y

ar
br
Li
or re
th tu
Rickettsiae:

au ec
L
Bacteriology and Taxonomy
by e
© lin
n
O
ID

Marina E. Eremeeva
M

Centers for Disease Control and Prevention, Atlanta, USA

C
ES

Post Graduate Educational Course: Intracellular bacteria – from biology to clinic

2-5 November 2010, Sousse-Tunisia
 y
Definition

ar
br
Li
• Taxonomy is the practice and science of classification

or re
• The word finds its roots in the Greek τάξις, taxis

th tu
(meaning 'order' or 'arrangement') and νόμος,

au ec
nomos (meaning 'law' or 'science')

L
by e
• Taxonomy uses taxonomic units, known as taxa
© lin
(singular taxon)
n
O

• Taxonomy, or taxonomic scheme, is also used as a

ID

particular classification ("the taxonomy of ..."),

M

arranged in a hierarchical structure

C
ES
 y
ar
Different Forms of Taxonomy

br
Li
• Biological classification (Linnaean taxonomy)

or re
a. The best known form of taxonomy

th tu
b. Applies empirical science to classifying - only the final step of

au ec
a process

L
c. Includes the prediction, discovery, description and

by e
(re)defining of taxa
© lin
n
d. Uses taxonomic ranks (Kingdom, Phylum, Class, Order,
O

Family, Genus, Species)

ID

• Phylogenetic systematics (Phylogenetics)

M
C

a. Predicts trees of descent

ES

b. Analyzes relationships among groups

 y
ar
Outline

br
Li
• Bacteriology

or re
th tu
Nature and properties of rickettsiae

au ec
• Phylogeny

L
by e
History of the evolution of a species or group
© lin
• Taxonomy
n
O

Classification of rickettsiae
ID
M
C
ES
 y
ar
It sounds very similar…

br
Li
or re
Rickettsiae = rickets

th tu
au ec
L
by e
© lin
Obligate Bone softening
n
intracellular due to vitamin
O

bacteria D deficiency
ID
M
C
ES
 y
ar
Howard Taylor Ricketts, M.D. (1871-1910)

br
Li
or re
• Described etiological agent of RMSF

th tu
• Recognized tick as a vector

au ec
• Discovered low prevalence of

L
infected ticks in nature

by e
• Suggested tick control program
© lin
• Proposed serum therapy or
n
O

vaccination
ID
M
C
ES
 y
ar
br
The term “rickettsia” has for many

Li
or re
years been loosely applied to a very wide

th tu
range of Gram-negative bacteria simply

au ec
because of their obligate association with

L
by e
arthropods and their hosts, their size and
© lin
their intracellular habitat…
n
O
ID
M
C
ES
 y
ar
However,

br
Li
The rickettsiae are

or re
a highly heterogeneous group that historically included:

th tu
au ec
Genera Rickettsia & Orientia

L
Genera Anaplasma, Ehrlichia, Neorickettsia,

by e
Wolbachia © lin
n
O

Genera Bartonella (Rochalimaea) & Grahamella

ID

Genera Haemobartonella & Eperythrozoon

M
C

Genera Coxiella, Rickettsiella & Piscirickettsia

ES
 y
16S rRNA gene

ar
Dr.

br
Brouqui

Li
or re
th tu
au ec
L
Dr.

by e
Birtles
© lin
n
Dr. Greub
O
ID
M
C
ES
 y
Formal Taxonomy of Rickettsia

ar
br
Li
Order: Rickettsiales

or re
th tu
au ec
Family: Rickettsiaceae

L
by e
© lin
Genus: Rickettsia
n
O
ID

Species: > 22 formally described species

M
C
ES
 y
ar
Further Division

br
Li
Genus Rickettsia In silico genome data

or re
th tu
typhus group typhus group

au ec
transition group

L
by e
spotted fever group spotted fever group
© lin
ancestral group
n
O
ID

•Antigene structure
M

•Temperature requirements
C

•Growth characteristics
ES

•DNA properties
 y
Rickettsia: Formal Description

ar
br
Li
• Obligate intracellular rod-shaped bacteria

or re
• 0.3-0.5 x 0.8-2.0 m

th tu
au ec
• Cell culture or chicken embryos for growth

L
• Slow growing

by e
• © lin
Surrounded by typical Gram –negative membrane
n
a. Not stained well by the Gram method
O

