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Journal of Bacteriology, August 1999, p. 4469-4475, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genomic Relatedness of Chlamydia
Isolates Determined by Amplified Fragment Length Polymorphism
Analysis
Adam
Meijer,1,*
Servaas A.
Morré,2
Adriaan J. C.
Van Den Brule,2
Paul H. M.
Savelkoul,3 and
Jacobus M.
Ossewaarde1
Research Laboratory for Infectious Diseases, National
Institute of Public Health and the Environment, 3720 BA
Bilthoven,1 and Section of Molecular
Pathology, Department of Pathology,2 and
Department of Clinical Microbiology and Infection
Control,3 University Hospital Vrije
Universiteit, 1081 HV Amsterdam, The Netherlands
Received 21 December 1998/Accepted 22 April 1999
 |
ABSTRACT |
The genomic relatedness of 19 Chlamydia pneumoniae
isolates (17 from respiratory origin and 2 from atherosclerotic
origin), 21 Chlamydia trachomatis isolates (all serovars
from the human biovar, an isolate from the mouse biovar, and a porcine
isolate), 6 Chlamydia psittaci isolates (5 avian isolates
and 1 feline isolate), and 1 Chlamydia pecorum isolate was
studied by analyzing genomic amplified fragment length polymorphism
(AFLP) fingerprints. The AFLP procedure was adapted from a previously
developed method for characterization of clinical C. trachomatis isolates. The fingerprints of all C. pneumoniae isolates were nearly identical, clustering together at
a Dice similarity of 92.6% (± 1.6% standard deviation). The
fingerprints of the C. trachomatis isolates of human,
mouse, and swine origin were clearly distinct from each other. The
fingerprints of the isolates from the human biovar could be divided
into at least 12 different types when the presence or absence of
specific bands was taken into account. The C. psittaci fingerprints could be divided into a parakeet, a pigeon, and a feline
type. The fingerprint of C. pecorum was clearly distinct from all others. Cluster analysis of selected isolates from all species
revealed groups other than those based on sequence data from single
genes (in particular, omp1 and rRNA genes) but was in
agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species
provided suggestions for a grouping of chlamydiae based on the analysis
of the whole genome. Furthermore, genomic AFLP analysis showed that the
genome of C. pneumoniae is highly conserved and that no
differences exist between isolates of respiratory and atherosclerotic origins.
| |
INTRODUCTION |
Chlamydiae are obligate
intracellularly growing bacteria. They are widespread throughout the
world and infect both humans and animals. Currently, four species,
Chlamydia pneumoniae, Chlamydia trachomatis,
Chlamydia psittaci, and Chlamydia pecorum,
belonging to the genus Chlamydia of the family
Chlamydiaceae within the order Chlamydiales, are
recognized (10, 11, 35). C. pneumoniae and
C. trachomatis are primarily human pathogens. C. pneumoniae has been recognized as a major cause of respiratory
infections. In addition, C. pneumoniae infection has been
associated with new-onset asthma, exacerbation of chronic asthma,
atherosclerotic disease, and, recently, Alzheimer's dementia (2,
22). C. trachomatis is a major cause of sexually
transmitted diseases and trachoma in humans (18). C. psittaci and C. pecorum are primarily animal pathogens,
but C. psittaci may cause zoonotic infections
(44).
Restriction fragment length polymorphism (RFLP) analysis of
PCR-amplified genes has been used to characterize chlamydial isolates. Using PCR-RFLP analysis of different genes, Chlamydia could
be differentiated at the species level, and C. trachomatis
and C. psittaci could be differentiated at a strain level
corresponding to serovars and types (4, 12-14, 24, 29, 31,
53). However, C. pneumoniae isolates originating from
all over the world could not be differentiated by this technique
(4, 13, 29). Furthermore, the available sequence data for
C. pneumoniae shows complete or nearly complete conservation
for omp1, omp2, 16S rRNA, domain I of the 23S
rRNA, RNase P RNA, the genes for the 53-kDa protein and the 76-kDa
protein, dnaK, and waaA (kdtA) and the
16S-23S ribosomal DNA intergenic spacer (7, 13, 15, 17, 21, 26,
30, 39, 40, 55).
By analysis of the whole genome using RFLP (1, 5, 9, 27, 28, 37,
38, 42), random amplification of polymorphic DNA (RAPD) (41,
45), or hybridization (5, 6, 9), the four species
could be differentiated, and subgroups could be recognized within the
C. trachomatis and C. psittaci species. These
findings are in agreement with the power of discrimination of RFLP and
RAPD at the species-to-strain level and of DNA-DNA hybridization at the
genus-to-subspecies level (52). RFLP analysis of the genome
of C. pneumoniae showed only two nearly identical patterns.
