Genetic Classification and Differentiation of Bartonella Species Based on Comparison of Partial ftsZ Gene Sequences

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Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Genetic Classification and Differentiation of

Bartonella

Species Based

on Comparison of Partial

ftsZ

Gene Sequences

Zaher Zeaiter,

1

_{Zhongxing Liang,}

2

_{and Didier Raoult}

1

_*

Unite´ des Rickettsies, CNRS UMR 6020 IFR 48, Faculte´ de Me´decine, Marseille, France,

1

_{and Division of Viral}

and Rickettsial Diseases, National Center for Infections Diseases, Centers for Disease Control

and Prevention, Atlanta, Georgia 30333

2

Received 25 April 2002/Returned for modification 9 June 2002/Accepted 15 July 2002

Currently, 19 species are recognized in the genus

Bartonella

, 7 of which are involved in an increasing variety

of human diseases. Development of molecular tools for detection, identification, and subtyping of strains and

isolates has promoted research on

Bartonella

spp. We amplified and sequenced the portion of the

ftsZ

gene

encoding the N-terminal region of the cell division protein for 13

Bartonella

species:

Bartonella alsatica

,

B. birtlesii

,

B. doshiae

,

B. elizabethae

,

B. grahami

,

B. koehlerae

,

B. schoenbuchensis

,

B. taylorii

,

B. tribocorum

,

Bartonella vinsonii

subsp.

arupensis

,

Bartonella vinsonii

subsp.

berkhoffii

,

Bartonella vinsonii

subsp.

vinsonii

, and

B. bovis

Bermond et al.(“

B. weissii

”). Phylogenetically derived trees revealed four statistically supported groups,

indicating that sequencing of the

ftsZ

gene is a useful tool for identifying evolutionary relationships among

Bartonella

species. Furthermore, we amplified and sequenced the portion of the

ftsZ

gene encoding the

C-terminal region of the protein for 4

B. bacilliformis

isolates, 14

B. clarridgeiae

isolates, 14

B. quintana

isolates,

and 30

B. henselae

isolates that were obtained from different geographic regions, hosts, and clinical specimens.

B. clarridgeiae

and

B. quintana

sequences were highly conserved, while those of the four

B. bacilliformis

isolates

differed from the type strain at 5 positions. Among

B. henselae

strains isolated from cats and patients, only two

genotypes were detected: Houston and Marseille. Among 80 clinical samples we detected

Bartonella

spp. in 35

(43.75%) and found the assay to be comparable to that of a combined intergenic-spacer-region- and

pap31

-based PCR assay. Our results show the usefulness of the portion of the

ftsZ

gene encoding the C-terminal

region for diagnosis of

Bartonella

infections. More samples should be tested to study its usefulness for

epidemiological investigations.

The genus

Bartonella

contains aerobic, fastidious,

gram-neg-ative bacilli belonging to the alpha-2 subgroup of the class

Proteobacteria.

Recently the number of

Bartonella

species

iso-lated has increased markedly (5, 6, 15, 27), and the bacteria are

considered emerging pathogens involved in an increasing

num-ber of recognized diseases (1, 28, 38). Currently, 19

Bartonella

species are recognized, and all are associated with mammalian

hosts.

Bartonella taylorii

,

B. elizabethae

,

B. tribocorum

, and

B. birtlesii

have been isolated from rats (6, 7, 11, 24);

B. grahamii

,

Bartonella vinsonii

subsp.

vinsonii

, and

B. doshiae

have been

recovered from voles (7, 11);

Bartonella vinsonii

subsp.

arupen-sis

has been isolated from mice (54);

B. alsatica

has been

isolated from rabbits (23);

B. koehlerae

,

B. clarridgeiae

,

B. henselae

, and “

B. weissii

,” recently described as

B. bovis

Ber-mond et al., have been found in cats (5, 17, 29, 31, 35); and

B. bovis

Bermond et al. has also been detected in cattle (5, 10).

Bartonella vinsonii

subsp.

berkhoffii

has been isolated from dogs

(10) and coyotes (13); “

B. washoensis

” has been demonstrated

in rodents (R. L. Regnery, personal communication);

B. quin-tana

and

B. bacilliformis

have been isolated from humans (22,

40), and

B. schoenbuchensis

and

B. capreoli

have been isolated

from wild roe deer (5, 15). To date, 7 of the 19 species have

been implicated in human disease (28).

