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Journal of Clinical Microbiology, December 1999, p. 4045-4047, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Bartonella Species Directly in
Clinical Specimens by PCR-Restriction Fragment Length
Polymorphism Analysis of a 16S rRNA Gene Fragment
Ghassan M.
Matar,1,*
Jane E.
Koehler,2
Georgia
Malcolm,3
Mary Ann
Lambert-Fair,3
Jordan
Tappero,2,3
Suzan B.
Hunter,3 and
Bala
Swaminathan3
Department of Microbiology and Immunology,
American University of Beirut, Beirut, Lebanon1;
Department of Medicine, Division of Infectious Diseases,
University of California at San Francisco, San Francisco,
California2; and Division of Bacterial
Diseases, National Center for Infectious Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia3
Received 18 June 1999/Returned for modification 3 August
1999/Accepted 24 August 1999
 |
ABSTRACT |
It is now established that two species of Bartonella,
namely, Bartonella henselae and B. quintana,
cause bacillary angiomatosis in human immunodeficiency virus-infected
patients. In addition, B. henselae causes cat scratch
disease and B. quintana, B. henselae, and
B. elizabethae can cause bacteremia and endocarditis in
immunocompetent persons. We have developed a PCR-restriction fragment
length polymorphism-based assay for direct detection and identification
to species level of Bartonella in clinical specimens. This
is accomplished by PCR amplification of Bartonella DNA
using primers derived from conserved regions of the gene carrying the
16S ribosomal DNA, followed by restriction analysis using
DdeI and MseI restriction endonucleases. We
amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from
digestion of B. henselae and B. quintana
amplicons with DdeI, whereas a different unique pattern was
seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical
patterns while B. quintana and B. elizabethae
gave a different pattern. By combining the restriction analysis data
generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the
Bartonella species associated with each tissue specimen.
| |
INTRODUCTION |
Twelve species of
Bartonella have been identified. Four, Bartonella
henselae, B. quintana, B. elizabethae, and
B. bacilliformis, are recognized as human pathogens, and
one, B. clarridgeiae, has been implicated as a human
pathogen by serology (8, 13, 15). B. bacilliformis, the first organism in the genus to be identified, causes bartonellosis (3). Recently, B. henselae
was detected by PCR (1) and isolated from the lymph nodes of
patients with cat scratch disease (CSD) (6), as well as from
the blood of pet cats (11, 12), providing strong evidence
for B. henselae as the causative agent of CSD. Both B. henselae and B. quintana cause bacteremia and bacillary
angiomatosis in both immunocompromised and immunocompetent persons
(10, 11, 18). B. quintana is also the etiologic
agent of trench fever (20, 21), and both B. quintana and B. elizabethae can cause endocarditis
(4, 7, 16, 20).
The implication of these Bartonella species in a variety of
human diseases and the difficulty in isolating them from clinical specimens underscore an urgent need for better detection and
identification methods. Birtles identified Bartonella
species by PCR-restriction fragment length polymorphism (RFLP) analysis
(2). We describe the development of a PCR-RFLP assay for the
identification of Bartonella to the species level and
demonstrate the application of this method directly to clinical
diagnostic specimens.
| |
MATERIALS AND METHODS |
Bartonella DNA fragments were amplified from 25 tissue specimens, including fresh or paraffin-embedded clinical
specimens (lymph node or lymph node aspirates, skin, subcutaneous
nodules, or other tissues), obtained from patients participating in
epidemiologic studies of CSD and bacillary angiomatosis
(19). Amplifications of control tissues were also run in
parallel. Control isolates were B. henselae B91-002000,
B. vinsonii B92-010225, B. elizabethae B92-002005, and B. quintana ATCC VR-358.
Bartonella species were grown on Trypticase soy agar with
5% sheep blood or on chocolate agar plates for 4 to 7 days at 37°C. A few colonies (8 to 10) were harvested in 1 ml of 0.1 M
phosphate-buffered saline, pH 7.0. Template DNA was prepared from
suspended cells or from fresh or paraffin-embedded clinical specimens
by the method described by Heller et al. (9). Positive and
negative controls were processed for DNA preparation and PCR
amplification along with the clinical specimens. The negative control
was a section of fresh or paraffin-embedded skin free of
Bartonella infection. The positive control was either
Bartonella organisms or paraffin slices that had been
artificially contaminated with Bartonella organisms.
