The document discusses the classification of bacteria in the family Enterobacteriaceae. It covers the historical methods of classification based on phenotypes as well as modern genetic methods using DNA-DNA hybridization and 16S rRNA gene sequencing. The key events in the classification of the family Enterobacteriaceae from the 1800s to present are outlined, noting changing taxonomy and increasing recognition of new species through molecular techniques.

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.