LECTURE TOPIC: TAXANOMY AND CLASSIFICATION OF BACTERIA BABCOCK UNIVERSITY COURSE CODE: MLSC 417 LECTURE TOPIC: TAXANOMY AND CLASSIFICATION OF BACTERIA

TAXANOMY It is likened to a skilful blend in which it is not easy to discern the ingredients (Cowan & Steele). Ingredients: Classification, Nomenclature and Identification.

Classification: Orderly arrangement of units into groups based on acceptable set of criteria. Nomenclature: Naming or labeling of the groups or units and individuals within the group. Identification: Entails the assignation of an unknown organism to a group within a scheme of classification.

Classification of Bacteria There is no official classification of bacteria. Taxanomy remains a matter of scientific judgment and a generally accepted standard. The commonly accepted classification is as listed in 2nd edition of Bergey’s Manual of Systematic Bacteriology.

Types of classification 1. Phylogenic classification Based on evolutionary (ancestry) origins. The ancestors of present day bacteria existed for 3 billion years. This system was used to recognise two domains of Bacteria and Archaea. It is the basis of naming (nomenclature) of bacteria.

2. Classification by phenotypic characteristics Morphological characteristics Biotyping Serotyping Antibiogram patterns Pyocin typing Phage typing

Morphology: Microscopic and macroscopic morphologies of bacteria were the first characteristics used to identify bacteria. Still the cornerstone for most identification algorithms used today, e.g. i) The Gram’s stain is used to classify bacteria based on their shape and staining reaction. ii) Macroscopic appearance of colonies on culture plates such as haemolysis on blood agar plates, pigmentation on culture media. ,

Biochemical typing is the most used method for definitive identification. Biotyping is also used for dividing group of organisms beyond species level. Serological typing is useful to identify organisms that are inert in biochemical testing e.g. Treponema pallidum, for rapid identification of some species e.g. Strep. pyogenes etc.

Antimicrobial pattern: pattern of susceptibility to antibiotics. Pyocin typing: Pyocins are a group of low molecular weight bactericidal proteins. Their bactericidal activity pattern can be used to classify bacteria. Phage typing: Susceptibility to phages (or bacterial viruses) is used to identify bacteria.

3. Classification by analytical characteristics. Cell wall analysis Whole cell lipid analysis Whole cell protein analysis Enzyme typing

Cell wall content of mycolic acid has been used to identify species of mycobacteria (ZN stain). Analysis of lipid content of the whole cell is used to characterise some bacteria. Commercial systems are available for performing this analysis.

Whole cell protein typing are handy tools in characterising bacteria especially at species level for epidemiological purposes. This is done in reference laboratories. Cellular enzyme typing is a handy tool in characterising bacteria especially at species level for epidemiological purposes.

4.Numerical classification: This a mathematical approach whereby all features are assigned equal values. This is the original Adansonian concept. The advent of computers has made this approach to taxanomy easier to apply. Used mainly for research

5. Genetic classification aka Molecular genetics: This is most precise and accurate. Includes systems such as: Guanine:Cytosine ratio: This is now obsolete and the method has been discarded.

b) DNA hybridization: Involves comparison of base sequence compatibility between two strains: to determine if two isolates were in the same genus or species. DNA probe: A probe from a marker strain is annealed with DNA from test strain. If the marker is bound to the DNA of test strain, the identity is confirmed.

Put in another way, DNA from the organism to be identified is extracted and exposed to the species specific probes. If the probes bind to the DNA, then the organism’s identity is confirmed. Can be applied directly to clinical specimens. It is definitive but tedious and requires expertise to handle.

c) Nuleic acid sequencing aka DNA amplification Extension of the hybridization method is Nucleic Acid sequencing or DNA amplification. Most recent and modern, probes are used to localise sequences that are unique to genus, species, sub-species.

Various methods are used but the commonest are PCR and LCR. This method is changing the face of laboratory diagnosis of infections. It requires expertise and it is pretty expensive. Very wide application.

d) Other genetic typing include i)Ribotyping and Ii) Plasmid analysis and Iii) Chromosomal DNA fragments. These are highly sophisticated systems and are not in routine use. .