B. bacilliformis

is the

agent of bartonellosis (Carrion’s disease), which is endemic in

Andean valleys in South America.

B. quintana

and

B. henselae

,

etiologic agents of trench fever and cat scratch disease (CSD),

respectively, have also been associated with endocarditis and

bacillary angiomatosis in immunocompromised patients (1).

B. elizabethae

and

B. vinsonii

subsp.

berkhoffii

cause endocarditis

(14, 46), and

B. vinsonii

subsp.

arupensis

was first isolated from

a febrile patient with heart valve disease in the United States

(54).

B. grahamii

has been implicated in cases of neuroretinitis

(30), and

B. clarridgeiae

is also suspected to be an agent of CSD

(32, 51). Because no distinguishing phenotypic characteristics

have been described for

Bartonella

species, their identification

and phylogenetic classification has been based mainly on

ge-netic studies. DNA hybridization and pulsed-field gel

electro-phoresis can be used for molecular characterization of

Bar-tonella

species (39, 47), but these techniques are not suitable

for routine use in a clinical laboratory. PCR-derived assays

allow detection and identification of the bacteria directly from

clinical samples even in conditions such as CSD, where

organ-isms are infrequently isolated in culture. Many DNA regions

and encoding gene sequences have been used in genetic

stud-ies: the 16S rRNA gene, the 16S–23S rRNA intergenic spacer

region (ITS) (26, 37), the citrate synthase gene (

gltA

) (8, 9, 25),

the riboflavin synthase alpha chain gene (

ribC

) (2), the heat

shock protein gene (

groEL

) (36, 55), the genes encoding the

PAP31 and 35-kDa proteins (33, 56), and the cell division

protein gene (

ftsZ

) (19, 29).

The FtsZ protein plays an important role in bacterial cell

division, and its gene sequence has been used to differentiate

three

Bartonella

species (29). Compared to other bacteria, the

* Corresponding author. Mailing address: Unite´ des Rickettsies,

CNRS UPRES-A 6020, Faculte´ de Me´decine, 27 boulevard Jean

Mou-lin, 13385 Marseille Cedex 05, France. Phone: (33) 4 91 32 43 75. Fax:

(33) 4 91 83 03 90. E-mail: Didier.Raoult@medecine.univ-mrs.fr.

3641

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FtsZ proteins of

Bartonella

species are nearly twice as large

and have an additional region at the C-terminal end (29, 42).

The C-terminal region has a higher degree of sequence

diver-gence than the N-terminal region and has recently been used

for

B. henselae

subtyping (19).

In our study we determined a partial 900-base nucleotide

sequence of

ftsZ

encoding the N-terminal region for the main

Bartonella

species and assessed its usefulness in species

differ-entiation and for inferring interspecies phylogenetic

relation-ships. Furthermore, we investigated PCR of the portion of the

ftsZ

gene encoding the C-terminal region as a means of

de-tecting and identifying

Bartonella

spp. in 80 clinical samples.

We also studied the usefulness of sequencing the portion of the

ftsZ

gene encoding the C-terminal end in subtyping

B. hen-selae

,

B. quintana

,

B. clarridgeiae

, and

B. bacilliformis

isolates

from patients and cats and for epidemiological investigations

of infections.

MATERIALS AND METHODS

Bartonellastrains, isolates, and DNA extraction.Strains and isolates used in this study are detailed in Tables 1 and 2.Bartonellaisolates were grown on 5% sheep blood agar (Biomerieux, Marcy l’E´toile, France) at 37°C under a 5% CO2-enriched atmosphere. Bacteria were harvested after 7 days of culture, and

DNA was extracted by the Chelex method (52). Genomic DNA was stored at 4°C until use as a template in PCR assays.

