MIC 206 Polyphasic Taxonomy PDF

# Polyphasic Taxonomy - Polyphasic taxonomy is a modern approach to microbial classification that combines various techniques to provide a more accurate and comprehensive understanding of a microorganism's characteristics. - Unlike traditional methods that relied solely on morphological features, p...

# Polyphasic Taxonomy - Polyphasic taxonomy is a modern approach to microbial classification that combines various techniques to provide a more accurate and comprehensive understanding of a microorganism's characteristics. - Unlike traditional methods that relied solely on morphological features, polyphase taxonomy integrates genotypic, phenotypic, and chemotaxonomic data to establish a robust classification system. - The term "polyphasic taxonomy" was introduced by Colwell (1970). - In practice, determination of the genus and species of a newly discovered prokaryote is based on polyphasic taxonomy. - This approach includes phenotypic, phylogenetic, and genotypic features. - Our recent understanding of the evolutionary relationships among microbes now serves as the theoretical underpinning for taxonomic classification. ## Classification of Microorganisms - Classification of microorganisms was earlier done on the basis of traditional microbiological methods (morphological, physiological and biochemical). - However, these techniques are time consuming as well as dependent upon many environmental factors. - The advanced techniques like sequence based, gel based and protein based systems have become advantageous due to their fast reactions, high specificity and less chance of error. - The methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. ## Key Components of Polyphase Taxonomy 1. **Genotypic Analysis:** - **Sequencing:** This involves determining the nucleotide sequence of a microorganism's genome, including its 16S rRNA gene, which is a commonly used marker for bacterial classification. - **Whole-Genome Sequencing:** This provides a complete picture of a microorganism's genetic makeup, allowing for in-depth analysis of its functional capabilities and evolutionary relationships. 2. **Phenotypic Analysis:** - **Morphological Characteristics:** This includes studying the shape, size, and arrangement of cells, as well as the presence of structures like flagella, endospores, and capsules. - **Physiological and Biochemical Properties:** This involves testing a microorganism's ability to utilize different carbon and nitrogen sources, produce enzymes, and tolerate various environmental conditions. 3. **Chemotaxonomic Analysis:** - **Cell Wall Composition:** This examines the composition of the cell wall, including the presence of peptidoglycan, teichoic acids, and lipopolysaccharides. - **Lipid and Fatty Acid Profiles:** This analyzes the types of lipids and fatty acids present in the microorganism's cell membrane. ## Advantages of Polyphase Taxonomy - Improved Accuracy: By combining multiple types of data, polyphase taxonomy provides a more accurate and reliable classification of microorganisms - Enhanced Resolution: It can distinguish between closely related microorganisms that may have similar morphological features but different genetic characteristics - Increased Confidence: The use of multiple techniques strengthens the confidence in the classification of a microorganism. - Discovery of New Species: Polyphase taxonomy can lead to the discovery of new microbial species that may have been overlooked using traditional methods. ## Classification Vs Identification | Criterion or Method | Used for Classification | Identification | |---|---|---| | Morphological characteristics | No (yes for cyanobacteria) | Yes | | Differential staining | Yes (for cell wall type) | Yes | | Biochemical testing | No | Yes | | Serology | No | Yes | | Phage typing | No | Yes | | Fatty acid profiles | No | Yes | | Flow cytometry | No | Yes | | DNA base composition | Yes | No | | DNA fingerprinting | No | Yes | | rRNA sequencing | Yes | No | | PCR | Yes | Yes | | Nucleic acid hybridization | Yes | Yes (DNA probes, DNA chips) | - A classification scheme provides a list of characteristics and a means for comparison to aid in the identification of an organism. - Once an organism is identified, it can be placed into a previously devised classification scheme. - Microorganisms are identified for practical purposes-for example, to determine an appropriate treatment for an infection. - They are not necessarily identified by the same techniques by which they are classified. - Most identification procedures are easily performed in a laboratory and use as few procedures or tests as possible. - Protozoa, parasitic worms, and fungi can usually be identified microscopically. - Most prokaryotic organisms don't have distinguishing morphological features or even much variation in size and shape. - Consequently, microbiologists have developed a variety of methods to test metabolic reactions and other characteristics to identify prokaryotes. - The observable characteristics-the phenotype-of a bacterium provide many traits that can be used to differentiate species. - Typically, for either describing a new species or identifying a bacterium, several of these traits are determined for the organism of interest. - The results are then compared with phenotypes of known organisms, either examined in parallel with the unknowns or from published information - The specific traits used depend on the kind of organism, and which traits are chosen for testing may arise from the investigator's purpose and from substantial prior knowledge of the bacterial group to which the new organism likely belongs. - For example, in applied situations, such as in clinical diagnostic microbiology, where identification may be an end in itself and time is of the essence, a well-defined subset of traits is typically used that quickly discriminates between likely possibilities. - 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 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. ## 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. ## Classical Characteristics - Classical approaches to taxonomy make use of morphological, physiological, biochemical, and