3 Classification, identification and typing of micro-organisms T. L. Pitt and M. R. Barer The algae (excluding the blue–green algae), the proto- KEY POINTS zoa, slime moulds and fungi include the larger eukary- otic (see Ch. 2) micro-organisms; their cells have the • Taxonomy is the classification, nomenclature and same general type of structure and organization as identification of microbes (algae, protozoa, slime that found in plants and animals. The bacteria, includ- moulds, fungi, bacteria, archaea and viruses). The ing organisms of the mycoplasma, rickettsia and naming of organisms by genus and species is chlamydia groups, together with the related blue– governed by an international code. green algae, comprise the smaller micro-organisms, • Bacteria can be separated into two major divisions with the form of cellular organization described as by their reaction to Gram’s stain, and exhibit a range prokaryotic. The archaea are a distinct phylogenetic of shapes and sizes from spherical (cocci) through group of prokaryotes that bear only a remote ances- rod shaped (bacilli) to filaments and spiral shapes. tral relationship to other organisms (see Ch. 2). As the • In clinical practice, bacteria are classified by algae, slime moulds and archaea are not currently macroscopic and microscopic morphology, their thought to contain species of medical or veterinary requirement for oxygen, and activity in phenotypic importance, they will not be considered further. Blue– and biochemical tests. green algae do not cause infection, but certain species • Various diagnostic test systems are used to detect produce potent peptide toxins that may affect persons specific bacteria in clinical systems, including specific or animals ingesting polluted water. gene probes, reaction with antibodies in ELISA The viruses are the smallest of the infective agents; formats, immunofluorescence and, increasingly, they have a relatively simple structure that is not com- PCR-based technology. parable with that of a cell, and their mode of repro- • Different bacterial species often exhibit different duction is fundamentally different from that of cellular population structures, highly diverse (panmictic) or organisms. Even simpler are viroids, protein-free relatively uniform (clonal) depending mainly on the fragments of single-stranded circular RNA that cause frequency of gene recombination (from external disease in plants. Another class of infectious particles sources). are prions, the causative agents of fatal neurodegen- • Typing of bacterial isolates is necessary for erative disorders in animals and man. These are pos- epidemiological investigations in outbreaks and for tulated to be naturally occurring host cell membrane surveillance, and a variety of phenotypic and genetic glycoproteins that undergo conformative changes to methods has evolved for the identification of strains. an infectious isoform (see Ch. 60). Micro-organisms may be classified in the following TAXONOMY large biological groups: 1. Algae Taxonomy consists of three components: classification, 2. Protozoa nomenclature and identification. Classification allows 3. Slime moulds the orderly grouping of micro-organisms, whereas 4. Fungi nomenclature concerns the naming of these organisms 5. Bacteria and requires agreement so that the same name is used 6. Archaea unambiguously by everyone. Changes in nomencla- 7. Viruses. ture may give rise to confusion and are subject to 24 CLASSIfication, idenTIfication and tyPInG of mICRo-oRGAnISmS 3 internationally agreed rules. In clinical practice, micro- Some genera, such as Acinetobacter, have been subdi- biologists are generally concerned with identification vided into a number of genomic species by DNA – the correct naming of isolates according to agreed homology analysis. Some are named and others are systems of classification. These components, together referred to only by a number. Many of the genomic with taxonomy, make up the overarching discipline of species cannot be differentiated with accuracy by phe- systematics, which is concerned with evolution, genet- notypic tests. Another subgenus grouping in current ics and speciation of organisms, and is commonly usage recognizes species complexes, which are differ- referred to as phylogenetics. entiated into genomovars by polyphasic taxonomic Protozoa, fungi and helminths are classified and methods. A good example of this is the B. cepacia named according to the standard rules of classifica- complex of organisms, which includes a very diverse tion and nomenclature that have been developed fol- group of organisms ranging from strict plant to lowing the pioneering work of the eighteenth century human pathogens. Swedish botanist Linnaeus (Carl von Linné). Large At present no standard