PCR amplification and DNA sequencing of the portion of theftsZgene en-coding the N-terminal region.Primers (Eurobio, Les Ulis, France) used for amplification and sequencing are shown in Table 3. PCRs were carried out in a PTC-200 automated thermocycler (MJ Research, Waltham, Mass.) using an Elongase DNA polymerase kit (Gibco-BRL, Cergy Pontoise, France) and prim-ers Bfp1 and Bfp2 (Table 3). Reaction mixtures (25␮l) contained the following (final concentrations): primers (0.5 pmol␮l⫺1_{each), deoxynucleoside}

triphos-phates (dATP, dCTP, dGTP, and dTTP) (0.2 mM␮l⫺1_{each), 1}_{␮l of buffer A,}

4␮l of buffer B, 0.6␮l of Elongase enzyme mix, 2.5␮l of DNA (150 to 200 ng), and sterile water. PCR amplifications were performed as follows: a 4-min

dena-turation at 94°C was followed by 44 cycles of denadena-turation for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 60 s at 68°C. Amplification was completed by holding the reaction mixture at 68°C for 10 min to ensure complete extension of the PCR products. These were separated by electrophoresis on 1% agarose gels, visualized by staining with ethidium bromide, and purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR products were sequenced in both directions using primers Bfp1, Bfp2, Bfs3, and Bfs4 and theD-Rhodamine Terminator

Cycle Sequencing Ready Reaction kit (Perkin-Elmer, Coignieres, France) ac-cording to the manufacturer’s instructions. Sequencing products were resolved using an ABI 3100 automated sequencer (Perkin-Elmer).

Analysis of sequences and construction of phylogenetic trees.Sequence anal-ysis was performed with ABI Prism DNA Sequencing Analanal-ysis Software, ver-sion 3.0 (Perkin Elmer), and multisequence alignment was performed with CLUSTAL W software, version 1.81 (53). DNA sequence similarities were cal-culated by use of MEGA 2.1 software (S. Kumar, K. Tamura, I. B. Jakobsen, and M. Nei, Molecular Evolutionary Genetics Analysis software, Tempe, Ariz., 2001). Phylogenetic trees were obtained from DNA sequences by using the maximum-parsimony method (DNAPARS software in PHYLIP) (20), distance methods (DNADIST [distance matrix with Kimura 2 parameters or Jukes-Can-tor parameters] and NEIGHBOR [neighbor joining]), and the maximum-likeli-hood method (DNAMLK software in PHYLIP). Bootstrap replicates were per-formed to estimate the node reliability of the phylogenetic trees obtained by the three methods (12). Bootstrap values were obtained from 100 trees (18) gener-ated randomly with SEQBOOT and CONSENSE in the PHYLIP software package. Only values above 90 were considered significant. Phylogenetic trees were established by using TreeView, version 1.5 (43). Only neighbor-joining trees are presented in this report. The phylogenetic trees we obtained were compared with those available forBartonellaspecies in GenBank, which were inferred from analyses of the 16S rRNA,gltA,rpoB, ITS, andgroELgene sequences.

PCR amplification and DNA sequencing of the portion of theftsZgene en-coding the C-terminal region.Primers used for amplification and sequencing of Bartonellaisolates and clinical samples are described in Table 3. PCR was carried out as described above by using primers BaftsZF and BaftsZR with 56°C as the annealing temperature. Sequencing was performed as described above by using primers BaftsZF, BaftsZR, BhftsZ 1393.n, Bh ftsZ 1247.p, Bq ftsZseqF, Bq ftsZseqR, Bb ftsZseqF, and Bb ftsZseqR. The resulting sequences from the differentBartonellaspecies were compared in order to investigate the usefulness of the C-terminal region in genotyping.

Clinical samples and DNA extraction.Eighty lymph node biopsy, lymph node aspirate, or valve samples from 79 patients with suspected CSD or endocarditis were sent to the Unite´ des Rickettsies to be tested for the presence ofBartonella spp. during December 2001. Thirty-nine samples had been found positive for Bartonellaspp. by use of ITS- andpap31-based PCR assays (47, 56). Ten to 25 mg of tissue or 200␮l of aspirate was used for extraction of total genomic DNA with the QIAamp tissue kit (Qiagen) according to the manufacturer’s instructions. Samples were handled under sterile conditions to avoid the risk of cross-con-tamination. Extracted DNA was suspended in 125␮l of elution buffer and stored at 4°C. DNAs from 10 bacterial strains and isolates were used as a negative control:Rickettsia helvetica,Escherichia coli,Mycobacterium tuberculosis, Pseudo-monas aeruginosa,Tropheryma whipplei,Afipia felis,Coxiella burnetii,Bosea mas-siliensis,Staphylococcus aureus, andEnterococcus faecalis.