ecological characteristics. - These characteristics have been employed in microbial taxonomy for many years and form the basis for phenetic classification. - When used in combination, they are quite useful in routine identification and may provide phylogenetic information as well. ## Morphological Characteristics - Morphological features are important in microbial taxonomy for many reasons. - Morphology is easy to study and analyze. - In addition, morphological comparisons are valuable because structural features depend on the expression of many genes, are usually genetically stable, and normally (at least in eucaryotes) do not vary greatly with environmental changes. - 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: - shape - size - surface characteristics and pigmentation - cell wall characteristics (Gram-staining) - sporulation characteristics - mechanisms of motility and - other cellular inclusions and ultrastructural characteristics - The transmission and scanning electron microscopes, with their greater resolution, have immensly aided the study of all microbial groups. - But many microorganisms look too similar to be classified by their structures alone. - Organisms that might differ in metabolic or physiological properties may look alike under a microscope. - Literally hundreds of bacterial species are small rods or small cocci. - Larger size and the presence of intracellular structures do not always mean easy classification, however. - Pneumocystis pneumonia is the most common opportunistic infection in AIDS and other immunocompromised patients. - Until the AIDS epidemic, the causative agent of this infection, _P. jirovecii_ [formerly "P. carinii"] was rarely seen in humans. - Pneumocystis lacks structures that can be easily used for identification, and its taxonomic position has been uncertain since its discovery in 1909 by Carlos Chagas in mice. - It was originally classified as a protozoan; however, in 1988 rRNA sequencing showed that Pneumocystis is actually a member of the Kingdom Fungi. - New treatments are being investigated as researchers consider this organism's relatedness to fungi. - Cell morphology tells us little about phylogenetic relationships. - However, morphological characteristics are still useful in identifying bacteria. - For example, differences in such structures as endospores or flagella can be helpful. - Many different morphological features are employed in the classification and identification of microorganism. ## Physiological and Metabolic Characteristics - Physiological and metabolic characteristics are very useful because they are directly related to the nature and activity of microbial enzymes and transport proteins. - Because proteins are gene products, analysis of these characteristics provides an indirect comparison of microbial genomes. ## Biochemical 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. ## Analysis of Fatty Acid Methyl Ester composition (FAME analysis) - Among the more useful biochemical characteristics used in microbial taxonomy are bacterial fatty acids, which can be analyzed using a technique called fatty acid methyl ester (FAME) analysis. - A fatty acid profile reveals specific differences in chain length, degree of saturation, branched chains, and hydroxyl groups. - Microbes of the same species will have identical fatty acid profiles, provided they are grown under the same conditions; this limits FAME analysis to only those microbes that can be grown in pure culture. - Finally, because a species is identified by comparing the results of the unknown microbe in question with the FAME profile of other, known microbes, identification is only possible if the species in question has been previously analyzed. - Nonetheless, FAME analysis is particularly important in public health, food, and water microbiology. - In these applications, microbiologists seek to identify specific microbial pathogens. ## Ecological Characteristics - The ability of a microorganism to colonize a specific environment is of taxonomic value. - Some microbes may be very similar in many other respects but inhabit different ecological niches, suggesting they may not be as closely related as first suspected. - Some examples of taxonomically important ecological properties are life cycle patterns; the nature of symbiotic relationships; the ability to cause disease in a particular host; and habitat preferences such as requirements for temperature, pH, oxygen, and osmotic concentration. - Many growth requirements are considered physiological characteristics as well. ## Molecular Characteristics - It is hard to overestimate how the study of DNA, RNA, and proteins has advanced our understanding of microbial evolution and taxonomy. - Evolutionary biologists studying plants and animals draw from a rich fossil record to assemble a history of morphological changes; in these cases, molecular approaches supplement such data. - In contrast, microorganisms have left almost no fossil record, so molecular analysis is the only feasible means of collecting a large and accurate data set that explores microbial evolution. - When scientists are careful to make only valid comparisons, phylogenetic inferences based on molecular approaches provide the most robust analysis of microbial evolution. - Since the first determination of the first whole genome sequence of _Haemophilus influenzae_ became available, high throughput sequencing (HTS) has been introduced and is more cost effective - At the same time, developments of user friendly bioinformatic analytical tools led to new insights in content and functioning of the sequenced prokaryotic genomes. - It is therefore not surprising that whole genome sequencing (WGS) data tend to underpin more and more prokaryotic systematics because it is undoubtedly the best resource to reveal natural relationships of prokaryotes. - 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.) ## Examples of molecular methods | Method | Technology | |---|---| | 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. | | 6S 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 foridentification 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. |