterococci, Rotavirus, Norovirus and Ascaris lumbricoides. Please note that the Fertilizer Safety Section may request additional indicator screening at the time of review. Pathogenicity tests should be done on the final product formulation. Controls utilizing any chemical filters and positive and negative controls must be provided along with the test data on the formulation.

4. Additional considerations for the taxonomic identification of Fungi

The classification and identification of fungi has relied on morphological criteria until advances in molecular techniques have allowed their application. Molecular techniques have become an important tool to establish the taxonomic status of different fungi. Section 2 (above) generally outlines the recommended polyphasic approach to taxonomic identification, which also applies to fungi.

Sexual dimorphism in fungi adds complexity to taxonomic identification. Many fungi have a teleomorph (sexual) and anamorph (asexual) form. These two forms have often been given different names and can even be in different a genus or family. Further, many anamorph-teleomorph pairs are yet unidentified and are only being discovered as sequencing data becomes available. Among the electrophoretic methods, restriction fragment length polymorphism (RFLP). RFLP-based typing methods have been used to reveal anamorph-teleomorph connections. Most commonly, the RFLP of PCR-amplified rRNA is used.

When using a public database to identify fungi based on nucleotide sequences, known relationships will be resolved, however bear in mind that different taxonomy information may result depending on the database used.

5. Assistance for taxonomic identification

We recommended that proponents seek assistance from a taxonomic identification service or professional. A number of the major culture collections/repositories and commercial laboratories offer fee-based identification and characterization services.

6. Taxonomic Classification and Nomenclature:

The taxonomic identification of the microorganism(s) should be based on the currently used and internationally accepted taxonomic classification system. The description of the microorganism(s) in the product and its characteristics must correspond to the characteristics described in standard resources and/or references that are commonly used by the scientific community to validate taxonomic classification. These can include but are not limited to:

  1. textbooks such as the Bergey's Manual of Systematic BacteriologyFootnote 7;
  2. The ProkaryotesFootnote 8;
  3. Applied Microbial SystematicsFootnote 9;
  4. Principles of fungal taxonomyFootnote 10;
  5. online resources such as the Catalogue of LifeFootnote 11;
  6. PubMed TaxonomyFootnote 12 and UniProt TaxonomyFootnote 13; and
  7. peer reviewed journals.

The taxonomic name should follow the nomenclature code officially recognized by the International Committee on Systematics of Prokaryotes (ICSP). Applicants should verify the "Approved List of Bacterial Names" to ensure that the nomenclature is in accordance with the latest Validation List developed and updated by the International Journal of Systematic and Evolutionary Microbiology (IJSEM)Footnote 14.

Please note that microbial taxonomic classification and nomenclature, particularly for bacteria, is in a constant state of flux as methodologies evolve to generate more reliable information to identify/classify and/or reclassify the current taxonomic scheme. Cross referencing more than one resource/reference will help in validating the current taxonomic designation and classification of a microorganism.

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