b. Retain basic fuschin when stained by Gimenez

ID
M
C
ES
 y
ar
Gimienez Staining of Rickettsia

br
Li
or re
th tu
au ec
L
by e
© lin
n
O
ID

•Rickettsia primarily infect endothelial cells in vivo

M

•Infect arthropod cells

C
ES

•Very promiscuous in cell culture

•Temperature range : 28-37oC
 y
Electron Microscopic Structure

ar
br
of Rickettsia

Li
or re
th tu
au ec
L
by e
© lin
n
O
ID
M
C

•Grow free in the cytoplasm but some can enter nucleus

ES

•Multiply by binary fission but have a slow generation time

•Surrounded by protein S-layer
 y
Intracellular Cycle of

ar
br
Rickettsia and Orientia

Li
or re
th tu
au ec
L
by e
© lin
n
O

Spotted fever
group rickettsiae
ID
M
C
ES
 y
Association with Cellular Cytoskeleton

ar
br
Spotted fever

Li
group

or re
th tu
Typhus group:

au ec
R. prowazekii

L
by e
© lin
n
O
ID
M
C
ES

Orientia
 y
Antigens and Proteins

ar
br
Li
or re
th tu
au ec
L
by e
© lin
n
O
ID

Serotyping antigens:
M

•OmpA & OmpB, Sca

C

•17 kDa lipoprotein

ES

•Heat shock proteins

OmpA and OmpB western blotting
• Smooth type LPS
 y
ar
Genetic Characteristics

br
Li
•

or re
32-33% G+C, spotted fever group

th tu
• 29% G+C, typhus group

au ec
• Genome 1.1-1.6 Mb

L
by e
• One circular chromosome
© lin
•
n
0-3 plasmids: 12,377-67,829 bp
O
ID
M
C
ES
 y
Different Systems for Classification

ar
br
Li
• Classic system

or re
a.Type of disease

th tu
b.Vector association

au ec
L
c.Geographic distribution

by e
d.Cell association characteristics
© lin
• Serotyping with mouse hyperimmune serum
n
O

• SDS-PAGE and PFGE

ID
M

• Comparison of nucleotide sequences

C

a.16S rRNA gene vs protein genes

ES
 y
Generic Characteristics Used for

ar
br
Classification of Rickettsiae

Li
or re
Geography &

th tu
Vector Disease
Impact

au ec
L
• Louse-borne • Typhus • Epidemic

by e
• Flea-borne • Spotted fever
© lin • Endemic
• Mite-borne • Scrub typhus • Sporadic
n
O

• Tick-borne
ID
M
C
ES
 y
Serotyping of Rickettsia

ar
br
Li
• LPS cross-reactive epitopes

or re
a.Proteus vulgaris OX19, OX2

th tu
au ec
b.Proteus mirabilis OXK

L
• Group-reactive LPS epitopes
by e
© lin
• Group and species specific protein epitopes
n
O

a. 17 kDa protein antigen (lipoprotein)

