Introduction to Phenotypic Bacterial Identification Techniques
Techniques of bacterial taxonomy - WDCM Dr. Yuhua Xin ( Techniques of... · Techniques of bacterial...
Transcript of Techniques of bacterial taxonomy - WDCM Dr. Yuhua Xin ( Techniques of... · Techniques of bacterial...
Techniques of bacterial taxonomy
China General Microbiological Culture Collection Center (CGMCC)
Institute of Microbiology
Chinese Academy of Sciences
Yuhua Xin
Hug et al. present a new view of the tree of life, revealing the existence of two extraordinarily
diverse and poorly characterized prokaryotic lineages: CPR bacteria (blue) and DPANN archaea
(purple).
• Microbiol taxonomy is a science of study and grouping of microorganisms.
• Bacterial Taxonomy concludes three separate but interrelated areas
– Classification
• Arrangement of organisms into groups (taxa) on the basis of similarities or relationships.
– Identification
• Process of characterizing organisms to determine an isolate as a member of an established taxon or a previously unidentified species.
– Nomenclature
• Assignment of a specific name according to international rules (International Code of Nomenclature of Bacteria[Sneath,1992]).
Bacterial Taxonomy
• Bacterial taxonomy incorporates multiple methods for
identification and description of new species
• The polyphasic approach to taxonomy uses three
methods
1) Phenotypic analysis
2) Genotypic analysis
3) Phylogenetic analysis
Bacterial Taxonomy
– Phenotypic characteristics• Morphological data• physiological and biochemical data• Chemotaxonomic characteristics
Fatty acid analysis The patterns of polar lipids present in the membrans Composition of cell wall
– Genotypic characteristics• DNA-DNA Hybridization • the guanine (G)+ cytosine (C) content (% GC). • Multilocus Sequence Typing (MLST)• DNA profiling
– Phylogenetic Analysis• 16S rRNA gene sequence analysis• Multi-gene sequence analysis• Whole-genome sequence analysis
Polyphasic Taxonomy
• No universally accepted concept of species for
prokaryotes
• Current definition of prokaryotic species
– Collection of strains sharing a high degree of
similarity in several independent traits
• Most important traits include: 70% or greater DNA-DNA
hybridization and 98.5 % or greater 16S rRNA gene
sequence identity
The Species Concept in Microbiology
Some Phenotypic Characteristics of Taxonomic Value
Some Phenotypic Characteristics of Taxonomic Value
Other Chemical characterization: Peptidoglycan, Polyamines, techoic acids,
mycolic acids, Lipopolysaccharides
Morphology
Phenotypic characteristics
Microscopic morphology
Cell morphology: rod, coccus, or spirillum
Cell arrangement: diplococcus, streptococcus,
tetrad, sarcina, irregular clusters (Micrococcus or
staphycoccus)
Special cell structures: flagellum, cilia, spore, capsule
cocci
• Morphology
• Morphological criteria
• Cell shape and size – supported by photographs
• Characteristic features (eg. stalks, prosthecae, budding or
branching, cell aggregates )
• Spore formation
• Location of flagella
• Motility (form, speed)
• Intracellular structures
• Colony shape and size
• Cellular pigments
5.0 μm
5.0 μm
5.0 μm
500 nm 200 nm
Scanning electron micrograph
Colony morphology :
Colonies can exhibit macroscopic
differences
colour, size,
shape, margin or edge,
surface feature etc.
Slant culture morphology
• Morphology
Staining
• Gram stain (the reaction may alter as the cells age)
• Acid-fast staining (strains containing mycolic acids)
• Sudan Black staining (stains containing lipophilic cellular
inclusions, eg. polyhydroxybutyric acid)
• Others (eg. spore staining, capsule staining)
physiological and biochemical data
Phenotypic characteristics
• Physiology and biochemistry
• The growth tolerance (eg. pH, temperature, NaCl
concentration)
• Enzyme activity, substrate utilization, antibiotics resistance,
etc. Fast methods: API and Biology test plates.
Note:
• To test with identical media and conditions or at least
comparable.
• To compare with type strain of type species of appropriate
genera.
• To analyze including strains of the most closely related taxa
rather than using the previously published data.
Nitrate Reduction Carbohydrate Fermentation Urease detection
Traditional methods
VP test MR test
Traditional methods
Nitrate reduction Citrate utilization Indole prudution
the ability of a microorganism to
withstand the effects of an antibiotic
on agar plates (Whether bacteria are
susceptible, intermediate, or resistant
depends on the amount of antibiotic
and the diameter of zone of inhibition).