Other approaches to classification Specific features: Based on specific , distinguishable features shared by all in the group. This classification is according to Bergey’s Manual of Determinative Bacteriology. The features that require difficult process or special apparatus are not listed.

NOMENCLATURE The term species is most commonly used in the pragmatic classifying system. It is the basic unit in the hierarchy of microbial world. The basis of this category is that members of the same species are able to produce others of their kind. The next higher category is genus.

bacteria with similar features are placed in The same genus comprises several species which may differ in some aspects. Further up the ladder of classification, bacteria with similar features are placed in successive larger categories. Thus similar genera are grouped into family; similar families are categorised as order; similar orders make up class and similar classes make up the phylum (kingdom).

Theoretical classification and Identification: This provides the bacteriologist with a relatively simple and pragmatic ways of placing bacteria into categories. Consideration is given to: Colonial morphology – This requires a measure of experience to be able to identify an organism by colonial appearance.

This is usually confirmed with a few rapid tests. Most isolates are divided into commensal, contaminants of no clinical interest, possible pathogen or probably pathogen based partially on this. Thus, it reduces the workload of a clinical laboratory.

Gram’s stain In 1884, a Danish scientist, Christian Gram, introduced a staining technique that indeed simplified and enhanced the classification of bacteria. The Gram’s stain has become the foundation on which bacterial identification is laid. The staining technique is cell wall dependent and simply divides bacteria into two groups: Gram positive and Gram negative.

The “key”: e.g. Coagulase test divides the species of staphylococcus into two groups: CPS and CNS Note that bacterial species are polythetic i.e. a property may vary within a species. Reaction pattern of an unknown isolate is compared to the “key” and its “goodness of fit” with each species is determined.

The key is now replaced by some identification systems such as the API system. Analytical Profile Index (API) The numerical profile of the unknown isolate is compared with an existing numerical pattern for a known species.

The highest index (best fit) as calculated for the unknown is the identity of the unknown. Unusual patterns may be due to many factors e.g. impure culture. Giving an isolate a species name: This requires careful consideration.

Once identified, the isolate is assumed to possess all the implied properties including biochemistry, pathogenicity capability etc. If unequivocal, state that the isolate is unusual. Routine identification of bacteria The diagnostic bacteriologist bases his identification strategy on what is termed Characterization tests

Routine identification of bacteria The diagnostic bacteriologist bases his identification strategy on what is termed Characterization tests These tests include : Microscopic morphology: Cell shape e.g. coccus

Size Arrangement .e.g. clusters, chain Staining e.g. Gram + or Gram - Capsule characteristic Spore morphology e.g. round, oval, terminal Flagellar arrangement e.g. monotrichous

Colonial morphology Solid media: For an experienced bench bacteriologist, data from this source is often sufficient for identification at genus level. Most clinical isolates discarded as non significant are identified usually by colonial appearance.

Action on media- Haemolysis - Swarming Further incubation may be necessary in order to ascertain the correct colonial morphology of a suspected pathogen. Action on media- Haemolysis - Pigmentation - Swarming

Biochemical tests Essential tool for bacterial classification and identification. Examples - Fermentation/oxidation of carbohydrates - Waste products - Metabolism of organic acids, lipids, proteins and amino acids.

- pH or redox range of growth -Tolerance of chemical agents In short, these tests collectively define nutritional and physiological interaction of the organism with its enviroment. - Usually technically simple and inexpensive

especially when adapted to multi point inoculating commercial test strips or micro titre plate formats. But they all have hidden complexities. - Most yield positive (+) or negative (-) results that are easy to read. Enzyme activity Characterization tests also include the action of various enyzmes or toxins produced by organisms.

Examples: - Coagulase for Staphylococcus species - Lecthinase for Clostridium species - Urease for Proteus species The above approach is pragmatic, simple and fast enough that the bench bacteriologist is able to play his role in the overall management of disease.

Systematic Bacteriology The systematic approach to the identification of an isolated bacterium is referred to as Systematic Bacteriology. The bench bacteriologist combines his theoretical knowledge of bacteriology, disease aetiology and clinical information as contained in request form, to initiate a systematic identification of an isolated bacterium.

With this, identification as set out above becomes easier. MERCI