For the PCR assays, samples were thawed at room temperature and amplified with primers FTS1p and FTS2p by using 56°C as the annealing temperature. Sterile distilled water was used in negative controls. A seminestedgroEL-derived assay was carried out (56) on samples for which discrepant results had been obtained in theftsZassay and the combined ITS/pap31assay, which was per-formed as previously described (47, 56).

Statistical analysis.Fisher’s exact test was used to compare the results of the combined ITS/pap31assay and theftsZassay. Observed differences were con-sidered significant when thePvalue was⬍0.05 for two-tailed tests.

RESULTS

Amplification of the portion of the

ftsZ

gene encoding the

N-terminal region for all the

Bartonella

species used in the

experiments yielded a single product of nearly 900 bp. Pairwise

comparison of these and the reported (Table 1)

ftsZ

sequences

revealed a sequence similarity ranging from 81.2 to 98.3%

(Table 4). When compared to the sequence similarities of

[image:2.587.42.285.92.307.2]

other genes of

Bartonella

species available in GenBank,

ftsZ

TABLE 1. Bacterial strains and sequences used for N-terminal

sequencing and phylogeny

Species (strain) Collection no._{or source}a

GenBank accession no. for: 16S rRNA ftsZ Bartonella alsatica(IBS382T₎ _{CIP 105477} _{AJ002139 AF467763}

Bartonella bacilliformis(KC584T₎ _{ATCC 35686} _Z11683 _AF007266

Bartonella birtlesii(IBS 325T₎ _{CIP 106294} _{AF204274 AF467762}

Bartonella clarridgeiae(Houston-2T_{) ATCC 51734} _U64691 _AF141018

Bartonella doshiae(R18T₎ _{NCTC 12862} _Z31351 _AF467754

Bartonella elizabethae(F9251T₎ _{ATCC 49927} _L01260 _AF467760

Bartonella grahamii(V2T₎ _{NCTC 12860} _Z31349 _AF467753

Bartonella henselae(Houston-1T₎ _{ATCC 49882} _M73229 _AF061746

Bartonella koehlerae(C-29T₎ _{ATCC 700693 AF076237 AF467755}

Bartonella quintana(OklahomaT₎ _CDC _M11927 _AF061747

Bartonella schoenbuchensis(R1T₎ _{NCTC 13165T AJ278187 AF467765}

Bartonella taylorii(M6T₎ _{NCTC 12861} _Z31350 _AF467756

Bartonella tribocorum(IBS 506T₎ _{CIP 104576} _{AJ003070 AF467759}

Bartonella vinsoniisubsp.arupensis

(OK 94-513T₎ ATCC 700727 AF214558 AF467758

Bartonella vinsoniisubsp.berkhoffii

(93-CO1T₎ ATCC 51672 U26258 AF467764

Bartonella vinsoniisubsp.vinsonii

(BakerT₎ ATCC VR-152 M73230 AF467757

Bartonella “weissi”(FC7049UT)b _{AF199502 AF467761}

a_{Abbreviations: CIP, Collection de l’Institut Pasteur; Paris, France; ATCC,} American Type Culture Collection, Manassas, Va.; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom; CDC, Centers for Disease Control and Prevention, Atlanta, Ga.

b_{Recently described as}_{B. bovis}_{Bermond et al.}

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sequence similarity was found to be similar to those of

gltA

(83.4 to 96.1%),

rpoB

(85.9 to 96%), and

groEL

(83.1 to 98%),

higher than that of the ITS (69.1 to 99.7%), and lower than

that of the 16S ribosomal DNA (rDNA) (97.7 to 99.8%)

(Fig. 1).

Bartonella

phylogeny derived from

ftsZ

sequences.

For each

of the 17

Bartonella

species, a sequence of 788 bp could be used

for alignment and comparison. Phylogenetic trees derived by

using parsimony and distance methods showed consistent

[image:3.587.55.538.84.648.2]

to-pologies and statistical support (Fig. 1). The

Bartonella

species

TABLE 2.