One extra band was observed in two of eight C. pneumoniae isolates (5). However, DNA-DNA hybridization experiments
showed 94 to 96% relatedness among C. pneumoniae isolates,
suggesting at least some genomic variation (6).
Recently, a novel high-resolution technique has been introduced for
whole-genome analysis: amplified fragment length polymorphism (AFLP)
(54). This technique requires relatively low amounts of
genomic DNA. The DNA is digested by a combination of a restriction enzyme that has a high number of restriction sites in DNA and a
restriction enzyme that has an average number of restriction sites in
DNA. Selected sets of restriction fragments are amplified and analyzed
on gels. This technique has proven its usefulness as a tool in
bacterial taxonomy and epidemiology (16, 20, 43) and has
also been applied in C. trachomatis research
(32).
Here, we report on the application of AFLP to analyze the differences
among Chlamydia species and, within the species, among subgroups.
| |
MATERIALS AND METHODS |
Chlamydia isolates.
The reference and laboratory
isolates studied are summarized in Table
1. The species, serovar, and biovar
information was obtained previously by established procedures (3,
10, 22).
Isolation and propagation of Chlamydia in cell
culture.
HeLa 229 (ATCC CCL 2.1) (for propagation of C. psittaci and C. trachomatis) and HEp2 (ATCC CCL 23)
(for propagation of C. pneumoniae and C. pecorum)
cell lines were maintained in Iscove's modified Dulbecco medium
(Gibco) supplemented with 10% fetal calf serum and antibiotics.
Isolation of chlamydiae from clinical samples or mouse lung homogenates
and propagation of Chlamydia isolates were carried out as
described previously (29). All isolates were tested for the
presence of Mycoplasma contamination by using a
Mycoplasma group-specific PCR (34). When
positive, the chlamydial isolates were decontaminated by Triton X-100
treatment as previously described (34) or by passage in mice
via intranasal infection followed by reisolation from the lungs 3 days postinfection.
Preparation of genomic DNA.
Elementary bodies (EBs) were
harvested by sonicating cell monolayers from four shell vials with
>75% infected cells in 1 ml of 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA. Next, 114 µl of the EB preparation was digested with 120 µg of DNase I (Boehringer Mannheim, Mannheim, Germany) in 10 mM
MgCl2 for 5 h at 37°C. After inactivation of the
DNase for 20 min at 70°C, RNA was digested with RNase A (Sigma, St.
Louis, Mo.) at a concentration of 0.1 mg/ml. Chlamydial DNA was
purified from the EBs with the High Pure PCR template preparation kit
(Boehringer Mannheim) according to the instructions of the
manufacturer. The DNA was eluted in 200 µl of 10 mM Tris-HCl, pH 8.5, and tested for the absence of human DNA by a 
-globin PCR assay as
described previously (23).
AFLP analysis.
Chlamydial DNA was digested with the
restriction enzymes EcoRI (Pharmacia LKB Biotechnology,
Uppsala, Sweden) and MseI (New England Biolabs [NEB] Inc.,
Beverly, Mass.). Simultaneously, generally applicable double-stranded
oligonucleotide adaptors, composed of a unique sequence and an overhang
complementary to the restriction sites in the genomic digest as
described previously (20, 32), were ligated to the
restriction fragments for 3 h at 37°C. This mixture consisted of
4 µl of chlamydial DNA, 1 U of EcoRI, 1 U of
MseI, 2 pmol of EcoRI adaptor, 2 pmol of
MseI adaptor, 1 µl of ligase buffer (NEB) (50 mM Tris-HCl
[pH 7.5] containing 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, and 25 µg of bovine serum albumin/ml), 0.5 M NaCl, 50 µg of
bovine serum albumin (NEB)/ml, 0.6 U of T4 DNA-ligase (NEB), and
H2O up to 10 µl. After restriction and ligation, the DNA
was diluted with H2O to a final volume of 100 µl and
stored at 
20°C until it was further analyzed. The ligation
products, now having unique sequences from the adaptors at both sites,
were amplified by PCR with adaptor-specific primers. The PCRs were
performed and optimized as described previously (20, 32).
