Chapter II Classification of bacteria.ppt

Editor's Notes

• #3: Approaches to Taxonomy Numerical Approach In their studies on members of the family Enterobacteriaceae, Edwards and Ewing established the following principles to characterize, classify, and identify organisms (Lennette et al., 1985): Classification and identification of an organism should be based on its overall morphologic and biochemical pattern. A single characteristic (pathogenicity, host range, or biochemical reaction), regardless of its importance, is not a sufficient basis for classifying or identifying an organism. A large and diverse strain sample must be tested to determine accurately the biochemical characteristics used to distinguish a given species. Atypical strains often are perfectly typical members of a given biogroup within an existing species, but sometimes they are typical members of an unrecognized new species. In numerical taxonomy (also called computer or phenetic taxonomy) many (50 to 200) biochemical, morphological, and cultural characteristics, as well as susceptibilities to antibiotics and inorganic compounds, are used to determine the degree of similarity between organisms. In numerical studies, investigators often calculate the coefficient of similarity or percentage of similarity between strains (where strain indicates a single isolate from a specimen). A dendrogram or a similarity matrix is constructed that joins individual strains into groups and places one group with other groups on the basis of their percentage of similarity. In the dendrogram in Figure 3-1, group 1 represents three Citrobacter freundii strains that are about 95 percent similar and join with a fourth C freundii strain at the level of 90 percent similarity. Group 2 is composed of three Citrobacter diversus strains that are 95 percent similar, and group 3 contains two E coli strains that are 95 percent similar, as well as a third E coli strain to which they are 90 percent similar. Similarity between groups 1 and 2 occurs at the 70 percent level, and group 3 is about 50 percent similar to groups 1 and 2. In some cases, certain characteristics may be weighted more heavily; for example, the presence of spores in Clostridium might be weighted more heavily than the organism's ability to use a specific carbon source. A given level of similarity can be equated with relatedness at the genus, species, and, sometimes, subspecies levels. For instance, strains of a given species may cluster at a 90% similarity level, species within a given genus may cluster at the 70 percent level, and different genera in the same family may cluster at the 50 percent or lower level (Fig. 3-1). When this approach is the only basis for defining a species, it is difficult to know how many and which tests should be chosen; whether and how the tests should be weighted; and what level of similarity should be chosen to reflect relatedness at the genus and species levels. Most bacteria have enough DNA to specify some 1,500 to 6,000 average-sized genes. Therefore, even a battery of 300 tests would assay only 5 to 20 percent of the genetic potential of a bacterium. Tests that are comparatively simple to conduct (such as those for carbohydrate utilization and for enzymes, presence of which can be assayed colorimetrically) are performed more often than tests for structural, reproductive, and regulatory genes, presence of which is difficult to assay. Thus, major differences may go undetected. Other types of errors may occur when species are classified solely on the basis of phenotype. For example, different enzymes (specified by different genes) may catalyze the same reaction. Also, even if a metabolic gene is functional, negative reactions can occur because of the inability of the substrate to enter the cell, because of a mutation in a regulatory gene, or by production of an inactive protein. There is not necessarily a one-to one correlation between a reaction and the number of genes needed to carry out that reaction. For instance, six enzymatic steps may be involved in a given pathway. If an assay for the end product is performed, a positive reaction indicates the presence of all six enzymes, whereas a negative reaction can mean the absence or non function of one to six enzymes. Several other strain characteristics can affect phenotypic characterization; these include growth rate, incubation temperature, salt requirement, and pH. Plasmids that carry metabolic genes can enable strains to carry out reactions atypical for strains of that species. The same set of "definitive" reactions cannot be used to classify all groups of organisms, and there is no standard number of specific reactions that allows identification of a species. Organisms are identified on the basis of phenotype, but, from the taxonomic standpoint, definition of species solely on this basis is subject to error.
• #5: Figure1, group 1 represents three Citrobacter freundii strains that are about 95 percent similar and join with a fourth C freundii strain at the level of 90 percent similarity. Group 2 is composed of three Citrobacter diversus strains that are 95 percent similar, and group 3 contains two E coli strains that are 95 percent similar, as well as a third E coli strain to which they are 90 percent similar. Similarity between groups 1 and 2 occurs at the 70 percent level, and group 3 is about 50 percent similar to groups 1 and 2. When this approach is the only basis for defining a species, it is difficult to know how many and which tests should be chosen; whether and how the tests should be weighted; and what level of similarity should be chosen to reflect relatedness at the genus and species levels. Most bacteria have enough DNA to specify some 1,500 to 6,000 average-sized genes. Therefore, even a battery of 300 tests would assay only 5 to 20 percent of the genetic potential of a bacterium. Tests that are comparatively simple to conduct (such as those for carbohydrate utilization and for enzymes, presence of which can be assayed colorimetrically) are performed more often than tests for structural, reproductive, and regulatory genes, presence of which is difficult to assay. Thus, major differences may go undetected. Other types of errors may occur when species are classified solely on the basis of phenotype. For example, different enzymes (specified by different genes) may catalyze the same reaction. Also, even if a metabolic gene is functional, negative reactions can occur because of the inability of the substrate to enter the cell, because of a mutation in a regulatory gene, or by production of an inactive protein.