Antibiotic sensitivity
Serological analysis
• Proteins and polysaccharides of some bacteria can function as
identifying markers
– Generally molecules on surface structures
• e.g., Cell wall, glycocalyx, flagella, pili
– Detection is based upon the
specific interaction between
antibodies and these
antigens
• e.g., Rapid detection of
Streptococcus pyogenes
How to perform and interpret the miniaturized,
multi-test technique for bacterial identification?
Disadvantages of traditional methods
Need experience
Complicated process
Labor-consuming
Time-consuming
Rapid Tests
• Commercial modifications of traditional biochemical tests
• APITM system
• Biolog Microbial ID System
• The API identification system is numerical taxonomy according to
the microbial physiological and biochemical characteristics.
• The API tests (kit) can identify a wide range of microorganisms.
• Have standardized and extensive databases of characteristic
biochemical reactions of microorganisms.
commercial products for bacteria identification
The API identification system is numerical taxonomy according to the microbial physiological and biochemical characteristics.
• API 50 CH – Performance of carbohydrate
metabolism tests
• API ZYM® – Semiquantification of enzymatic
activities
API 20E – 11 biochemical tests and enzymatic activities, 9 Fermentation/Oxidation
…….
15
eg. API 20E
Isolate Prepare Incubate Read
suspension
reaction strip
reagents
incubation chamber
Positive Negative
Bacteria, Yeast and Fungi Identification
The Biolog Microbial ID System can rapidly identify over 2,500 species of aerobic and anaerobic bacteria, yeasts and fungi.
Tetrazolium redox dyes are used to colorimetrically indicate utilization of the
carbon sources or resistance to inhibitory chemicals.
1. Isolate pure culture on agar media
2. Prepare inoculum at specified cell density
3. Inoculate the Biolog MicroPlate
4. Incubate the plate, observe and enter the reaction
pattern to obtain ID result
simple, straightforward procedure
• Commercial systems are very accurate for the more
common species and provide quick test results in a cost-
effective manner.
• The MicroStation System has extensive applications also
for microbial community analysis in soil, water and other
environments.
Cornerstone of microbial taxonomy
• Bacterial identification
• Microbial ecology
• Evolution
• Cultivate more unknown bacteria
The importance of growth phenotypes
• With the publication of the first edition of the Bergey's Manual of Determinative Bacteriology in 1923, microbiologists began to systematically describe and define bacterial species based on lists of phenotypes, primarily growth related.
• 1926, L.E. den Dooren de Jong showed that bacteria could be
readily distinguished by growth assays on agar media with
several hundred C- and N-sources.
The importance of growth phenotypes
1. As the number of newly described taxa increased substantially, a problem with
commercial systems is the construction of databases
Challenges in phenotypic identification
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2. Phenotypic properties do not accurately reflect the entire extent of the
genomic complexity of a given species
3. Phenotypic properties can be unstable at times and expression can be
dependent upon changes in environmental conditions, e.g., growth
substrate, temperature, and pH levels
4. Can be used only for organisms that can be cultivated in vitro
M.J. Janda & S.L. (2002), J. Clin. Microbiol.
Challenges in phenotypic identification
Chemical characterization
Phenotypic characteristics
Fatty Acid Analyses
– Relies on variation in type and
proportion of fatty acids present in
membrane lipids for specific prokaryotic
groups
– Requires rigid standardization because
FAME profiles can vary as a function of
temperature, growth phase, and growth
medium
MIDI Sherlock® Microbial Identification System
Procedure of fatty acids analysis
Cultivation of bacteria
The growth temperature and growth media effect the fatty acids compositions,
culture conditions must be standardized for all strains and strains were collected at the same logarithm growth period, when comparing the fatty acid composition within a group of bacteria
Preparation of fatty-acid methyl esters (FAMEs)
Fatty acids Saponification /Methylation FAMES
Identification of fatty acids
GC analysis and identified by MIDI system
Respiratory quinones
• Respiratory quinones:a group of non protein, lipid-soluble electron carriers in the respiratory electron-transport system.
• Function:promote the transfer of electrons between the proteins of the electron-transport chain.
• Distribution:in both anaerobic and aerobic organisms within the Bacteria and Archaea.
• Type:divided into two basic structural classes, benzoquinones and naphthoquinones .