ID

b. S-layer proteins and SCA proteins

M
C

c. Heat shock proteins

ES
 y
Indirect Immunofluorescence Assay:

ar
br
Mouse Serum Serotyping, Philip et al. 1978

Li
or re
th tu
SPecificity Difference= (Aa + Bb) – (Ab + Ba)

au ec
L
SPD > 3 : different serotypes

by e
SPD < 3 : the same serotypes
© lin
n
Aa & Bb, titer of serum with homologous antigen
Ab & Ba, titer of serum with heterologous antigen
O
ID
M
C
ES
 y
Antigenic Diversity of Rickettsia

ar
br
Li
or re
th tu
au ec
L
by e
© lin
n
O
ID
M
C
ES

A: Antigenic diversity estimated based on

monoclonal antibody panel reactivity
Xu and Raoult, 1998 B. ompA sequence diversity
 y
ar
Limitations

br
Li
• Hard to standardize

or re
• Subjective

th tu
au ec
• Need multiple controls

L
by e
© lin
n
O
ID
M
C
ES
 y
Chromosome Typing: RFLP and PFGE

ar
br
• Infrequent cutter restriction endonuclease analysis

Li
• Pulsed field gel electrophoresis

or re
th tu
au ec
L
by e
© lin
n
O
ID
M
C
ES

Roux and Raoult 1995; Eremeeva et al. 1994

 y
ar
Limits

br
Li
or re
Not applicable to uncultivated Rickettsia

th tu
au ec
L
by e
© lin
n
O
ID
M
C
ES
 y
Taxonomy: Molecular Criteria

ar
br
Li
Criteria Free living bacteria

or re
th tu
DNA-DNA hybridization Cut off 70%

au ec
L
Average nucleotide identity Cut off 94%

by e
© lin
16S rRNA gene sequence
n
Cut off 97%
O
similarity
ID
M

Common standards of species definition are not

C

useful for classification of rickettsiae

ES
 y
Comparison of Nucleotide Sequences

ar
br
Genus level

Li
•16S rRNA gene (rrs)

or re
•Citrate synthase gene (gltA)

th tu
•17 kDa protein gene

au ec
Species level

L
by e
•Citrate synthase gene (gltA)
© lin
n
•OmpA protein gene (ompA)
•OmpB protein gene (ompB)
O

•Sca4 protein gene (sca4)

ID

Isolate level Intergenic regions

M
C
ES
 y
ar
Multiple Gene Sequence Analysis

br
Li
gltA ompA ompB sca4 Group

or re
R. rickettsii, R. conorii

th tu
Strain S, R. africae
R. parkeri, R. sibirica
R. mongolotimonae
RrickG

au ec
RrickG RrickG RrickG R. slovaca, R. honei

Rjapn Rjapn R. japonica

Rjapn Rjapn

L
Rmont Rmont R. montanenesis
Rmont Rmont

by e
RmasG RmasG R. massiliae, Bar 29
RmasG RmasG R. rhipicephali
Rhelv

Rfel
© lin
Rfel
n Rhelv

Rfel
Rhelv

Rfel
R. aeschlimannii

R. felis
O
RakrG RakrG RakrG RakrG R. akari
R. australis
RproG RproG RproG R. prowazekii
ID

R. typhi
AB
AB AB bacterium
Rcan
M

Rcan R. canadensis
Rbel
C

Rbel R. bellii
ES

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

 y
Phylogeny of Individual Coding Sequences Match

ar
Phylogeny of Whole Genome

br
Li
or re
th tu
au ec
L
by e
© lin
n
O
ID
M
C
ES

Concatenated Genes for Type Four Secretion System (left) and Whole Genome (right)
ES
C
M
ID
O
n
© lin
by e
L
au ec
th tu
or re
Li
br
ar
y
 y
ar
Multiple Locus Sequence Analysis

br
Li
or re
Gene target Cut off Classification

th tu
16S rRNA gene 98.1% Rickettsia sp.

au ec
gltA 86.5% Rickettsia sp.

L
by e
99.9% New or validated Rickettsia sp.
© lin
n
ompA 98.8% New or validated Rickettsia sp.
O

ompB 99.2% New or validated Rickettsia sp.

ID
M

sca4 99.3% New or validated Rickettsia sp.

