Rickettsiae:Bacteriology and Taxonomy

Marina E. Eremeeva Centers for Disease Control and Prevention, Atlanta, USA

Post Graduate Educational Course: Intracellular bacteria – from biology to clinic2-5 November 2010, Sousse-TunisiaESCMID

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Definition

• Taxonomy is the practice and science of classification

• The word finds its roots in the Greek τάξις, taxis(meaning 'order' or 'arrangement') and νόμος, nomos (meaning 'law' or 'science')

• Taxonomy uses taxonomic units, known as taxa(singular taxon)

• Taxonomy, or taxonomic scheme, is also used as a particular classification ("the taxonomy of ..."), arranged in a hierarchical structure

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Different Forms of Taxonomy• Biological classification (Linnaean taxonomy)

a. The best known form of taxonomy

b. Applies empirical science to classifying - only the final step of a process

c. Includes the prediction, discovery, description and (re)defining of taxa

d. Uses taxonomic ranks (Kingdom, Phylum, Class, Order, Family, Genus, Species)

• Phylogenetic systematics (Phylogenetics)a. Predicts trees of descentb. Analyzes relationships among groupsESCMID

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Outline

• Bacteriology

Nature and properties of rickettsiae

• Phylogeny

History of the evolution of a species or group

• Taxonomy

Classification of rickettsiae

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It sounds very similar…

Rickettsiae = rickets

Obligate intracellular

bacteria

Bone softening due to vitamin

D deficiency

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Howard Taylor Ricketts, M.D. (1871-1910)

• Described etiological agent of RMSF• Recognized tick as a vector• Discovered low prevalence of

infected ticks in nature• Suggested tick control program• Proposed serum therapy or

vaccination

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The term “rickettsia” has for many years been loosely applied to a very wide range of Gram-negative bacteria simply because of their obligate association with arthropods and their hosts, their size and their intracellular habitat…

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However,

The rickettsiae are

a highly heterogeneous group that historically included:

Genera Rickettsia & Orientia

Genera Anaplasma, Ehrlichia, Neorickettsia,

Wolbachia

Genera Bartonella (Rochalimaea) & Grahamella

Genera Haemobartonella & Eperythrozoon

Genera Coxiella, Rickettsiella & Piscirickettsia

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16S rRNA gene

Dr. Birtles

Dr. Greub

Dr. Brouqui

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Formal Taxonomy of Rickettsia

Order: Rickettsiales

Family: Rickettsiaceae

Genus: Rickettsia

Species: > 22 formally described species

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Further Division

Genus Rickettsia

typhus group

spotted fever group

In silico genome data

typhus group

transition group

spotted fever group

ancestral group

•Antigene structure•Temperature requirements•Growth characteristics•DNA propertiesESCMID

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Rickettsia: Formal Description

• Obligate intracellular rod-shaped bacteria

• 0.3-0.5 x 0.8-2.0 m

• Cell culture or chicken embryos for growth

• Slow growing

• Surrounded by typical Gram –negative membrane

a. Not stained well by the Gram method

b. Retain basic fuschin when stained by Gimenez

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Gimienez Staining of Rickettsia

•Rickettsia primarily infect endothelial cells in vivo•Infect arthropod cells•Very promiscuous in cell culture •Temperature range : 28-37oCESCMID

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Electron Microscopic Structure of Rickettsia

•Grow free in the cytoplasm but some can enter nucleus•Multiply by binary fission but have a slow generation time•Surrounded by protein S-layerESCMID

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Intracellular Cycle of Rickettsia and Orientia

Spotted fever group rickettsiae

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Association with Cellular CytoskeletonSpotted fever

group

Typhus group:

R. prowazekii

OrientiaESCMID

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Antigens and Proteins

Serotyping antigens:

•OmpA & OmpB, Sca

•17 kDa lipoprotein

•Heat shock proteins

• Smooth type LPSOmpA and OmpB western blotting

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Genetic Characteristics

• 32-33% G+C, spotted fever group

• 29% G+C, typhus group

• Genome 1.1-1.6 Mb

• One circular chromosome

• 0-3 plasmids: 12,377-67,829 bp

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• Classic system

a.Type of disease

b.Vector association

c.Geographic distribution

d.Cell association characteristics

• Serotyping with mouse hyperimmune serum

• SDS-PAGE and PFGE

• Comparison of nucleotide sequences

a.16S rRNA gene vs protein genes

Different Systems for Classification

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Generic Characteristics Used for Classification of Rickettsiae

Vector

• Louse-borne

• Flea-borne

• Mite-borne

• Tick-borne

Disease

• Typhus

• Spotted fever

• Scrub typhus

Geography & Impact

• Epidemic

• Endemic

• Sporadic

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Serotyping of Rickettsia

• LPS cross-reactive epitopes

a.Proteus vulgaris OX19, OX2

b.Proteus mirabilis OXK

• Group-reactive LPS epitopes

• Group and species specific protein epitopes

a. 17 kDa protein antigen (lipoprotein)

b. S-layer proteins and SCA proteins

c. Heat shock proteinsESCMID

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Indirect Immunofluorescence Assay:Mouse Serum Serotyping, Philip et al. 1978

SPecificity Difference= (Aa + Bb) – (Ab + Ba)

SPD > 3 : different serotypesSPD < 3 : the same serotypes

Aa & Bb, titer of serum with homologous antigenAb & Ba, titer of serum with heterologous antigen

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Antigenic Diversity of Rickettsia

Xu and Raoult, 1998

A: Antigenic diversity estimated based on monoclonal antibody panel reactivityB. ompA sequence diversity

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Limitations

• Hard to standardize

• Subjective

• Need multiple controls

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Chromosome Typing: RFLP and PFGE• Infrequent cutter restriction endonuclease analysis