Fig. 1. Nature Reviews Molecular Cell Biology 2002,3, 836-847
Ubiquinone (Q-n)
Plastoquinone
Rhodoquinone (RQ)
n
Sulfolobusquinone
Caldariellaquinone
Epoxyubiquinone
Menaquinone (MK-n)
Demethylmenaquinone (DMK) (DMMK-)
Thermoaquinone
Chlorobiumquinone (CK )
Methionaquinone
Partially hydrogenated menaquinone (MK-n(Hm))
Benzoquinones Naphthoquinones
Menaquinone: MK-n
--- Most Gram-positive bacteria and anaerobic Gram-negative bacteria,
--- Archeae: dementhylmenaquinone (DMK),
--- Actinomycetes: partially hydrogenated MK,
--- A variable number of isoprenoid residues: MK-5-15.
Ubiquinone ( Coenzyme Q, Co Q or Q )
---Ubiquinones: aerobic Gram-negative, α-, β-, γ- group of Proteobacteria&rodshaped acetic acid bacteria;
---Rhodoquinone (RQ): photosynthetic bacteria,
---the number of isoprenoid units in side-chain arevariable (Q-7-Q-14).
Ubiquinone and Menaquinone
Ubiquinone and menaquinone are abbreviated as Q-n and MK-n(Hm);
n is the number of isoprene units and m is the number of hydrogen atoms substituting unsaturated bond.
Ubiquinone: Q-n Menaquinone: MK-n(Hm)
Respiratory quinones profiles of bacteria
Taxon Main Quinone System
Proteobacteria
α-SubcalssAgrobacteriumRhodomicrobium vannieliiRhodopseudomonas acidophila
Q-10Q-10+RQ-10Q-10+RQ-10+MK-10
β- SubclassAlcaligenesBrachymonas, ZoogloeaRhodocyclus, Rubrivivax
Q-8Q-8+RQ-8Q-8+ MK-8
γ- SubclassAcinetobacter, PseudomonasAzotobacterChromatiaceaeEctothiorhodospiraceaeEnterobacteriaceaeVibrio
Q-9Q-8,Q-8+MK-8Q-7+MK-7, Q-8+ MK-8Q-8+MK-8 +DMK-8Q-8+MK-8( +DMK-8)
δ- SubclassDesulfobulbusDesulfococcusDesulfovibrio
MK-5(H2)MK-7MK-6, MK-6(H2)
Taxon Gram (+) bacteria Main Quinone system
Low G+C content groupBacillusEnterococcus
MK-7MK-8 , DMK-9
High G+C content groupArthrobacterAureobacteriumCorynebacteriumKribbellaStreptomyces
MK-9(H2)MK-11+MK12MK-8(H2), MK-9(H2)MK-9(H4)MK-9(H6), MK-9(H8)
CyanobacteriaNostoc PQ-9+K1
Spirosoma group Spirosoma MK-7
Bacteroides/Flavobacteria group BacteroidesCapnocytophagaFlavobacterium/CytophagaSphingobacterium
MK-10+MK-11MK-6MK-6+MK-7MK-7
OthersChlorobiumChloroflexusDeinobacter
MK-7+CKMK-10+MK-8MK-8
Respiratory quinones profiles of the bacteria
Identification of quinones (HPLC)
Polar lipids analysis
There is a vast diversity of polar lipids now known to be present in prokaryotes.
Phospholipids form an essential component of the cell membrane.
Be related to permeability of the membrane and regulation at the membrane.
bacause they possess not only a hydrophobic region but also a hydrophilic region on the molecule. They show a distinctive amphipathic characteristics.
The polar lipids known to occur in bacteria:
---phospholipids,
---glycolipids,
---glycophospholipids,
---aminophospholipids,
---amino acid derived lipids,
---capnines,
---sphingolipids,
---sulfur-containing lipids. Structure of PC (phosphatidylcholine )
The mainly kinds of polar lipids in bacterial cell
Phospholipids: PC (phosphatidylcholine),
PE (phosphatidylethanolamine),
PI (phosphatidylinositol),
PG (phosphatidyglycerol),
PS (phosphatidylserine),
PME (phosphatidylmethylethanolamine),
PIMs (phosphatidylinositol mannosides),
DPG (diphosphatidylglycerol),
PB (phosphatidylbutanediol),
PA (phosphatidic acid, phosphatidate)
Glycolipids:
PI (phosphatidylinositol),
PIMs (phosphatidylinositol mannosides) etc.