• #6: Phylogenetic Approach The ideal means of identifying and classifying bacteria would be to compare each gene sequence in a given strain with the gene sequences for every known species. This cannot be done, but the total DNA of one organism can be compared with that of any other organism by a method called nucleic acid hybridization or DNA hybridization. This method can be used to measure the number of DNA sequences that any two organisms have in common and to estimate the percentage of divergence within DNA sequences that are related but not identical. DNA relatedness studies have been done for yeasts, viruses, bacteriophages, and many groups of bacteria. Five factors can be used to determine DNA relatedness: genome size, guanine-pluscytosine (G+C) content, DNA relatedness under conditions optimal for DNA reassociation, thermal stability of related DNA sequences, and DNA relatedness under conditions supraoptimal for DNA reassociation. Because it is not practical to conduct these genotypic or phylogenetic evaluations in clinical laboratories, the results of simpler tests usually must be correlated with known phylogenetic data. For example, yellow strains of Enterobacter cloacae were shown, by DNA relatedness, to form a separate species, Enterobacter sakazakii, but were not designated as such until results of practical tests were correlated with the DNA data to allow routine laboratories to identify the new species.
• #11: DNA Relatedness under Conditions Optimal for DNA Reassociation DNA relatedness is determined by allowing single-stranded DNA from one strain to reassociate with single-stranded DNA from a second strain, to form a double-stranded DNA molecule (Figure 3-2). This is a specific, temperature-dependent reaction. The optimal temperature for DNA reassociation is 25 to 30°C below the temperature at which native double-stranded DNA denatures into single strands. Many studies indicate that a bacterial species is composed of strains that are 70 to 100 percent related. In contrast, relatedness between different species is 0 to about 65 percent. It is important to emphasize that the term "related" does not mean "identical" or "homologous." Similar but nonidentical nucleic acid sequences can reassociate. Thermal Stability of Related DNA Sequences Each 1 percent of unpaired nucleotide bases in a double-stranded DNA sequence causes a 1 percent decrease in the thermal stability of that DNA duplex. Therefore, a comparison between the thermal stability of a control double-stranded molecule (in which both strands of DNA are from the same organism) and that of a heteroduplex (DNA strands from two different organisms) allows assessment of divergence between related nucleotide sequences. DNA Relatedness under Supraoptimal Conditions for DNA Reassociation When the incubation temperature used for DNA reassociation is raised from 25-30° C below the denaturation temperature to only 10-15° C below the denaturation temperature, only very closely related (and therefore highly thermally stable) DNA sequences can reassociate. Strains from the same species are 60 percent or more related at these supraoptimal incubation temperatures. Defining Species on the Basis of DNA Relatedness Use of these five factors allows a species definition based on DNA. Thus, E coli can be defined as a series of strains with a G+C content of 49 to 52 moles percent, a genome molecular weight of 2.3 X 109 to 3.0 X 109, relatedness of 70 percent or more at an optimal reassociation temperature with 0 to 4 percent divergence in related sequences, and relatedness of 60 percent or more at a supraoptimal reassociation temperature. Experience with more than 300 species has produced an arbitrary phylogenetic definition of a species to which most taxonomists subscribe: "strains with approximately 70% or greater DNA-DNA relatedness and with 5° C or less divergence in related sequences." When these two criteria are met, genome size and G+C content are always similar, and relatedness is almost always 60 percent or more at supraoptimal incubation temperatures. The 70 percent species relatedness rule has been ignored occasionally when the existing nomenclature is deeply ingrained, as is that for E coli and the four Shigella species. Because these organisms are all 70 percent or more related, DNA studies indicate that they should be grouped into a single species, instead of the present five species in two genera. This change has not been made because of the presumed confusion that would result. DNA relatedness provides one species definition that can be applied equally to all organisms. Moreover, it cannot be affected by phenotypic variation, mutations, or the presence or absence of metabolic or other plasmids. It measures overall relatedness, and these factors affect only a very small percentage of the total DNA.