Bartonella

strains and isolates used for C-terminal amplification and sequencing

Species (strain) Clinical sourcea _{Geographic origin} _{B. henselae}_genotype

B. quintana(FullerT₎ _{Trench fever patient} _Yugoslavia

B. quintana(OklahomaT₎ _{Bacteremia, HIV-positive patient} _{United States}

B. quintana(URBQMTF 20) Blood culture France

B. quintana(URBQMTF 47) Bacteremia, homeless patient France

B. quintana(URBQMIE 48) Endocarditis patient France

B. quintana(URBQMTF 95) Bacteremia, homeless patient France

B. quintana(URBQMNHP 90) Body louse France

B. quintana(URBQMTF 108) Body louse France

B. henselaeMarseille (U8) Lymph node, CSD patient France Marseille

B. henselaeMarseille (U9) Endocarditis patient France Marseille

B. henselaeHouston-1 HIV-positive patient United States Houston

B. henselae(SA-2) Suspected CSD patient United States Houston

B. henselae(90-615) Lymph node, CSD patient United States Houston

B. henselae(CAL-1) Septicemia United States Marseille

B. henselaeEN1 Endocarditis patient France Marseille

B. henselae1129 Endocarditis patient France Houston

B. henselae5327 Endocarditis patient France Marseille

B. henselae5097 Endocarditis patient France Houston

B. henselaeC20 Cat France Houston

B. henselaeC51 Cat France Marseille

B. henselaeNZ1 Cat of patient with CSD Australia Marseille

B. henselaeNZ7 Cat of patient with CSD Australia Houston

B. bacilliformisT _{Bartonellosis patient} _Peru

B. bacilliformisCuzco 8 Bartonellosis patient Peru

B. clarridgeiae Cat United States

B. clarridgeiaeC23 Cat France

a_{HIV, human immunodeficiency virus.}

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were divided into two clades with significant bootstrap values

(99%); the first contained

B. birtlesii

,

B. schoenbuchensis

, and

B. bovis

Bermond et al. in one arm and

B. clarridgeiae

and

B. bacilliformis

in the second arm. The second clade contained

three clusters. All three

B. vinsonii

subspecies (

B. vinsonii

subsp.

vinsonii

,

B. vinsonii

subsp.

arupensis

, and

B. vinsonii

subsp.

berkhoffii

) grouped together in the first cluster (100%);

the second contained

B. henselae

,

B. koehlerae

, and

B. quintana

(99%); the third contained

B. elizabethae

,

B. tribocorum

, and

B. grahamii

(99%). The branching of

B. taylorii

,

B. alsatica

, and

B. doshiae

in the different groups was not reliable (70, 46, and

32%, respectively).

Comparison of the sequences of the

ftsZ

gene encoding the

C-terminal region for subtyping

Bartonella

species isolates.

A

fragment of nearly 885 bp of the portion of the

ftsZ

gene

encoding the C-terminal region was amplified from four

iso-lates of

B. bacilliformis

, 14 of

B. clarridgeiae

, 14 of

B. quintana

,

and 30 of

B. henselae

. The sequences of all the fragments were

compared with one another and with those available in

Gen-Bank (Table 2). The sequences of each of the 14

B. quintana

isolates were identical to one another and to that previously

described for

B. quintana

(Oklahoma) (29). The sequences of

all

B. clarridgeiae

isolates used were identical to that reported

for

B. clarridgeiae

(GenBank accession no. AF141018). While

the sequences of all four of our

B. bacilliformis

isolates were

identical (accession no. AF467752), they differed from that of

B. bacilliformis

T

_{at 5 positions: 1071, 1279, 1490, 1587, and}

1676 (numbered relative to the

ftsZ

gene of

B. bacilliformis

,

accession no. AF007266). Variation at the first and fourth of

these positions yielded silent mutations. Two previously

de-scribed genotypes (4, 16, 19, 49, 50, 55, 56) were detected

among the

B. henselae

isolates we tested (Table 2). The

Hous-ton sequence (accession no. AF161249) was found in 43.3% of

our isolates; the remainder were of the Marseille genotype

(accession no. AF161251).

Use of

ftsZ

C-terminal-derived primers for detection and

identification of

Bartonella

spp. directly from clinical samples

and comparison of their efficiency with that of the combined

ITS/

pap31

PCR assay.