Ten microliters of bacterial or tissue lysates was used to amplify the
16S ribosomal DNA (rDNA) fragment by the method of Relman et al.
(14). PCR amplification was carried out in 100-µl reaction
mixtures consisting of 10 µl of DNA and 90 µl of the amplification
mix, which contained the following components: 20 pmol each of p12B and
p24E primers, 0.5 mM MgCl2, 200 µM each deoxynucleoside
triphosphate, 10 µl of Gene Amp PCR buffer (Perkin-Elmer, Norwalk,
Conn.), and 2.5 U of Taq DNA polymerase (Perkin-Elmer).
The PCR amplification was performed in a PC-100 Thermal controller (MJ
Research, Watertown, Mass.) for 35 cycles. Each cycle consisted of
120 s at 94°C, 60 s at 60°C, and 90 s at 72°C, and a final extension of 10 min at 72°C was done. The amplified products were detected by electrophoresis on a 1% agarose gel (14 by 14 cm) in
1× Tris-borate-EDTA buffer at 100 V for 60 min. Gels were stained with
ethidium bromide and photographed. PCR controls included a known
positive DNA extract and a reagent blank.
For digestion of PCR-amplified DNA from cultures and clinical
specimens, 10 to 15 µl of PCR-amplified DNA was restricted with 10 U
of DdeI and MseI in a total volume of 15 to 20 µl, respectively, according to the manufacturer's specifications
(New England Biolabs, Inc., Beverly, Mass.). The restriction fragments
were separated by electrophoresis on agarose gels (4% NuSieve agarose
[3:1] [FMC Bioproducts, Rockland, Maine] at 60 V for 2 h. Gels
were photographed and fragment sizes were determined with interpolation
by using the BioImage system whole-band software analysis (Millipore
Corp., Ann Arbor, Mich.).
For clinical specimens with PCR-amplified DNA revealing more than one
band, the 296-bp DNA in the agarose gel was detected with ethidium
bromide, excised, and placed in a 1.5-ml microcentrifuge tube. The
agarose slice was washed once with 500 µl of Tris-EDTA buffer and
twice with enzyme buffer. The buffer was discarded, and the agarose
plug was cut into small pieces. Ten units of the restriction enzymes
(DdeI and MseI) was added in a 100-µl solution (10× buffer [10 µl], bovine serum albumin [2.5 µl], and
deionized water to a volume of 100 µl). DNA was precipitated from the
supernatant with 3 M sodium acetate and 2.5 volumes of 95% cold
ethanol. The pellet was washed once in cold 70% ethanol, dried in a
vacuum dessicator, and dissolved in 20 µl of water. Fragments were
detected by electrophoresis on 4% NuSieve agarose gels and were
analyzed as described previously.
| |
RESULTS |
Figure 1 shows the 296-bp fragment
as well as the various RFLP patterns derived from digestion of
amplicons of the four species with DdeI and MseI,
and Table 1 lists the observed and
predicted sizes of the fragments from the digested amplicons. B. henselae and B. quintana gave nearly identical patterns
with DdeI, whereas B. vinsonii and B. elizabethae gave a different and unique pattern. With
MseI, B. henselae and B. vinsonii had
nearly identical patterns, and B. quintana and B. elizabethae had patterns different from those of B. henselae and B. vinsonii but nearly identical to each other. Composite types generated from restriction analysis using DdeI and MseI were unique for each species (Table
1).
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FIG. 1.