C
ES

Fournier et al., 2003

 y
ar
Defining a Status of New Rickettsia

br
Li
• New sequences, but it does not grow –

or re
Candidatus Rickettsia sp.

th tu
• New sequences, sustained isolate, unique

au ec
phenotypic features – NEW Rickettsia sp.

L
by e
• Sequences similar to a validated species, but the
© lin
isolate is antigenically and epidemiologically different
n
O

from type strain –

ID

New subspecies of the known Rickettsia sp.

M

• Sequences and antigenic characteristics are similar to

C

a known Rickettsia sp. – NEW isolate (strain)

ES
 y
ar
Characterizing New Rickettsia

br
Li
• Sequence 16S rDNA and gltA

or re
• Sequence ompA, ompB, sca4

th tu
• Determine genetic similarity to the nearest neighbors

au ec
•

L
Obtain sustained cell culture isolate

by e
• Describe phenotypic characteristics
© lin
•
n
Deposit type strain into two culture collections
O

• Publish in International Journal of Systematic and

ID

Evolutionary Microbiology
M
C

New species name should appear on the

ES

Approved List of Bacterial Species

ES
C
M
ID
O
n
© lin
by e
L
au ec
th tu
or re
Li
br
ar
y
 y
ar
New Rickettsial Disease

br
Li
• New disease can be named with a

or re
th tu
description of single patient case

au ec
• No requirements that agent is

L
by e
characterized or cultivated
© lin
n
• Specific name should be given to each
O

disease caused by each rickettsial species

ID
M

• Each disease should have a single name

C
ES
 R.sibirica

Genus Rickettsia:

y
R. parkeri

ar
R. africae

R. conorii

R. marmionii

16S rRNA gene

br
R. japonica

R. heilongjiangii

Li
R. peacockii

R. rickettsii

R. honei

Pre-molecular time:

or re
R. slovaca

Endosymbiont of Dermacentor variabilis

Rickettsia sp. RpA4

12 formally described species

th tu
Rickettsia sp. DnS14

R. amblyommii 2 unnamed isolates

au ec
R. aeschlimanii

R. montanensis

R. monacensis
Molecular era:

L
R. rhipicephali

R. helvetica

Candidatus R. tarasevichiae
+ 11 newly described species

by e
R. akari
+ description of subspecies for
© lin
R. bellii

Rickettsia sp. PAR

Endosymbiont of Tetranychus urticae

n
Endosymbiont of Dermacentor andersonii
R. conorii (4) and R. sibirica (2)
+ recognition of Candidatus species
Endosymbiont of Onychiurus sinensis

+ many new isolates with unsettled status

O
R. massiliae

R. prowazekii

R. typhi
ID

R. australis

R. canadensis

R. limonae
M

Endosymbiont of Heclepsis marginata

Endosymbiont of Torix tagoi

C

Endosymbiont of Torix tukubana

Anaplasma phagocytophilum
ES

Ehrlichia chaffeensis

Ehrlichia muris
Classic known Classic unknown
Rickettsial endosymbiont "Montezuma" pathogens pathogenicity
Endosymbiont of Acanthamoeba UWC36

Endosymbiont of Acanthamoeba UWC8 Classic Recently described

Escherichia coli pathogenicity
0.02 established recently
 y
Take Home Lesson

ar
br
 Taxonomy is never finished and is

Li
or re
dynamic, but it is always a necessary

th tu
communication tool to promote research

au ec
L
in the scientific community

by e
© lin
 Standard for the creation of new taxa
n
O

varies with investigators; depending on

ID

the available data, resources and current

M

state of the art of laboratory research

C
ES

 Usefulness of taxons
 y
ar
br
Li
or re
th tu
au ec
L
by e
© lin
n
O
ID

For more information, please, contact Centers for Disease Control and Prevention
1600 Clifton Road NE, Atlanta GA 30333
M

Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348

Email:cdcinfo@cdc.gov Web:www.cdc.gov
C

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

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