All negative controls gave no PCR

products. When the 80 clinical samples which had previously

been tested with the combined ITS/

pap31

PCR assay (41, 48)

[image:4.587.49.540.84.239.2]

were assayed, C-terminal

ftsZ

amplicons were detected in 35

TABLE 3. Primers used for PCR and/or sequencing

Primera _Bartonella_species _{Primer sequence} Source or

reference

Bfp1 (ap,s)

All

5⬘-ATTAATCTGCAYCGGCCAGA-3⬘

This study

Bfp2 (ap,s)

All

5⬘-ACVGADACACGAATAACACC-3⬘

This study

Bfs3 (s)

All

5⬘-TTACAAAAATCYGTTGATAC-3⬘

This study

Bfs4 (s)

All

5⬘-GTATCAACRGATTTTTGTAA-3⬘

This study

BaftsZF (ap,s)

All

5⬘-GCTAATCGTATTCGCGAAGAA-3⬘

This study

BaftsZR (ap,s)

All

5⬘-GCTGGTATTTCCAAYTGATCT-3⬘

This study

BhftsZ 1393.n (s)

B. henselae, B. clarridgeiae

5⬘-GCGAACTACGGCTTACTTGC-3⬘

19 Bh ftsZ 1247.p (s)

B. henselae, B. clarridgeiae

5⬘-CGGTTGGAGAGCAGTTTCGTC-3⬘

19 Bq ftsZseqF (s)

B. quintana

5⬘-GCACATATTCTTGATGAGAT-3⬘

This study

Bq ftsZseqR (s)

B. quintana

5⬘-CCCCTATCATCTCATCAAG-3⬘

This study

Bb ftsZseqF (s)

B. bacilliformis

5⬘-GCGCATGTTCTTAGTGAAAT-3⬘

This study

Bb ftsZseqR (s)

B. bacilliformis

5⬘-CCTGTATACGTGATGCATTT-3⬘

This study

FTS1p (ap,s)

All

5⬘-GCCTTCTCATCCTCAACTT-3⬘

This study

FTS2p (ap,s)

All

5⬘-CAGCCTCTTCACGATGTG-3⬘

This study

a_{ap, amplification primer; s, sequencing primer.}

TABLE 4. Level of

ftsZ

DNA sequence similarity for

Bartonella

species

Taxon Similarity (%) with taxon:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1)B. alsatica

2)B. vinsoniisubsp.arupensis 92.9 3)B. bacilliformis 86.8 87.9 4)B. vinsoniisubsp.Berkhoffii 92.6 98.0 88.4

5)B. birtlesii 98.3 88.6 88.6 88.7

6)B. clarridgeiae 87.5 88.3 88.9 88.2 88.2

7)B. doshiae 90.5 91.4 86.7 91.2 88.4 86.9

8)B. elizabethae 89.5 91.6 86.9 91.2 87.2 86.8 89.6 9)B. grahamii 90.9 91.7 86.7 91.5 87.5 87.8 88.9 95.4 10)B. henselae 92.0 93.1 87.7 92.2 88.9 87.9 91.5 90.5 90.1 11)B. koehlerae 91.2 92.2 87.7 91.6 88.8 88.2 90.3 90.5 90.0 95.9 12)B. quintana 91.5 92.9 81.2 92.1 88.9 88.3 91.6 91.6 91.3 94.4 93.8 13)B. schoenbuchensis 89.3 88.7 88.6 88.6 98.0 87.7 88.3 87.2 87.4 88.7 88.8 88.8 14)B. taylorii 91.6 92.9 87.3 93.0 87.5 87.7 90.3 91.0 92.4 91.0 90.1 91.0 87.5 15)B. tribocorum 89.7 91.2 87.8 91.1 87.2 87.3 89.8 96.3 95.2 90.2 90.0 90.3 87.2 91.0 16)B. vinsoniisubsp.vinsonii 93.8 98.3 87.8 97.7 89.1 87.8 91.6 91.1 92.0 92.8 91.6 92.5 89.2 93.3 91.1 17)“B. weissii”(B. bovisBermond et al.) 89.2 88.1 88.4 87.9 94.4 86.9 86.8 86.7 87.3 88.9 88.2 88.6 94.3 87.5 87.7 88.6

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samples (43.75%) from 34 patients. The overall correlation

between the C-terminal

ftsZ

assay results and those of the

ITS-and

pap31

-derived assay was 89.7%, but this was not significant

(

P

⫽

0.052). Four samples were negative by the

ftsZ

assay but

positive with the ITS-

pap31

assay; three of these samples were

also positive in the seminested

groEL

-derived assay (56).