classification of bacteria is subdivisions (class, order, family, etc.) are finally clas- universally accepted and applied, although Bergey’s sified into individual species designated by a Latin Manual of Determinative Bacteriology is widely used binomial, the first term of which is the genus, e.g. as an authoritative source. Bacterial nomenclature is Plasmodium (genus) falciparum (species). Occasion- governed by an international code prepared by the ally it is useful to recognize a biological variant with International Committee on Systematic Bacteriology particular properties: thus, Trypanosoma (genus) and published as Approved Lists of Bacterial Names brucei (species) gambiense (variant) differs from the in the International Journal of Systematic and Evolu- variant T. brucei brucei in being pathogenic for man. tionary Microbiology; most new species are also first Bacteria are similarly classified, but bacterial diver- described in this journal, and a species is considered sity encompasses more variety than all the rest of to be validly published only if it appears on a valida- cellular life put together and the natural capacity of tion list in this journal. bacteria for genetic and phenotypic variation and The International Committee on Taxonomy of adaptation make rigid classifications difficult. To Viruses (ICTV) classifies viruses and publishes its date, identification has predominantly been performed reports in the journal Archives of Virology. Latin by the use of keys that allow the organization of bac- names are used wherever possible for the ranks family, terial traits based on growth or activity in systems that subfamily and genus, but at present there are no test their biochemical properties. Some tests are defin- formal categories higher than family and binomial itive of a genus or species, for example the universal nomenclature is not used for species. Viruses do not production of catalase enzyme and cytochrome c, lend themselves easily to classification according to respectively, by Staphylococcus spp. and Pseudomonas Linnaean principles, and vernacular names still have aeruginosa. Other characters may be unique to indi- wide usage among medical virologists. Readers are vidual species and serve to differentiate them from referred to the standard work on virus taxonomy organisms with closely similar biochemical activity Classification and Nomenclature of Viruses and the profiles. Some bacteria do not grow in the laboratory ICTV database website. (leprosy bacillus, treponemes), and identification by genetic methods may be necessary. As the technolo- gies for genetic analysis become more readily applica- METHODS OF CLASSIFICATION ble in clinical labs, so they and other rapid analytical methods, such as those based on mass spectrometry, Adansonian or numerical classification are coming to replace the traditional biochemical methods to achieve identification. The taxonomic In most systems of bacterial classification, the major ranks used in the classification of bacteria are (example groups are distinguished by fundamental characters in parentheses): such as cell shape, Gram-stain reaction and spore for- mation; genera and species are usually distinguished • Kingdom (Prokaryotae) by properties such as fermentation reactions, nutri- • Division (Gracilicutes) tional requirements and pathogenicity. The relative • Class (Betaproteobacteria) ‘importance’ of different characters in defining major • Order (Burkholderiales) and minor groupings is often purely arbitrary. The • Family (Burkholderiaceae) uncertainties of arbitrary choices are avoided in the •( Genus Burkholderia) Adansonian system of taxonomy. This system deter- •( Species Burkholderia cepacia). mines the degrees of relationship between strains by a 25 3 mICRoBIAL BIoLoGy Percentage similarity component of bacterial DNA, varying from about 40 50 60 70 80 90 100 25–80% mol in different genera. However, for any one species, the G + C content is relatively fixed, or falls A within a very narrow range, and this provides a basis B for classification. E I Strains C DNA homology F Another approach to classification is to arrange indi- G vidual organisms into groups on the basis of the J homology of their DNA base sequences. This exploits D the fact that double strands re-form (anneal) from H separated strands during controlled cooling of a heated preparation of DNA. This process can be XY readily demonstrated with suitably heated homolo- Fig. 3.1 Hierarchical taxonomic tree (dendrogram) prepared from gous DNA extracted from a single species, but it can similarity matrix data. The dashed lines X and Y indicate levels of also occur with DNA from two related species, so that similarity at which separation into genera and species might be hybrid pairs of DNA strands