Aminolipids ( free amino groups):
PE (phosphatidylethanolamine),
PS (phosphatidylserine),
PME (phosphatidylmethylethanoamine),
PE (phosphatidylethanolamino)
PI (phosphatidylinositol)
Fatty acids
Glycerol backbone
Inositolhead group
(Phosphatidyl ethanolamine, PE) ( Phosphatidyl methy ethanolamine, PME)
(Phosphatidyl choline, PC) (Phosphatidyl glycerol, PG)
(Phospholipids of unknown structure containing glucosamine,
GluNu)
polar lipids for taxonomy (Lechevallier, 1980)
Common polar lipids appeared in bacteria
α-, β-, γ- group of Proteobacteria: generally posses three major phospholipids: PG, PE and DPG.
Firmicutes and Actinobacteria: complex mixtures of polar lipids.
Bacillus, Rhodococcus, Nocardia, Mycobacterium, Planococcus and Sporosarcina contain PE.
Corynebacterium, Micrococcus and Staphlococcus do not contain PE.
• Peptidoglycan Types
• Amino acid
– Diamino acid
meso-DAP, LL-DAP, Lysine, Ornithine, OH-lysine, OH-ornithine, OH-DAP, DAB, Lanthionine, Diaminopimelic acid
– Composition
– Sequence
• Acyl type
• Cell wall sugar (family, genus, species)
arabinose, galactose, xylose, madurose(3-O-methyl-D-
galactose) etc. Actinomycetes in the 3-O-methylrhamnose,
2-O-methylrhamnose, etc.
Bacterial Cell Wall
IJSEM, 2002, 52(3): 1049-1070
References:
1. Schleifer, K. H. and Kandler, O. (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Reviews. 36, 407-477 .
2. Minikin, D. E. et al. (1984 ) An intergrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids J. Microbiol Methods. 2, 233-241.
3. Tindall, B. J., Rossello-Mora, R., Busse, H,-J., Ludwig, W. and Kampfer, P. (2010) Note on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60, 249-266.
4. Komagta, K. and Suzuki K.-I. (1987) Lipid and cell-wall analysis in bacterial systematics Methods Miceobiol 19, 161-203.
5. Michael Goodfellow and Anthony G. O’ Donnell Chemical Methods in Prokaryotic Systematics.
Genetic-based characterization
• Several methods of genotypic analysis are available and
used
• DNA-DNA Hybridization
• the guanine (G)+ cytosine (C) content (% GC).
• Multilocus Sequence Typing (MLST)
• DNA profiling
Genotypic Methods
• Genomes of two organisms are hybridized to
examine proportion of similarities in their gene
sequences
– Provides rough index of similarity between two
organisms
– Useful complement to SSU rRNA gene sequencing
– Useful for differentiating very similar organisms
– Hybridization values 70% or higher suggest strains
belong to the same species
DNA-DNA hybridization
• G+C content- percentage of Guanine (G) and Cytosine (C) base
pairs in the genome;
• One of the required characteristics of the minimum list of data for a description of a new species;
– Vary between 20 and 75% among Bacteria and Archaea;
– Generally accepted that if GC content of two strains differ by ~ 5% they are unlikely to be closely related.
G+C content
• paper chromatography method
• thermal denaturation temperature method
• HPLC method
Methods
Reference
• Marmur, J. & Doty, P. (1962). Determination of the base composition of
deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5,
109-118.
• Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by high-performance
liquid chromatography. Int J Syst Bacteriol 39, 159-167.
• De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of
DNA hybridization from renaturation rates. Eur J Biochem 12, 133-142.