PCR-RFLP of B. henselae, B. vinsonii, B. elizabethae, and B. quintana.
Lane 1, 50-bp ladder; lanes 2 to 4, B. henselae B91-002000;
lanes 5 to 7, B. vinsonii B92-010225; lanes 8 to 10, B. elizabethae B92-002005; lanes 11 to 13, B. quintana ATCC VR-358; lane 14, 50-bp ladder. PCR products were
uncut (lanes 2, 5, 8, and 11) or cut with DdeI (lanes 3, 6, 9, and 12) or MseI (lanes 4, 7, 10, and 13).
|
|
Table 2 shows the distribution of
Bartonella species among 25 clinical specimens. Twenty-one
of 26 (81%) of the isolates gave the restriction pattern for B. henselae, 4 (15.38%) gave that for B. quintana, and 1 (4%) gave that for B. elizabethae. The RFLP patterns of
B. henselae and B. quintana obtained from clinical specimens are shown in Fig. 2.
All 21 B. henselae amplified PCR products have the same
composite restriction pattern with DdeI and MseI.
This pattern is identical to that found for the B. henselae
type strain (B91-002000). All four B. quintana amplified PCR
products have the same composite restriction pattern, which is
identical to that of the B. quintana type strain (ATCC
VR-358). It should be noted that minor differences in fragment lengths among isolates within the same species may be encountered
(5). The sizes of digested DNA fragments are shown in Table
1.
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|
FIG. 2.
PCR-RFLP of B. quintana and B. henselae from clinical specimens. Lane 1, 50-bp ladder, lanes 2 to
4, B. quintana H93-176; lanes 5 to 7, B. henselae
B92-007003; lane 8, blank; lanes 9 to 11, B. quintana
B93-007356; lane 12, 50-bp ladder. PCR products were left uncut (lanes
2, 5, and 9) or were cut with DdeI (lanes 3, 6, and 10) or
MseI (lanes 4, 7, and 11).
|
|
| |
DISCUSSION |
We were able to differentiate among the different
Bartonella species implicated in human disease by extending
the 16S rDNA-based detection method for Bartonella species
by using restriction analysis of the 296-bp amplicons. This was
achieved by generating species-specific composite patterns based on
digestion of 16S rDNA amplicons with DdeI and
MseI restriction enzymes. The RFLP method was applied to
amplicons from clinical specimens, allowing direct species identification for these specimens. The predicted and observed fragment
lengths for all four species matched each other. Predicted fragments 28 for B. vinsonii and B. elizabethae cut with
DdeI were not observed because they were small and were not
visualized on gels.
Our method was shown to be more advantageous than a previously
described method (1) because only one set of primers was used in this study, followed by restriction analysis, allowing for
direct identification in a variety of clinical specimens of the
Bartonella species implicated in human disease (Table 2). Although the method described by Anderson et al. (1) allows for the detection of B. quintana and B. henselae
in lymph nodes and lymph node aspirates, it requires multiple steps and
the use of radioactive probes.
Our PCR-RFLP-based assay may offer the advantage of early diagnosis of
suspected Bartonella species infections and can be used to
differentiate among three species causing human disease in North
America. This rapid and specific test is an alternative to culture that
provides more-timely information to clinicians, who can then direct
antibiotic therapy and suggest prevention strategies to their patients
based on species-specific test results (11).
| |
ACKNOWLEDGMENTS |
This work was initiated in 1993 while G.M.M. was a Visiting
Associate at the Centers for Disease Control and Prevention (CDC).
This study is supported in part by NIH R29 AI36075 and the California
Universitywide AIDS Research Program (to J.E.K.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, American University of Beirut, 850 Third Ave., New York, NY 10022. Phone: 961-1-340460, ext. 5128. Fax: (212)
583-7650. E-mail: gmatar{at}aub.edu.lb.
| |
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Journal of Clinical Microbiology, December 1999, p. 4045-4047, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
