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Classification of Enterobacteriaceae family
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Classification of Enterobacteriaceae family

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  • 1. Enterobacteriaceae: Classification. Dr Abhijit Chaudhury
  • 2. Basis of Bacterial Classification  Taxonomy –the principles and practice of classifying bacteria (OR) The orderly classification of organisms based on their presumed natural relationships  Classification –arrangement of strains into natural groups (taxa)-phenetic(phenotypic and genetic) and phylogenetic (OR) The theory and process of ordering the organisms, on the basis of shared properties, into groups.
  • 3.  Nomenclature –allocation of names to these groups  Identification-processes by which unknowns are referred to known taxa. Species – a collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly .
  • 4.  Genus: represent natural evolutionary groups as defined by techniques that actually measure evolutionary distance and these natural groups share phenotypic similarities that differentiate them from other genera. OR "a group of species which are grouped together for convenience rather than because of a close evolutionary relationship“.  A biogroup (synonym,biovar) is defined to be a group of strains that have a common biochemical reaction pattern, which is often unusual for the particular species.
  • 5. • Classification • Organization into groups • Car • Truck • SUV • Van • Identification • Distinguishing features • Engine size • Mileage • Number of passengers • Type of transmission  Nomenclature  Providing a formal name  Genus & species  Honda City  Maruti 800  Ambassador Nova  Fiat 1800D Trinity of Classification, Nomenclature, and Identification = TAXONOMY ( S.T. Cowan)
  • 6. PHENETICS  A method of natural classification. It is based on empirical classification of general characters.  It may or may not include genetic information.  When it excludes genetic information it is called PHENOTYPIC CLASSIFICATION.
  • 7. Methods of Classification  Historically, prokaryotes were classified on the basis of their phenotype (morphology, staining reactions, biochemistry, substrates/products, antigens etc). In other words a phenotypic characterization was based on the information carried in the products of the genes. These classification systems were artificial.  Modern characterization is based on the information carried in the genes i.e. the genome. This is genetic information and can also tell us something about the evolution of the organism. In other words phylogenetics.
  • 8. Phenotypic Classification: Numerical Taxonomy  Adanson(1763), PHA Sneath (1957)  Mathematical and statistical methodology  A large number of tests (~100) are carried out and the results are scored as positive or negative. Several control species are included in the analysis.  1 = trait is present, 0=absent  All characteristics are given equal weight and a computer based analysis is carried out to group the bacteria according to shared properties.
  • 9. Numerical Taxonomy  It gives results that broadly coincide with non-numerical classification.  Has been found useful for the study of certain groups thought to be difficult to classify like Rhodococcus group.
  • 10. Genetic/Molecular Methods  DNA Study 1. DNA Base Composition 2. DNA Homology  16 s rRNA gene sequence
  • 11. DNA Base Composition  It denotes the relative amounts of G=C and A=T amounts. Conventionally GC base composition is used.
  • 12. Melting curve for a double-stranded DNA molecule. As the temperature is raised during the experiment, the double- stranded DNA is converted to the single-stranded form and the UV absorbance of the solution increases. The midpoint temperature, Tm, can be calculated from the curve.
  • 13. Graph showing the direct relationship between mol % G + C and midpoint temperature (Tm) of purified DNA in thermal denaturation experiments.
  • 14. DNA Homology  DNA-DNA pairing ( Schildkraut 1961) provide a great deal of information about the relationship between organisms at species level.  Not found useful in revealing broader groups among bacteria.  Strains with values of 70% or greater are considered to be the same species.
  • 15. 16s rRNA Gene Sequence ADVANTAGES  Universal presence  16S rRNA gene is present in all bacteria  Large Subunit (LSU) gene is present in all fungi  Gene structure  Conserved regions  identical in all microorganisms  used for PCR primer location  Divergent regions  different in many microorganisms  used for identification (sequencing)
  • 16. Advantages  High content of information  500 bp sequence with 4 different bases → 4500 = 1 x 10301 variants  15 biochemical tests with “yes/no” result → 215 = 3 x 104 variants  16S rDNA has become the Standard for Taxonomic Classification. ## Gold Standard for species identification: DNA-DNA homology.
