Enterobacteriaceae: Classification.

Dr Abhijit Chaudhury

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.

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 .

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.

• 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)

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.

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.

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.

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.

Genetic/Molecular Methods

DNA Study

1. DNA Base Composition 2. DNA Homology

16 s rRNA gene sequence

DNA Base Composition It denotes the relative amounts of G=C and

A=T amounts. Conventionally GC base composition is used.

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.

Graph showing the direct relationship between mol % G + C and midpoint temperature (Tm) of purified DNA in thermal denaturation experiments.

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.

16s rRNA Gene SequenceADVANTAGES 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)

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.

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

Methodology

Genomic DNA extraction

Universal (specific) primer design

PCR reaction

PCR product purification

Directed sequencing

(Full length SEQ)

Data analysis

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

Bacterial Species 1. If there is >70% homology based

on hybridization 2. Usually have 99%-100% rRNA

sequence identity 3. Less than 50C difference in thermal

stability Organisms with less than 98% 16S

rRNA sequence and < 70% DNA:DNA are likely to be different species.

Phylogenetic treesTwo different formats of phylogenetic trees used to show relatedness among species.

Universal phylogenetic tree as determined from comparative ribosomal RNA sequencing.

Detailed phylogenetic tree of the major lineages (phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons

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).

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.

Enterobacteriaceae

Domain: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae

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)

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).

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.

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.

1900-1950

They proposed 8 genera in this family:Genus I Serratia Genus V ShigellaGenus II ColobactrumGenus VI Paracolobactrum

Genus III Proteus Genus VII ErwiniaGenus IV SalmonellaGenus VIII Proshigella

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.

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.

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.

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

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.

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.

Proposed ChangesSALMONELLA 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).

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.

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.

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)

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.

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.

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.”

References1. 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.