• #14: Polyphasic Approach In practice, the approach to bacterial taxonomy should be polyphasic (Fig. 3-3). The first step is phenotypic grouping of strains by morphological, biochemical and any other characteristics of interest. The phenotypic groups are then tested for DNA relatedness to determine whether the observed phenotypic homogeneity (or heterogeneity) is reflected by phylogenetic homogeneity or heterogeneity. The third and most important step is reexamination of the biochemical characteristics of the DNA relatedness groups. This allows determination of the biochemical borders of each group and determination of reactions of diagnostic value for the group. For identification of a given organism, the importance of specific tests is weighted on the basis of correlation with DNA results. Occasionally, the reactions commonly used will not distinguish completely between two distinct DNA relatedness groups. In these cases, other biochemical tests of diagnostic value must be sought.
• #18: Phenotypic Characteristics Useful in Classification and Identification Morphologic Characteristics Both wet-mounted and properly stained bacterial cell suspensions can yield a great deal of information. These simple tests can indicate the Gram reaction of the organism; whether it is acid-fast; its motility; the arrangement of its flagella; the presence of spores, capsules, and inclusion bodies; and, of course, its shape. This information often can allow identification of an organism to the genus level, or can minimize the possibility that it belongs to one or another group. Colony characteristics and pigmentation are also quite helpful. For example, colonies of several Porphyromonas species autofluoresce under long-wavelength ultraviolet light, and Proteus species swarm on appropriate media. Growth Characteristics A primary distinguishing characteristic is whether an organism grows aerobically, anaerobically, facultatively (i.e., in either the presence or absence of oxygen), or microaerobically (i.e., in the presence of a less than atmospheric partial pressure of oxygen). The proper atmospheric conditions are essential for isolating and identifying bacteria. Other important growth assessments include the incubation temperature, pH, nutrients required, and resistance to antibiotics. For example, one diarrheal disease agent, Campylobacter jejuni, grows well at 42° C in the presence of several antibiotics; another, Y enterocolitica, grows better than most other bacteria at 4° C. Legionella, Haemophilus, and some other pathogens require specific growth factors, whereas E coli and most other Enterobacteriaceae can grow on minimal media. Antigens and Phage Susceptibility Cell wall (O), flagellar (H), and capsular (K) antigens are used to aid in classifying certain organisms at the species level, to serotype strains of medically important species for epidemiologic purposes, or to identify serotypes of public health importance. Serotyping is also sometimes used to distinguish strains of exceptional virulence or public health importance, for example with V cholerae (O1 is the pandemic strain) and E coli (enterotoxigenic, enteroinvasive, enterohemorrhagic, and enteropathogenic serotypes). Phage typing (determining the susceptibility pattern of an isolate to a set of specific bacteriophages) has been used primarily as an aid in epidemiologic surveillance of diseases caused by Staphylococcus aureus, mycobacteria, P aeruginosa, V cholerae, and S typhi. Susceptibility to bacteriocins has also been used as an epidemiologic strain marker. In most cases recently, phage and bacteriocin typing have been supplanted by molecular methods. Biochemical Characteristics Most bacteria are identified and classified largely on the basis of their reactions in a series of biochemical tests. Some tests are used routinely for many groups of bacteria (oxidase, nitrate reduction, amino acid degrading enzymes, fermentation or utilization of carbohydrates); others are restricted to a single family, genus, or species (coagulase test for staphylococci, pyrrolidonyl arylamidase test for Gram-positive cocci). Both the number of tests needed and the actual tests used for identification vary from one group of organisms to another. Therefore, the lengths to which a laboratory should go in detecting and identifying organisms must be decided in each laboratory on the basis of its function, the type of population it serves, and its resources. Clinical laboratories today base the extent of their work on the clinical relevance of an isolate to the particular patient from which it originated, the public health significance of complete identification, and the overall cost-benefit analysis of their procedures. For example, the Centers for Disease Control and Prevention (CDC) reference laboratory uses at least 46 tests to identify members of the Enterobacteriaceae, whereas most clinical laboratories, using commercial identification kits or simple rapid tests, identify isolates with far fewer criteria.
• #21: Phage typing (determining the susceptibility pattern of an isolate to a set of specific bacteriophages) has been used primarily as an aid in epidemiologic surveillance of diseases caused by Staphylococcus aureus, mycobacteria, P aeruginosa, V cholerae, and S typhi. Susceptibility to bacteriocins has also been used as an epidemiologic strain marker. In most cases recently, phage and bacteriocin typing have been supplanted by molecular methods.
• #23: For example, the Centers for Disease Control and Prevention (CDC) reference laboratory uses at least 46 tests to identify members of the Enterobacteriaceae, whereas most clinical laboratories, using commercial identification kits or simple rapid tests, identify isolates with far fewer criteria.
