Microbial Taxonomy

• Taxonomy (Greek taxis, arrangement or order, and nomos, law, or nemein, to distribute or govern) is defined as the science of biological classification.
• Consists of three separate but interrelated parts: classification, nomenclature, and identification.
• Once a classification scheme is selected, it is used to arrange organisms into groups called taxa (s., taxon) based on mutual similarity.

• Nomenclature is the branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with published rules.
• Identification is the practical side of taxonomy, the process of determining if a particular isolate belongs to a recognized taxon.
• The term systematics is often used for taxonomy.

Binomial System of Nomenclature

Binomial system was devised by Carolus Linnaeus

• Each organism has two names
• genus name – italicized and capitalized (e.g., Escherichia)
• species epithet – italicized but not capitalized (e.g., coli)
• can be abbreviated after first use (e.g., E. coli)
• a new procaryotic species cannot be recognized until it has been published in the International Journal of Systematic and Evolutionary Microbiology

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TAXONOMIC RANKS

• The basic taxonomic group in microbial taxonomy is the species.
• A procaryotic species is a collection of strains that share many stable properties and differ significantly from other groups of strains
• A strain consists of the descendents of a single, pure microbial culture.
• Biovars are variant strains characterized by biochemical or physiological differences,
• Morphovars differ morphologically, and
• serovars have distinctive antigenic properties

• Bacteria are classified and identified to distinguish one organism from another and to group similar organisms by criteria of interest to microbiologists or other scientists.
• The classification of bacteria serves a variety of different functions.
• Because of this variety, bacteria may be grouped using many different typing schemes.

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.

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. F

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
• 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.

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 Grampositive cocci).

Classification of Bacteria on the Basis of Shape

• A) Cocci:These types of bacteria are unicellular, spherical or elliptical shape. Either they may remain as a single cell or may aggregate together for various�configurations.
• B) Bacilli: – These are rod shaped or cylindrical bacteria which either remain singly or in pairs. Example: –Bacillus cereus.
• C) Vibro: – The vibro are the curved, comma shaped bacteria and represented by a single genus. Example: – Vibro cholerae.
• D) Spirilla: – These type of bacteria are spiral or spring like with multiple curvature and terminal flagella. Example: –Spirillum volutans.

On the Basis of Number of Flagella

1. Atrichos: – These bacteria has no flagella. Example: Corynebacterium diptherae.
2. Monotrichous: – One flagellum is attached to one end of the bacteria cell.�Example: – Vibro cholerae.
3. Lophotrichous: – Bunch of flagella is attached to one end of the bacteria cell. Example: Pseudomonas.
4. Amphitrichous: – Bunch of flagella arising from both end of the bacteria cell. Example: Rhodospirillum rubrum.
5. Peritrichous : – The flagella are evenly distributed surrounding the entire bacterial cell. Example: E. coli.

Spore Formation

1. Spore forming bacteria:�Those bacteria that produce spore during unfavorable condition.�These are further divided into two groups: �i) Endospore forming bacteria: Spore is produced within the bacterial cell.�Examples. Bacillus, Clostridium, Sporosarcina etc�ii) Exospore forming bacteria: Spore is produced outside the cell. Example. Methylosinus �

2. Non sporing bacteria:�Those bacteria which do not produce spores.�Eg. E. coli, Salmonella.

On the Basis of Mode of Nutrition

• 1. Phototrophs:�Those bacteria which gain energy from light.�Phototrops are further divided into two groups on the basis of source of�electron.�Photolithotrophs: these bacteria gain energy from light and uses reduced�inorganic compounds such as H2S as electron source. Eg. Chromatium�okenii.�Photoorganotrophs: these bacteria gain energy from light and uses organic�compounds such as succinate as electron source. �

• 2. Chemotrophs: �Those bacteria gain energy from chemical compounds.�They cannot carry out photosynthesis.�Chemotrops are further divided into two groups on the basis of source of electron.�
• Chemolithotrophs: they gain energy from oxidation of chemical compound and reduces inorganic compounds such as NH3 as electron source. Eg. Nitrosomonas.�
• Chemoorganotrophs: they gain energy from chemical compounds and uses organic compound such as glucose and amino acids as source of electron.�eg. Pseudomonas pseudoflava.