DISCUSSION

In the past decade, a number of new

Bartonella

species have

been described (6, 15, 27) and comparisons of 16S rDNA

sequences have led to many taxonomic changes in the genus

Bartonella

(7, 11). Although comparison of 16S rDNA gene

sequences has been useful in phylogenetic studies at the genus

level (41), its use has been questioned in studies at the species

level (21; M. Hasegawa and T. Hashimoto, Letter, Nature

361:

23, 1993). Other genes have been used empirically in

at-tempts to classify the

Bartonella

species: the

gltA

gene (9), the

rpoB

gene (45), the 16S–23S rRNA ITS (26), and the

groEL

(55) gene. The FtsZ protein plays an important role in

bacte-rial cell division, and recently its sequence was established for

four

Bartonella

species (29). In our study we sequenced the

900-base sequence encoding the N-terminal region (partial) of

the

ftsZ

gene for all recognized

Bartonella

species. The

se-quences were generally well conserved (81.2 to 98.3% [Table

4]) between species, but the sequence divergence present

al-lowed us to develop a phylogenetic tree (Fig. 1) which was well

supported for most of the strains studied. We compared this

tree with those inferred from sequences of the 16S rDNA,

gltA

,

rpoB

, ITS, and

groEL

genes of

Bartonella

species available in

GenBank (Fig. 1). With the

ftsZ

sequences, the

Bartonella

species were divided into two well supported clades which were

also obtained with the

groEL

and

rpoB

sequences. Within these

clades, various supported clusters could be found with the

different DNA sequences. The statistically supported cluster

formed by the subspecies of

B. vinsonii

in the

ftsZ

tree was also

found in the

rpoB

-, ITS-, and

groEL

-derived trees. A cluster

containing

B. henselae

and

B. koehlerae

was found in the

ITS-derived tree, while a cluster containing

B. henselae

and

B. quintana

was obtained in the

rpoB

- and

groEL

-derived trees. A

cluster including

B. tribocorum

,

B. elizabethae

, and

B. grahamii

was present in the phylogenetic trees established by using the

gltA

,

rpoB

, ITS, and

groEL

sequences.

B. taylorii

was included

in this cluster in the ITS- and

groEL

-derived trees. A cluster

formed by

B. bovis

Bermond et al. and

B. birtlesii

was found in

the

groEL

-derived tree, and a cluster of

B. bovis

Bermond et al.

and

B. schoenbuchensis

was found in the 16S rDNA-inferred

tree. The similarities we found between the phylogenetic trees

derived with the

ftsZ

gene sequences and those derived with

other genes shows that

fstZ

gene sequencing should be

[image:5.587.57.532.75.342.2]

consid-ered a useful tool to be included in phylogeny studies.

We believe that it is important to consider the sequences of

several genes in phylogeny studies. Although each

gene-de-rived tree will differ from the others and will have different

FIG. 1. Comparison of neighbor-joining trees based on 16S rDNA,

gltA

,

rpoB

, ITS,

groEL

, and

ftsZ

partial or complete sequences. Bootstrap

values at tree nodes are based on 100 replicates; values of

⬎90 are boldfaced. Trees were unrooted, and only topology was shown for these trees.

a

_,

_gltA

_{, citrate synthase;}

_rpoB

_{, beta subunit of RNA polymerase; ITS, 16S–23S rRNA ITS;}

_groEL

_{, heat shock protein;}

_ftsZ

_{, cell division protein.}

b

_{, range of the level of DNA sequence similarity for each gene used. Designations for species and subspecies consist of the letter}

_B

_(for

_Bartonella

₎

and the following abbreviations:

ber

,

vinsonii

subsp.

berkhoffii

;

vin

,

vinsonii

subsp.

vinsonii

;

aru

,

vinsonii

subsp.

arupensis

;

tri

,

tribocorum

;

eli

,

elizabethae

;

gra

,

grahamii

;

tay

,

taylorii

;

als

,

alsatica

;

dos

,

doshiae

;

hen

,

henselae

;

qui

,

quintana

;

koe

,

koehlerae

;

cla

,

clarridgeiae

;

bir

,

birtlesii

;

sho

,

schoenbuchensis

;

bac

,

bacilliformis

;

bov

,

bovis

Bermond et al.