• #24: Classification Below and Above the Species Level Below the Species Level Particularly for epidemiological purposes, clinical microbiologists must distinguish strains with particular traits from other strains in the same species. For example, serotype O157:H7 E coli are identified in stool specimens because of their association with bloody diarrhea and subsequent hemolytic uremic syndrome. Below the species level, strains are designated as groups or types on the basis of common serologic or biochemical reactions, phage or bacteriocin sensitivity, pathogenicity, or other characteristics. Many of these characteristics are already used and accepted: serotype, phage type, colicin type, biotype, bioserotype (a group of strains from the same species with common biochemical and serologic characteristics that set them apart from other members of the species), and pathotype (e.g., toxigenic Clostridium difficile, invasive E coli, and toxigenic Corynebacterium diphtheriae). Above the Species Level In addition to species and subspecies designations, clinical microbiologists must be familiar with genera and families. A genus is a group of related species, and a family is a group of related genera. An ideal genus would be composed of species with similar phenotypic and phylogenetic characteristics. Some phenotypically homogeneous genera approach this criterion (Citrobacter, Yersinia, and Serratia). More often, however, the phenotypic similarity is present, but the genetic relatedness is not. Bacillus, Clostridium, and Legionella are examples of accepted phenotypic genera in which genetic relatedness between species is not 50 to 65 percent, but 0 to 65 percent. When phenotypic and genetic similarity are not both present, phenotypic similarity generally should be given priority in establishing genera. Identification practices are simplified by having the most phenotypically similar species in the same genus. The primary consideration for a genus is that it contain biochemically similar species that are convenient or important to consider as a group separate from other groups of organisms. The sequencing of ribosomal RNA (rRNA) genes, which have been highly conserved through evolution, allows phylogenetic comparisons to be made between species whose total DNAs are essentially unrelated. It also allows phylogenetic classification at the genus, family, and higher taxonomic levels. The rRNA sequence data are usually not used to designate genera or families unless supported by similarities in phenotypic tests.
• #25: Designation of New Species and Nomenclatural Changes Species are named according to principles and rules of nomenclature set forth in the Bacteriological Code. Scientific names are taken from Latin or Greek. The correct name of a species or higher taxon is determined by three criteria: valid publication, legitimacy of the name with regard to the rules of nomenclature, and priority of publication (that is, it must be the first validly published name for the taxon). To be published validly, a new species proposal must contain the species name, a description of the species, and the designation of a type strain for the species, and the name must be published in the International Journal for Systematic Bacteriology (IJSB). Once proposed, a name does not go through a formal process to be accepted officially; in fact, the opposite is true a validly published name is assumed to be correct unless and until it is challenged officially. A challenge is initiated by publishing a request for an opinion (to the Judicial Commission of the International Association of Microbiological Societies) in the IJSB. Designation of New Species and Nomenclatural Changes Species are named according to principles and rules of nomenclature set forth in the Bacteriological Code. Scientific names are taken from Latin or Greek. The correct name of a species or higher taxon is determined by three criteria: valid publication, legitimacy of the name with regard to the rules of nomenclature, and priority of publication (that is, it must be the first validly published name for the taxon). To be published validly, a new species proposal must contain the species name, a description of the species, and the designation of a type strain for the species, and the name must be published in the International Journal for Systematic Bacteriology (IJSB). Once proposed, a name does not go through a formal process to be accepted officially; in fact, the opposite is truea validly published name is assumed to be correct unless and until it is challenged officially. A challenge is initiated by publishing a request for an opinion (to the Judicial Commission of the International Association of Microbiological Societies) in the IJSB. This occurs only in cases in which the validity of a name is questioned with respect to compliance with the rules of the Bacteriological Code. A question of classification that is based on scientific data (for example, whether a species, on the basis of its biochemical or genetic characteristics, or both, should be placed in a new genus or an existing genus) is not settled by the Judicial Commission, but by the preference and usage of the scientific community. This is why there are pairs of names such as Providencia rettgeri/Proteus rettgeri, Moraxella catarrhalis/Branhamella catarrhalis, and Legionella micdadei/Tatlockia micdadei. More than one name may thus exist for a single organism. This is not, however, restricted to bacterial nomenclature. Multiple names exist for many antibiotics and other drugs and enzymes. A number of genera have been divided into additional genera and species have been moved to new or existing genera, such as Arcobacter (new genus for former members of Campylobacter) and Burkholderia species (formerly species of Pseudomonas). Two former Campylobacter species (cinaedi and fennelliae) have been moved to the existing genus Helicobacter in another example. The best source of information for new species proposals and nomenclatural changes is the IJSB. In addition, the Journal of Clinical Microbiology often publishes descriptions of newly described microorganisms isolated from clinical sources. Information, including biochemical reactions and sources of isolation, about new organisms of clinical importance, disease outbreaks caused by newer species, and reviews of clinical significance of certain organisms may be found in the Annals of Internal Medicine, Journal of Infectious Diseases, Clinical Microbiology Reviews, and Clinical Infectious Diseases. The data provided in these publications supplement and update Bergey's Manual of Systematic Bacteriology, the definitive taxonomic reference text.