• DNA Profiling
– Several methods can be used to generate DNA
fragment patterns for analysis of genotypic similarity
among strains, including
• Ribotyping
• RFLP, AFLP
• AP-PCR, RAPD
• ARDRA, rep-PCR
– Method in which several different
“housekeeping genes” from an
organism are sequenced (~450-bp)
– Has sufficient resolving power to
distinguish between very closely
related strains
Multilocus Sequence Typing (MLST)
“Nothing in Biology makes sense except in the light of evolution” (T. Dobzhansky, 1900-1975)
“Nothing in evolution makes sense except in the light of phylogenetics” (many phylogenetists)
Phylogenetics
Phylogenetic Analysis
• 16S rRNA gene sequence analysis
• Multi-gene sequence analysis
• Whole-genome sequence analysis
Arcticibacter Hh36T(JX949238)
Arcticibacter svalbardensis MN12-7T (AQPN01000042)
Pedobacter tournemirensis TF5-37.2-LB10T (GU198945)
Pedobacter xinjiangensis 12157T (EU734803)
Pedobacter zeaxanthinifaciens TDMA-5T (AB264126)
Pedobacter lentus DS-40T (EF446146)
Pedobacter daechungensis Dae 13T (AB267722)
Pedobacter terricola DS-45T (EF446147)
Pedobacter koreensis WPCB189T (DQ092871)
Pedobacter insulae DS-39T (EF100697)
Pedobacter boryungensis BR-9T (HM640986)
Pedobacter westerhofensis WB3.3-22T (AM491369)
Pedobacter africanus DSM 12126T (AJ438171)
Pedobacter duraquae WB2.1-25T (AM491368)
Pedobacter caeni LMG 22862T (AJ786798)
Pedobacter steynii WB2.3-45T (AM491372)
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16S rRNA gene sequence analysis
• The most widely used molecular clocks are small
subunit ribosomal RNA (SSU rRNA) genes
– Found in all domains of life
• 16S rRNA in prokaryotes and 18S rRNA in eukaryotes
– Functionally constant
– Sufficiently conserved (change slowly)
– Sufficient length
• Carl Woese
– Pioneered the use of SSU rRNA for
phylogenetic studies in 1970s
– Established the presence of three
domains of life:
• Bacteria, Archaea, and Eukarya
– Provided a unified phylogenetic
framework for Bacteria
• 16S rRNA gene sequences are useful in taxonomy; serve
as “gold standard” for the identification and description of
new species
– Proposed that a bacterium should be considered a new
species if its 16S rRNA gene sequence differs by more than
3% from any named strain, and a new genus if it differs by
more than 5%
– Less than 98.5% 16S similarity indicates different species, but
greater than 98.5% does not indicate the same species.
Phylogenetic Analysis——16S rRNA gene
• Phenetic Methods—
Distance based
– UPGMA
– Minimum Evolution
– Neighbor Joining
– Bayesian analyses
Phylogenetic Analysis——Tree Building methods
• Cladistic Methods—
Character Based
– Maximum likelihood
– Maximum Parsimony
Phylogenetic Analysis——Tree Building methods
RAxML
EzTaxon-e
Phylogenetic Analysis——Tree Building methods
MLSA is a method for the genotypic characterization of a diverse group of prokaryotes by comparing
sequences of multiple housekeeping genes. Multiple genes provide more informative nucleotide sites
and buffers against the distorting effects of recombination of one of the loci. The best approach is to
concatenate the sequences of at least 12 genes from a set of strains and to use the concatenated
sequences to reconstruct a phylogenetic tree which can identify deeply branching clusters and help to
delineate genotypic clustering within a genus or species.
Multilocus sequence analysis——MLSA
Schleifer, Karl Heinz. Syst Appl Microbiol 32.8 (2009): 533-542.
Multilocus sequence analysis——MLSA
Maiden, et al., Nat Rev Micro. 2013, 11(10): 728-736.
• Whole-genome sequence analyses are becoming
more common
— ANI (average nucleotide identity) has been demonstrated to
correlate with DDH, where the range of ~95–96% similarity
may reflect the current boundary of 70% DDH similarity (Goris
et al., 2007). ANI may substitute for DDH analyses in the near
future.
Whole-genome sequence analyses
Richter M & Rosselló-Móra R. Proc Natl Acad Sci U S A, 2009, 106(45): 19126-19131.
– Nomenclature
• Assignment of a specific name according to international rules (International Code of Nomenclature of Bacteria[Sneath,1992]).
Nomenclature
http://www.bacterio.net/
• Major references in bacterial diversity
– Bergey’s Manual of Systematic Bacteriology (Springer)
Bergey’s Manual of Determinative Bacteriology
Bergey’s Manual of Systematic Bacteriology
– The Prokaryotes (Springer)
Taxonomy References
• NCBI Taxonomyhttp://www.ncbi.nlm.nih.gov/Taxonomy/
• TOBAhttp://www.taxonomicoutline.org/
• Bergey’s Taxonomyhttp://www.bergeys.org/outlines.html
• List of Prokaryotic Names with Standing in Nomenclaturehttp://www.bacterio.cict.fr/index.html
• Bacterial Nomenclature Up-to-Date http://www.dsmz.de/microorganisms/bacterial_nomenclature.php
• The International Code of Nomenclature of Prokaryotes: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=icnb
• EzTaxon-e Database
http://eztaxon-e.ezbiocloud.net/
Taxonomy References
Some National Microbial Culture Collections