The primers had, in addition to the adaptor-specific sequence, no
(Eco-0 and Mse-0) or one (Eco-A and Mse-C) additional selective
nucleotide at the 3'-terminal end of the primer. This selective
nucleotide allowed the amplification of only a subset of restriction
fragments when the banding pattern, obtained after PCR with primers
without a selective nucleotide, was too complex. The Eco-0 primer or
the Eco-A primer was fluorescently labeled with Texas red (Isogen
Bioscience BV, Maarssen, The Netherlands). The PCR products were
separated in a denaturing polyacrylamide sequencing gel with a Vistra
725 automated sequencer (Amersham Life Science, Cleveland, Ohio).
Fluoroimages of the banding pattern saved by the sequencer in a
computer TIFF file were analyzed with GelCompar version 4.0 software
(Applied Maths, Kortrijk, Belgium). The fluoroimages were normalized by
alignment of the AFLP patterns by using molecular size markers included
at regular intervals in each gel, and background fluorescence was
subtracted with mathematical algorithms included in the GelCompar
software. Fluorescent amplification fragments between 100 and 500 bp
were included in the cluster analysis. Levels of correlation between
fingerprints were calculated with the curve-based Pearson product
moment correlation coefficient (46) and the band-based Dice
similarity coefficient (SD), which is equal to
the ratio of twice the number of bands common for fingerprints A and B
and the total number of bands in fingerprints A and B (46).
To calculate SD, bands were assigned to the
fluoroimages automatically by the GelCompar software, using a minimal
elevation of 5% of a band relative to the surrounding area and a
minimal area of 0.5% of a band relative to the total area of the
banding pattern, and to match bands in two compared fingerprints, a
position tolerance of 0.8% relative to the total length of the pattern was allowed. Cluster analysis was carried out by the unweighted pair
group method using arithmetic averages (UPGMA) algorithm (46) included in the GelCompar software.
DNA-DNA hybridization data.
DNA-DNA hybridization data from
previous studies (6, 9, 10) were used to infer a phylogram
by the UPGMA method of the PHYLIP program package, version 3.5c
(8). The final phylogram was visualized with the TreeView
program, version 1.30 (36).
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RESULTS |
All chlamydial isolates were free of Mycoplasma
contamination as determined by PCR, or, if positive, they were
decontaminated by Triton X-100 treatment or mouse passage prior to AFLP
analysis. All chlamydial DNA preparations were free of human DNA as
assessed by the -globin PCR assay. This ensures that the
fingerprints obtained by AFLP analysis are specific for chlamydia.
Using a selection of C. trachomatis and C. pneumoniae isolates, the most discriminatory primer combination in
the AFLP reaction was determined. The primer pair Eco-0-Mse-C was
selected, since AFLP fingerprints of different C. trachomatis serovars showed different banding patterns
(32). With a selection of C. pneumoniae isolates,
no variation was observed in the AFLP fingerprints with either of the
primer combinations. However, since the primer combination Eco-0-Mse-C
showed a discrete banding pattern and a sufficient number of bands to
be informative, and since different AFLP fingerprints were observed for
closely related C. trachomatis isolates (32), this primer pair was used in all subsequent experiments.
The results of cluster analyses with matrices of Pearson product moment
correlation coefficients and of Dice similarity coefficients were
identical regarding the grouping of chlamydiae, as described below.
AFLP fingerprints of selected isolates with or without Triton X-100
treatment or mouse passage (C. trachomatis isolate R19 and
C. pneumoniae isolates CWL-029, CWL-050, PS-32, and 2023) were identical regarding the number and location of bands, clustering at an SD of 
96.8%. Only minor variation was
observed in the intensities of some bands.
The AFLP fingerprints of all 19 C. pneumoniae isolates were
nearly identical, clustering at an SD of 92.6%
(±1.6% standard deviation [SD]) (Fig.
1). Only minor variation in the
intensities of some bands could be noted, while the number and location
of all bands were identical.
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FIG. 1.
Digitized AFLP fingerprints and phylogram of 19 C. pneumoniae isolates. The phylogram was inferred by the UPGMA
method with a band-based Dice similarity coefficient
(SD) matrix. SD values
(in percentages) and molecular sizes (Mw) (in base pairs) are shown
above the phylogram and fingerprints, respectively. The names of the
isolates as presented in Table 1 are shown on the right.
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|
The AFLP fingerprints of C. trachomatis showed considerable
heterogeneity (Fig. 2). The mouse and
swine isolates were different from the human isolates. The clusters
separated from each other at an SD of 64.9%
(±2.4% SD) (Fig. 2). Also, the mouse and swine isolates were
different from each other (SD = 73.5%) (Fig.
2). The human isolates clustered at an SD of
88.3% (±2.5% SD) (Fig. 2). Within the human cluster, at least 12 different fingerprint types could be observed, based on the number and
locations of specific bands.
