T-4- 126 – Identification and taxonomic classification of microorganism(s) represented for use as supplements under the Fertilizers Act
The purpose of this document is to provide guidance on acceptable scientific methodologies and techniques for the identification of microorganisms intended for use as active ingredients in microbial supplements.
This document focuses on the taxonomic identification of pure cultures of microorganisms belonging to the kingdoms of: 1) bacteria, 2) archaea, 3) fungi; either naturally occurring or those modified by genetic manipulation. It also includes guidance on identification methods for microbial consortia.
2. Microbial identification
Knowledge of the phenotypic, genotypic and biological characteristics of a microorganism is essential 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. 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 or animal pathogens may be sourced from the Public Health Agency of Canada's Centre for Biosecurity website and the Public Health Agency's ePATHogen Risk Group Database.
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 CFIA recommends that applicants include classical microbiological and phenotypic analysis along with molecular tools to ensure accuracy of identification. 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. Methods commonly used in identification and substantiation of taxonomic classification of microorganisms are summarized below.
2.1 Phenotypic analysis
Phenotypic methods are suitable for microorganisms that can grow as pure culture on artificial media and have well-established growth parameters, physiological and biochemical profiles.
The expression of microbial phenotypes is highly dependent on environmental variables (for example, culture pH, temperature, selective vs non-selective media, depletion of nutrients, presence of stressors, etc.), and thus, may introduce inconsistencies in the identification process. 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. To be accepted, phenotypic analysis requires supporting data from other methods as part of a polyphasic approach.
2.1.1 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:
- surface characteristics and pigmentation
- cell wall characteristics (Gram-staining)
- sporulation characteristics
- mechanisms of motility and
- other cellular inclusions and ultrastructural characteristics
2.1.2 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.
2.1.3 Analysis of Fatty Acid Methyl Ester composition (FAME analysis)
Microorganisms can be identified by analyzing the fatty acid profiles of whole cells or cell membranes using gas-liquid chromatography or mass spectrometry. Fatty acid profiles (type, content, proportion and variation) are compared against those of known organisms to identify genus and species.
2.2 Molecular methods
Molecular biology contributes a set of powerful new tools. 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 (for example, soil, peat, water etc.).
2.2.1 Pattern- and sequence-based genotypic methods
In pattern-based techniques, a series of fragments from an organism's chromosomal DNA are generated. The fragments are then separated based on size to generate a profile, or a fingerprint that is unique to that organism and its very close relatives. This information is compiled to generate a database of fingerprints for known organisms to which the profile of test micro-organism can be compared. 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 based techniques are:
|Repetitive element PCR (polymerase chain reaction)||Primers target specific repetitive elements distributed in chromosomes at random|
|Amplified fragment length polymorphism (AFLP)||Chromosomal DNA is digested by restriction enzymes followed by PCR using adapters coupled to the restriction sites|
|Riboprinting||Chromosomal DNA is digested by restriction enzymes followed by probing for ribosomal genes|
|Random amplification of polymorphic DNA||Short stretches of chromosomal DNA are randomly amplified by a set of arbitrary short primers|
|Pulsed-field gel electrophoresis||Rare –cutting restriction enzymes are used to cut chromosomal DNA into large fragments and fragments are determined|
|Multiplex PCR||Diagnostic genes targeted by PCR primers|
In sequence-based techniques, the sequence of a specific stretch of DNA is determined. The degree of similarity, or match, between a DNA sequence from a database and a test sequence is a measurement of how closely related the two organisms are. Computer algorithms are then used to compare multiple sequences to one another and build a phylogenetic tree based on the results. Some examples of sequence- based techniques are:
|16S ribosomal RNA (16S rRNA) gene sequencing analysis||Conserved primers are used to amplify then sequence the rRNAgene, sequences are then compared with a database for genus and species identification of microorganisms.|
|Internal transcribed spacer (ITS) rRNA gene sequencing analysis||Specific primers are used to amplify, then sequence internal transcribed spacers between the 16S and 23S rRNA gene loci. Sequences are then compared with a database for identification of microorganisms at the species and sub-species levels.|
|Multilocus sequence typing (MLST) and Multilocus sequence analysis (MLSA)||DNA sequencing of a specific subset of conserved and semiconserved genes for a given species followed by a comparison of concatenated sequences|
Both fingerprinting techniques and sequence-based methods have strengths and weaknesses. 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. Considering these limitations, it is important to validate the results of genotypic microbial identification methods with data from other sources (for example, 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:
- Ribosomal Database Project (RDP)
- Europe's collection of nucleotide sequence data (EMBL) and
- Universal Protein Resource (UniProt)
Product proponents are not limited to using the reference materials listed above; these are intended as guidance only.
2.2.2 Proteomics-based methods
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. Proteomics tools offer 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:
- Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDITOF-MS)
- Electro Spray Ionization Mass Spectrometry (ESI-MS)
- Surface-Enhanced Laser Desorption/Ionization (SELDI)
- Mass Spectrometry
- One- or two-dimensional Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)
- The combination of mass spectrometry, gel electrophoresis, and bioinformatics, etc.
Serological methods such as Western blotting, Immuno-precipitation (IP) and Enzyme Linked Immunosorbant Assay (ELISA) use antibodies to detect specific proteins that are unique to and/or characteristic of a microorganism. The applicability of serological methods is dependent on the availability, sensitivity and specificity of the antibodies used.
2.3 Whole genome sequencing
Whole-genome sequencing (WGS) is a comprehensive method for analyzing entire genomes. The ability to produce large volumes of data with today's sequencers make whole-genome sequencing a powerful tool for genomics research. WGS provides a high-resolution, base-by-base view of the entire genome, captures both large and small variants and delivers large volumes of data in a short amount of time. This approach allows investigation of all genes from single organism culture, providing a comprehensive analysis of the microbial genome including precise strain identification. WGS can detect single nucleotide variants, insertions/deletions, copy number changes, and large structural variants.
Whole genome sequencing can be considered the ultimate tool in strain characterization. As such, WGS results will be accepted as a stand-alone substantiation of microbial identification as part of an application for registration under the Fertilizers Act.
3. Identification methods for microbial consortia
A microbial consortium is a complex community of microorganisms taken from a single natural environment whose composition is maintained without further manipulation. The CFIA recommends one of the following approaches for taxonomic identification of microbial consortia:
3.1 Identification of all individual species through a polyphasic approach
Where possible, this approach should be chosen. A combination of molecular, biochemical and microbiological methods may be used.
3.2 Identify taxonomic groupings and screening for hazardous microbial components
Where precise taxonomic designation is not possible, 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.
Where taxonomic groupings are identified, the consortium should be screened for hazardous species relevant to the product in question. 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. 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).
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.
Fertilizer Safety Section
Canadian Food Inspection Agency
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