Taxonomy of microorganisms
Taxonomy is comprised of three elements: characterisation; classification; and nomenclature. The reliability of microbial typing, identification and the establishment of an effective microbiological resource centre (mBRC) is dependent upon a reliable and established taxonomy. Characterisation of a microbial strain may be argued to be the key element in microbial taxonomy. Microorganisms should be analysed and described as comprehensively as is reasonably possible, with the object of obtaining complete pictures of distinguishing traits, enabling stable classifications. Bacterial characterisation may follow two basic modes of analyses, i.e., characterisation of: 1) phenotypic traits; and 2) genotypic traits. Phenotypic traits are the observable features that result from the expression of the genes of an organism. Genotypic traits of an organism are those encoded within its genetic material, the genome.
Phenotypic characterisation of microorganisms
Phenotypic characterisations have been the traditional basis of bacterial descriptions and classifications. The methods employed have included descriptions of morphology, both cell and colony features. Physiological testing generates profiles of characteristic reactions for the utilisation of substrates or other features that offer insight into basic metabolic activities of bacteria. Various commercial test panels have been developed which have proven to be useful for automation and standardisation of testing. The protocols utilising physiological test panels are sensitive, often effective for discriminating the most closely related bacterial taxa, i.e., at the level of the strain.
Chemical characterisation (chemo-taxonomy) exploits differences in structural elements of bacterial cells, i.e., the cell wall, the cell membrane or the cytoplasm. Such differences may be detected in fatty acids, cell peptidoglycan, teichoic acids, mycolic acids, polar lipids, respiratory quinones, pigments and polyamines. MALDI-TOF Mass Spectrometry, targeting cell proteins on the basis of their masses, offers new high-resolution approaches to obtain rapid identifications of microorganisms.
Genotypic characterisation of microorganisms
The last nearly 50 years have witnessed an explosion in the application of DNA-based analyses for the characterisation of microorganisms. DNA sequence determinations and analyses have rapidly become the “methods of choice” among bacterial systematists, as those that offer the highest level of resolution and differentiation. With the application of targeted PCR-amplification and sequencing of ribosomal RNA (rRNA) genes, particularly the small subunit or 16S rRNA genes (Medlin et al., 1988), phylogenetic relationships of bacteria are able to be estimated rapidly, reliably and with reproducibility in different laboratories. More recently, gene sequencing, targeting selected “house-keeping” genes (i.e., genes of conserved enzymes essential for cellular function), as well as combinations of gene targets in “multi-locus” analyses, offer opportunities to exploit the varying degrees of conservation contained within genomes for elucidating bacterial taxa at increasingly higher levels of resolution (Bishop et al., 2009; Maiden et al., 1998).
The complete genome sequence has been accepted to be the reference standard to determine phylogeny and phylogeny should determine bacterial taxonomy (Wayne et al., 1998). With the increasing speed and the decreasing costs of DNA sequencing in recent years, the ability to cover complete genomes of bacteria has become reality with the recent introduction of “next-generation” sequencing technology.
Identification of microoganisms
The identification of microorganisms is directly dependent upon the characterisation of strains and the classification of strains, based upon the characterisation, and the categorisation of strains, dependent upon established nomenclature.
Characterisation and Identification in the CCUG
In the CCUG, we use phenotyping of strains, since 1968. Most strains held in the CCUG have been characterised phenotypically, to some degree. Since 2005, the CCUG has added genotypic characterisation to its identification protocols, predominantly through comparative 16SrRNA gene sequence analyses but also through developing and employing sequence analyses of other housekeeping genes for bacterial strain comparisions and species identifications.
16S rRNA gene comparative sequence analysis
The most common gene used for bacterial genotyping is the gene encoding the 16S rRNA. This ribosmal RNA is a part of the small subunit of the ribsome and is highly conserved among the whole eubacterial kingdom. The gene is approximately 1,500 base-pairs long and has highly conserved regions and regions of sequence variability that can be used for species discrimination and identification.
The degree of similarity of this gene is an important factor in the definition of bacterial species and a sequence difference greater than 1.5% is considered to indicate different species. There are many examples of different bacterial species exhibiting 16S rRNA gene sequence similarities greater than 1.5%, demonstrating a limited variation of this gene and capability for species-level differentiation. At the CCUG. 16S rRNA gene sequence-based genotyping is an accredited analysis, documented by the Swedish authorities overseeing the protocols used in hospital clinical laboratories.
Housekeeping genes comparative sequence analyses
When the 16S rRNA gene sequences of microorganisms are not able to provide reliable identifications, we often use other conserved, functional genes for analyses. For different bacterial genera, we have selected different genes as the best for resolving the species. Frequently we use genes known to provide good separation within a certain genus but we also The CCUG is able to carry out multi-locus sequence analysis (MLSA), employing targeted PCR-amplification and comparative sequence analyses for identification of species of approximately 15 – 20 different bacterial genera.
Whole-genome sequence determination and analyses
Since 2015, the CCUG has applied approaches of whole-genome sequence determinations, to draft levels or to closed-genome levels, for in depth analyses …