• Pulsed field gel electrophoresis

Roux and Raoult 1995; Eremeeva et al. 1994

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Limits

Not applicable to uncultivated Rickettsia

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Taxonomy: Molecular Criteria

Common standards of species definition are not useful for classification of rickettsiae

Criteria Free living bacteria

DNA-DNA hybridization Cut off 70%

Average nucleotide identity Cut off 94%

16S rRNA gene sequence similarity

Cut off 97%

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Comparison of Nucleotide Sequences

•16S rRNA gene (rrs)•Citrate synthase gene (gltA)•17 kDa protein gene

Genus level

Species level

•Citrate synthase gene (gltA)•OmpA protein gene (ompA)•OmpB protein gene (ompB)•Sca4 protein gene (sca4)

Isolate level Intergenic regions

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Multiple Gene Sequence Analysis

Adapted from Z. Sekeyova, V. Roux & D. Raoult, 2001

gltA ompA ompB sca4 Group

RrickG

Rjapn

Rmont

RmasG

Rhelv

Rfel

RakrG

RproG

AB

Rcan

Rbel

RrickG

Rjapn

Rmont

RmasG

Rhelv

Rfel

RakrG

RproG

RrickG

Rjapn

Rmont

RmasG

Rfel

RakrG

RrickG

Rjapn

Rmont

RmasG

Rhelv

Rfel

RakrG

RproG

AB

Rcan

Rbel

R. rickettsii, R. conoriiStrain S, R. africaeR. parkeri, R. sibiricaR. mongolotimonaeR. slovaca, R. honei

R. japonica

R. montanenesis

R. massiliae, Bar 29R. rhipicephaliR. aeschlimannii

R. felis

R. akariR. australis

R. prowazekiiR. typhi

AB bacterium

R. canadensis

R. bellii

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Phylogeny of Individual Coding Sequences Match Phylogeny of Whole Genome

Concatenated Genes for Type Four Secretion System (left) and Whole Genome (right)ESCMID

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Multiple Locus Sequence Analysis

Gene target Cut off Classification

16S rRNA gene 98.1% Rickettsia sp.

gltA 86.5% Rickettsia sp.

99.9% New or validated Rickettsia sp.

ompA 98.8% New or validated Rickettsia sp.

ompB 99.2% New or validated Rickettsia sp.

sca4 99.3% New or validated Rickettsia sp.

Fournier et al., 2003

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Defining a Status of New Rickettsia

• New sequences, but it does not grow –

Candidatus Rickettsia sp.

• New sequences, sustained isolate, unique phenotypic features – NEW Rickettsia sp.

• Sequences similar to a validated species, but the isolate is antigenically and epidemiologically different from type strain –

New subspecies of the known Rickettsia sp.

• Sequences and antigenic characteristics are similar to a known Rickettsia sp. – NEW isolate (strain)ESCMID

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Characterizing New Rickettsia• Sequence 16S rDNA and gltA

• Sequence ompA, ompB, sca4

• Determine genetic similarity to the nearest neighbors

• Obtain sustained cell culture isolate

• Describe phenotypic characteristics

• Deposit type strain into two culture collections

• Publish in International Journal of Systematic and Evolutionary Microbiology

New species name should appear on the

Approved List of Bacterial SpeciesESCMID O

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New Rickettsial Disease

• New disease can be named with a description of single patient case

• No requirements that agent is characterized or cultivated

• Specific name should be given to each disease caused by each rickettsial species

• Each disease should have a single name

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Genus Rickettsia:16S rRNA gene

R.sibirica

R. parkeri

R. africae

R. conorii

R. marmionii

R. japonica

R. heilongjiangii

R. peacockii

R. rickettsii

R. honei

R. slovaca

Endosymbiont of Dermacentor variabilis

Rickettsia sp. RpA4

Rickettsia sp. DnS14

R. amblyommii

R. aeschlimanii

R. montanensis

R. monacensis

R. rhipicephali

R. helvetica

Candidatus R. tarasevichiae

R. akari

R. bellii

Rickettsia sp. PAR

Endosymbiont of Tetranychus urticae

Endosymbiont of Dermacentor andersonii

Endosymbiont of Onychiurus sinensis

R. massiliae

R. prowazekii

R. typhi

R. australis

R. canadensis

R. limonae

Endosymbiont of Heclepsis marginata

Endosymbiont of Torix tagoi

Endosymbiont of Torix tukubana

Anaplasma phagocytophilum

Ehrlichia chaffeensis

Ehrlichia muris

Rickettsial endosymbiont "Montezuma"

Endosymbiont of Acanthamoeba UWC36

Endosymbiont of Acanthamoeba UWC8

Escherichia coli

0.02

Classic known pathogens

Classic unknown pathogenicity

Classic pathogenicityestablished recently

Recently described

Pre-molecular time:12 formally described species2 unnamed isolates

Molecular era:+ 11 newly described species+ description of subspecies for

R. conorii (4) and R. sibirica (2)+ recognition of Candidatus species+ many new isolates with unsettled status

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Taxonomy is never finished and is dynamic, but it is always a necessary communication tool to promote research in the scientific community

Standard for the creation of new taxavaries with investigators; depending on the available data, resources and current state of the art of laboratory research

Usefulness of taxons

Take Home Lesson

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The findings and conclusions in this report are those of the author and do not necessarily represent the official

position of the Centers for Disease Control and Prevention

For more information, please, contact Centers for Disease Control and Prevention1600 Clifton Road NE, Atlanta GA 30333Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348Email:cdcinfo@cdc.gov Web:www.cdc.gov

MEremeeva@cdc.gov (404-639-4612)

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