  • 17. 16s r RNA Methodology  First step: Determination of the ribosomal RNA gene sequence of an unknown microorganism  Second step: Comparison of the generated sequence with the sequences of known microorganisms present in a database
  • 18. Methodology Genomic DNA extraction Universal (specific) primer design PCR reaction PCR product purification Directed sequencing (Full length SEQ) Data analysis
  • 19. Methodology  NCBI Genebank webpage  Nucleotide-Nucleotide BLAST (Basic Local Alignment Search Tool) : Paste in the linear sequence data and submit. Search is performed and list of matches provided  ~ 99%-100%: Species confirmed ~97% -99% : Genus confirmed, new species < 97% : New Genus, New species
  • 20. Bacterial Species  1. If there is >70% homology based on hybridization 2. Usually have 99%-100% rRNA sequence identity 3. Less than 50 C difference in thermal stability  Organisms with less than 98% 16S rRNA sequence and < 70% DNA:DNA are likely to be different species.
  • 21. Phylogenetic trees Two different formats of phylogenetic trees used to show relatedness among species.
  • 22. Universal phylogenetic tree as determined from comparative ribosomal RNA sequencing.
  • 23. Detailed phylogenetic tree of the major lineages (phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons
  • 24. 16s rRNA Sequencing- Conclusion  Can better discriminate bacterial isolates than many phenotypic methods.  Can identify novel, poorly described, rarely isolated, or phenotypically aberrant strains  Can define relatedness of organisms + evolutionary distance.  Can be used for organisms that have not been cultured (Uncultivable bacteria).
  • 25. International groups  International Committee for Systematic Bacteriology (ICSB) supervises the Bacteriological Code. It regularly provides list of recent validly published species names and proposed changes in nomenclature First in Int J of Systematic Bacteriology Then in Int J Of Systematic and Evolutionary Microbiology  The status of the scheme is reviewed every 10 years in Bergey’s Manual of Systematic Bacteriology (Latest edition 2001, Edition 2; 5 volumes. Vol 2 (2005) The Proteobacteria.
  • 26. Enterobacteriaceae  Domain: Bacteria  Phylum: Proteobacteria  Class: Gamma Proteobacteria  Order: Enterobacteriales  Family: Enterobacteriaceae
  • 27. Enterobacteriaceae: the 1800s  The first member Serratia marcescens was discovered by Bizio in 1823 on a dish of Italian barley (Polenta).  After more than 50 years, Klebsiella and Proteus were discovered in 1880s.  Theobald Smith in 1893: Lactose Fermenter ( Benign Organisms/Coliforms) and Non lactose fermenters (Dangerous pathogens)  In 1897, third group- Paracolon bacilli (Delayed lactose fermentation)
  • 28. Enterobacteriaceae: 1900-1950  Gram negative facultative bacilli were being discovered and named arbitrarily based on place/person/some unique character. ( Bathesda –Ballerup, Providence groups/ Morgan’s bacilli/ Proteus etc) and designated LF/NLF/ or Paracolon bacilli.  During the same time, two nondescript genera were being used to house the bacteria: Bacterium ( B.coli) and Bacillus ( B.cloacae).
  • 29. 1900-1950  Otto Rahn in 1937 first proposed the name Enterobacteriaceae family for a group of biochemically and morphologically similar organisms with a single genus Enterobacter. It was used to put together 112 species.  The first publication of the Kauffmann- White scheme (Salmonella Subcommittee, 1934,)listed 44 serovars of the Salmonella.
  • 30. 1900-1950  Borman, Stuart, Wheeler (1944) defined the family as :Gram-negative, non-sporogenic rods widely distributed in nature. Grow well on artificial media. All species attack glucose, forming acid or acid and visible gas (H2 present). Characteristically, nitrites are produced from nitrates. When motile,the flagella are peritrichous.
  • 31. 1900-1950 They proposed 8 genera in this family: Genus I Serratia Genus V Shigella Genus II ColobactrumGenus VI Paracolobactrum Genus III Proteus Genus VII Erwinia Genus IV SalmonellaGenus VIII Proshigella
  • 32. 1950-1970  Cowan (1956, 1957): Six genera: Salmonella, Escherichia, Shigella, Citrobacter, Klebsiella, Proteus.  Ewing (1960, 1966): 4 tribes, 10 genera. Tribe 1: Escherichiae: Escherichia, Shigella Tribe 2: Salmonellae: Salmonella, Arizona, Citrobacter Tribe 3: Klebsiellae: Klebsiella, Enterobacter, Serratia Tribe 4: Proteae: Proteus, Providencia.
  • 33. 1950 1970  During this period various other methods were used for identification and classification: 1. Chemotaxonomy (Gas liquid chromatography for Fatty acids) 2. Carbon utilization assay 3. Phage typing 4. Antigenic types etc.