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levels of statistical support, it has been found that groupings

obtained with two different sequences at bootstrapping values

over 90% are stable and reliable (48). In previous phylogenetic

studies

B. bacilliformis

was chosen to be the outgroup, but

because new

Bartonella

species have been described recently

(6, 15) we chose to draw an unrooted tree.

Because

Bartonella

species are implicated in an increasing

variety of human diseases, the development of species-specific

tools for their detection and identification in clinical samples is

becoming more crucial, especially in light of the difficulties in

culturing these bacteria (34). In 1996, Drancourt et al. (16)

reported two serotypes of

B. henselae

(Houston and Marseille),

and later Bergmans et al. (4) confirmed by 16S rDNA gene

sequence analysis that there were two genotypes of

B. henselae

,

genotypes I and II, corresponding to the Houston and

Mar-seille serotypes, respectively. More recently, further studies

have confirmed the presence of these two subspecies (3, 4, 19,

33, 49, 50, 56). Many genes have been used to characterize

Bartonella

isolates (2, 8, 9, 27, 36, 37, 55), and the cell division

protein (FtsZ) has also been used for detection (29) and

sub-typing of

Bartonella

species (19). In our study we amplified and

sequenced the

ftsZ

sequence corresponding to the C-terminal

region for 4

B. bacilliformis

isolates, 14

B. clarridgeiae

isolates,

14 B. quintana

isolates, and 30

B. henselae

isolates from

differ-ent geographic regions, hosts, and clinical samples. The

se-quences of the

B. clarridgeiae

and

B. quintana

isolates were

identical to those of the type strains. Similarly, sequencing of

the

groEL

gene could not be used to differentiate

B. quintana

isolates (55). When the ITS sequence was used for subtyping,

however,

B. quintana

isolates were found to belong to three

genotypes and different sequences were found for all the

B. clarridgeiae

isolates studied (26). This difference may be

ex-plained by the high degree of variability of ITS sequences. The

ftsZ

sequence data may show the homogeneity of the

B. clar-ridgeiae

and

B. quintana

isolates. The sequences of the four

B. bacilliformis

isolates we studied were identical to one another

but different from that of

B. bacilliformis

T

_{at 5 positions, only}

3 of which yielded significant amino acid substitutions. Among

the 30

B. henselae

isolates we studied, only Houston and

Mar-seille genotypes were found and there was no evidence of

genotype III, detected by Ehrenborg et al. (19).

We also tested whether the C-terminal

ftsZ

assay could

de-tect the DNAs of

Bartonella

species in clinical samples and

compared its sensitivity with that of a combined ITS-

pap31

assay. We believe that false-positive PCR results due to

con-tamination problems may be prevented by using a number of

primer pairs which target different genes. Addition of the

ftsZ

gene to the panel of genes available for diagnosis of infections

by PCR may be useful, and it may be a good tool for the

“suicide” PCR application (44).

Conclusion.

We confirmed that using one pair of primers

enables the comparison of partial

ftsZ

sequences for all

Bar-tonella

species and that this is a useful tool for detection and

identification, which should facilitate routine work on clinical

samples. The sequences obtained were also useful in

phyloge-netic analyses at the species level, and the results obtained

correlated closely with those obtained in previous studies using

other markers. Furthermore, we showed that

Bartonella

spe-cies occur in two clades and that

B. bacilliformis

belongs to a

robust and well-defined clade. Using multiple DNA sequences

seems to be the most suitable way to reliably infer phylogeny.

We also showed the usefulness of

ftsZ

C-terminal region

se-quencing in the direct detection and identification of

Bar-tonella

species in clinical samples and for subtyping

B. henselae

and

B. bacilliformis

isolates. Its usefulness for epidemiological

studies should be further investigated by using a diverse range

of clinical samples.

ACKNOWLEDGMENTS

We thank Yves Piemont for providing the

B. schoenbuchensis

strain,

Jennifer Robson for providing

B. henselae

isolates from Australian

cats, and Pat Kelly for reviewing the manuscript.

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