• 3. Autotrophs:�Those bacteria which uses carbondioxide as sole source of carbon to prepare its own food.�Autotrophs are divided into two types on the basis of energy utilized to assimilate carbondioxide. ie. Photoautotrophs and chemoautotrophs.�
• Photoautotrophs: they utilized light to assimilate CO2. They are further�divided into two group on the basis of electron sources. Ie. Photolithotropic autotrophs and Photoorganotropic autotrophs
• Chemoautotrophs: They utilize chemical energy for assimilation of CO2.

• 4. Heterotrophs:�Those bacteria which uses organic compound as carbon source.�They lack the ability to fix CO2.
• Most of the human pathogenic bacteria are heterotropic in nature.�Some heterotrops are simple, because they have simple nutritional requirement.
• However there are some bacteria that require special nutrients for their growth; known as fastidious heterotrophs.

On the Basis of Temperature Requirement

• 1.Psychrophiles:�Bacteria that can grow at 0°C or below but the optimum temperature of growth is 15 °C or below and maximum temperature is 20°C are called psychrophiles
• Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid nature to the cell membrane even at lower temperature.
• Examples: Vibrio psychroerythrus, vibrio marinus, Polaromonas vaculata,�Psychroflexus.

• 2. Psychrotrops (facultative psychrophiles):�Those bacteria that can grow even at 0°C but optimum temperature for growth is (20-30)°C�
• 3. Mesophiles:�Those bacteria that can grow best between (25-40) C but optimum temperature for growth is 37C�Most of the human pathogens are mesophilic in nature.�Examples: E. coli, Salmonella, Klebsiella, Staphylococci.

• 4. Thermophiles: Those bacteria that can best grow above 45C.
• Thermophiles capable of growing in mesophilic range are called facultative thermophiles.
• True thermophiles are called as Stenothermophiles, they are obligate thermophiles,
• Thermophils contains saturated fattyacids in their cell membrane so their cell membrane does not become too fluid even at higher temperature.
• Examples: Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus.

Basis of Oxygen Requirement

• Obligate Aerobes:�Require oxygen to live.�Example: Pseudomonas, common nosocomial pathogen.�
• Facultative Anaerobes:�Can use oxygen, but can grow in its absence.�They have complex set of enzymes.�Examples: E. coli, Staphylococcus, yeasts, and many intestinal bacteria.�
• Obligate Anaerobes:�Cannot use oxygen and are harmed by the presence of toxic forms of oxygen.�Examples: Clostridium bacteria that cause tetanus and botulism.�
• Aerotolerant Anaerobes:�Cannot use oxygen, but tolerate its presence.�Can break down toxic forms of oxygen.�Example: Lactobacillus carries out fermentation regardless of oxygen�presence.�
• Microaerophiles:�Require oxygen, but at low concentrations. Sensitive to toxic forms of oxygen.�Example: Campylobacter.

Basis of pH of Growth

1. Acidophiles:�These bacteria grow best at an acidic pH.�The cytoplasm of these bacteria are acidic in nature.�Some acidopiles are thermophilic in nature, such bacteria are called Thermoacidophiles.�Examples: Thiobacillus thioxidans, Thiobacillus, ferroxidans, Thermoplasma, Sulfolobus

2. Alkaliphiles:�These bacteria grow best at an alkaline pH.�Example: Vibrio cholerae optimum ph of growth is 8.2.

3. Neutrophiles:�These bacteria grow best at neutral pH (6.5-7.5).�Most of the bacteria grow at neutral pH.�Example: E. coli

Basis of Osmotic Pressure Requirement

• Halophiles:�Require moderate to large salt concentrations.�Cell membrane of halophilic bacteria is made up of glycoprotein with high content of negatively charged glutamic acid and aspartic acids. So high concentration of Na+ ion concentration is required to shield the –ve charge.

Ocean water contains 3.5% salt. Most such bacteria are present in the oceans.�Archeobacteria, Halobacterium, Halococcus.

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• Extreme or Obligate Halophiles:�Require a very high salt concentrations (20 to 30%).�Bacteria in Dead Sea, brine vats.

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• Facultative Halophiles:�Do not require high salt concentrations for growth, but tolerate upto 2% saltor more.

TECHNIQUES FOR DETERMINING MICROBIAL TAXONOMY AND PHYLOGENY

Divided into two groups: classical and molecular.

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A. Classical Characteristics

• make use of morphological, physiological, biochemical, ecological, and genetic characteristics.