  • 34. 1970- Till Date  Don Brenner at CDC in early 1970s pioneered the use of DNA-DNA hybridization as the gold standard for defining relatedness. This, together with Numerical taxonomy had two important effects: 1. A number of organisms regarded as separate species were found to be single genomic species. EX: E. coli and Shigella, All salmonella 2. Recognition of numerous new species previously thought to be aberrant biotypes of particular species.
  • 35. 1970- Till date  The advent of 16s rRNA sequencing helped in identifying many clinical and environmental isolates to species level, unidentifiable by conventional methods.  In 1972, there were 11 genera and 26 species.  In 1985, 22 genera, 69 species  In 2004, 40 genera, and 200 species.  At present, 47 genera (http://www.bacterio.cict.fr)  Type Genus: Escherichia  Type Species: E.coli
  • 36. 1970- Till date  Farmer JJ et al in 1985 reviewed all the existing genera and species of the family and described their phenotypic characters.  It has a series of differential charts to assist in identification and a large chart with the reactions of 98 different organisms for 47 tests often used in identification.
  • 37. Proposed Changes  Inclusion of the Genus Plesiomonas : Based on 16s rRNA sequence, it is closer to Enterobacteriaceae than Vibrionaceae family. It also contains the common enterobacterial antigens.  Klebsiella to Raoultella : Three species K.terrigena, K.ornithinolytica, K.planticola .  Calymmatobacterium granulomatis to genus Klebsiella: This is an un-cultivable bacteria . Shares nucleotide sequences with Klebsiella, and the disease Granuloma inguinale resembles rhinoscleroma.
  • 38. Proposed Changes SALMONELLA  Good agreement on some issues, but still with several problem areas.  All serotypes of Salmonella probably belong to on DNA hybridization group.  The genospecies was named S. cholerasuis, and later changed to S. enterica. (1982)  Seven subgroups (subspecies): enterica, salamae, arizonae, diarizonae, houtenae, bongori, and indica.  Subgroup Bongori should be elevated to species level based on DNA hybridization and MLEE studies. (1989).
  • 39. Proposed Changes  Citrobacter diversus and C. koseri  Both the names have been used, but C. diversus have been used more frequently.  In 1980, C. diversus became the correct name for this organism, but in 1993 ICSB issued an opinion that C. koseri should be used.  Both have similar properties, but different type strains exist.
  • 40. Proposed Changes Enterobacter sakazakii  Enterobacter sakazakii was defined as a new species in 1980  In the original study fifteen biogroups of E. sakazakii were described  Full length 16S rRNA gene sequences, comprising greater than 1300bp has been done along with DNA hybridization.
  • 41. Proposed changes  These organisms are a microbiological hazard  Occurring in the infant food chain with historic high morbidity and mortality in neonates.  Therefore Cronobacter gen. nov. has been proposed after the Greek mythological god, Cronos, who was described as swallowing his children at birth. (Iversen et al. BMC evolutionary Biology, 2007)
  • 42. What the future holds?  Taxonomy is a dynamic and ongoing process.  New species and genera will continue to be added.  A number of named organisms are known to contain multiple species although the phenotypic methods cannot unambiguously separate them. Ex. E.cloacae, H.alvei,Rahnella aquatilis, Serratia liquefaciens.
  • 43. Future  The increasing knowledge concerning Enterobacteriaceae will continue to challenge the microbiologists to redefine and re evaluate the concepts regarding the biochemical characteristics, ecologic relationships, biosphere distribution, and disease producing potential of this family.
  • 44. The Silver Lining  Many of the new organisms may never be seen in a given clinical microbiology laboratory, but will be encountered more frequently by reference laboratories.  80 to 95% of all isolates seen in a general hospital setting will be Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis.  Over 99% of all clinical isolates will belong to only 23 species.  Keep this distribution in mind and not be overwhelmed with the large number of new species. Adage: "When you hear hoofbeats, think horses, not zebras.”
  • 45. References 1. Topley and Wilson’s Microbiology: 8th and 10 th edition. 2. The Enterobacteria By J. Michael Janda, Sharon L. Abbott. 2006. ASM. 3. Borman EK , Stuart CA AND Wheeler KM. Taxonomy of the family Enterobacteriaceae. J Bacteriol 1944;48:351-367. 4. Farmer JJ III, Davis BR, Hickman-Brenner FW. Biochemical Identification of New Species and Biogroups of Enterobacteriaceae Isolated from Clinical Specimens. J Clin Microbiol 1985;21:46-76.