Ecological Characteristics

• The ability of a microorganism to colonize a specific environment is of taxonomic value
• life cycle patterns;
• the nature of symbiotic relationships;
• the ability to cause disease in a particular host;
• habitat preferences
• such as requirements for temperature, pH, oxygen, and osmotic concentration

Genetic Analysis

• Although procaryotes do not reproduce sexually, the study of chromosomal gene exchange through transformation, conjugation, and transduction is sometimes useful in their classification
• E.g. plasmids

B. Molecular Characteristics

Uses study of the DNA, RNA, and proteins

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• Nucleic Acid Base Composition
• G C content
• The G C content often is determined from the�melting temperature (Tm) of DNA.
• they can confirm a taxonomic scheme
• variation within a genus is usually less than 10%
• Higher G + C gives a higher melting temperature

Nucleic Acid Hybridization

• The similarity between genomes can be compared
• common procedure for hybridisation:
• bind nonradioactive DNA to nitrocellulose filter
• incubate filter with radioactive single-stranded DNA
• The quantity of radioactivity bound to the filter reflects the amount of hybridisation between the 2 DNA and thus similarity of the 2 sequences.

• The small subunit rRNAs (SSU rRNAs) are used microbial evolution and relatedness because they play the same role in all microorganisms.
• Comparative analysis of 16S rRNA sequences - These are short, conserved nucleotide sequences that are specific for a phylogenetically defined group of organisms.
• complete chromosomes can now be sequenced and compared

Nucleic Acid Sequencing

Multilocus sequence typing (MLST)

• The use of DNA sequences to determine species and strain (as opposed to genus) identity requires the analysis of genes that evolve more quickly than those that encode rRNA. �
• Multiple genes are usually examined to avoid misleading results that can arise through lateral gene transfer. E.g. 5 to 7 housekeeping genes

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• MLST is helpful for differentiating isolates at the strain and species levels.

Genomic Fingerprinting

• A group of techniques is used to used to classify microbes and help determine phylogenetic relationships

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restriction fragment length polymorphism (RFLP) analysis

• it employs the capacity of restriction endonucleases to recognize specific nucleotide sequences.
• pattern of DNA fragments generated by endonuclease cleavage (called restriction fragments) is a direct representation of nucleotide sequence

PCR- highly conserved and repetitive DNA sequences are amplified and resolved and visualized on an agarose gel

Amino Acid Sequencing

• amino acid sequences of proteins directly reflect mRNA sequences and therefore represent the genes coding for their synthesis.
• transport proteins, histones and heat-shock proteins, transcription and translation proteins, and a variety of metabolic enzymes have been used in taxonomic and phylogenetic studies. �

• The sequences of nucleic acids and proteins change with time and are considered to be molecular chronometers.
• the sequences of many rRNAs and proteins gradually change over time without destroying or severely altering their functions.
• Highly conserved molecules such as rRNAs are used to follow large-scale evolutionary changes, whereas rapidly changing molecules are employed to follow speciation.

Molecular Chronometers / Molecular clocks

Molecular phylogeny

• Phyletic system: compares organisms based on evolutionary relationships.
• Evolution is the change in a line of descent (e.g. heritable change) over time leading to new species or varieties.
• Ribosomal RNAs (rRNAs) and its respective genes (DNA) are excellent descriptors of microbial taxa based on phylogeny.

Why ribosomal RNAs?

• Found among all living organisms (for 3.8 of the last 4.5 billion years). Integral part of protein synthesis machinery.
• rRNAs offer a type of sequence information that makes them excellent descriptors of an organism's evolutionary history.
• No detectable horizontal gene transfer, especially important for the prokaryotes.
• Large and growing database; RDP contains ~100K SSU rRNAs

Phylogenetic Trees

• A phylogenetic tree is a graph made of branches that connect nodes
• The nodes represent taxonomic units such as species or genes; the external nodes at the end of the branches represent living (extant) organisms. ��

• Phylogenetic trees are developed by comparing nucleotide or amino acid sequences.
• To compare two molecules, their sequences�must first be aligned so that similar parts match up.
• Evolutionary distance: This is simply a quantitative indication of the number of positions that differ between two aligned macromolecules.
• Parsimony analysis. In this approach, relationships are determined by estimating the minimum number of sequence changes required to give the final sequences being compared.