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FIG. 2.
Digitized AFLP fingerprints and phylogram of 21 C. trachomatis isolates. The phylogram was inferred by the UPGMA
method with a band-based Dice similarity coefficient
(SD) matrix. SD values
(in percentages) and molecular sizes (Mw) (in base pairs) are shown
above the phylogram and fingerprints, respectively. The names of the
isolates as presented in Table 1 are shown on the right.
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|
A cluster analysis with representatives of all species and some
subgroups is shown in Fig. 3 for AFLP
fingerprints and in Fig. 4 for DNA-DNA
hybridization relatedness. The matrices of corresponding
SD values and previously published DNA-DNA
hybridization relatedness percentages are summarized in Tables
2 and 3,
respectively. Only one C. psittaci parakeet isolate was
included in the cluster analysis of AFLP fingerprints, since C. psittaci isolates 6BC, ORNI, P635, and P650 were identical in the
number, locations, and intensities of bands, clustering at an
SD of 95.4% (±1.2% SD) (data not shown).
Within the species C. psittaci, three different AFLP
fingerprints, a parakeet, a pigeon, and a feline type, could be
observed, corresponding to the hosts from which the isolates originated. Regarding the human C. trachomatis isolates as
one operational taxonomic unit (OTU), eight different OTUs could
be recognized in the phylogram of AFLP fingerprint types
(C. pneumoniae, human C. trachomatis,
mouse C. trachomatis, swine C. trachomatis, feline C. psittaci, parakeet C. psittaci,
pigeon C. psittaci, and C. pecorum) (Fig. 3).
Using DNA-DNA hybridization data to infer a phylogram, six different
OTUs could be recognized (C. pneumoniae, human C. trachomatis, mouse C. trachomatis, feline C. psittaci, parakeet and pigeon C. psittaci, and
C. pecorum) (Fig. 4). These OTUs were identical to those of
the corresponding AFLP fingerprint types, except for the C. psittaci isolates of parakeet and pigeon origin. These were
regarded as one OTU based on their high relatedness (93.4%).
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FIG. 3.
Digitized AFLP fingerprints and phylogram of
representative isolates of all four Chlamydia species. The
phylogram was inferred by the UPGMA method with a band-based Dice
similarity coefficient (SD) matrix.
SD values (in percentages) and molecular sizes
(Mw) (in base pairs) are shown above the phylogram and fingerprints,
respectively. The names of the isolates as presented in Table 1 are
shown on the right, with prefixes indicating the hosts (Bo, bovine; Fe,
feline; Hu, human; Mu, murine; Pgn, pigeon; Prk, parakeet; Sw, swine)
and species codes (Cpe, C. pecorum; Cpn, C. pneumoniae; Cps, C. psittaci; Ctr, C. trachomatis).
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FIG. 4.
Phylogram inferred by the UPGMA method with the DNA-DNA
hybridization data matrix in Table 3. The relatedness between C. pneumoniae and C. trachomatis (murine) is missing and
has been estimated as minimal ( 5%).
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| |
DISCUSSION |
In this study, we demonstrated the use of AFLP for the analysis of
chlamydial genomic DNA. The C. pneumoniae isolates,
including respiratory and atherosclerotic isolates, showed identical
AFLP fingerprints, while those of human C. trachomatis
isolates could be divided into several groups. The AFLP fingerprints of
C. trachomatis mouse and swine isolates were different from
each other and from those of the human C. trachomatis
isolates. AFLP fingerprints of C. psittaci isolates could be
differentiated into three types, a parakeet, a pigeon, and a feline
type. The AFLP fingerprint of the C. pecorum isolate was
clearly distinct from those of the other Chlamydia species.
Some Chlamydia species or subspecies harbor plasmids of
approximately 7.5 kbp that may interfere with the banding pattern of
AFLP. Analysis of plasmid sequences derived from GenBank revealed that,
theoretically, one or two bands in the fingerprint could originate from
plasmids. Since plasmid sequences are highly conserved within human
C. trachomatis and subgroups of C. psittaci
(25, 49), the interference of plasmid-derived fragments in
the cluster analysis was probably negligible.
The genome of C. pneumoniae is highly conserved, since the
AFLP fingerprints of world-wide-derived isolates were almost identical, in agreement with previously reported genomic RFLP analysis results (5). However, considering the DNA-DNA hybridization
relatedness of 94 to 96% among C. pneumoniae isolates
(6), some differences might be expected. Other restriction
enzyme combinations might improve the differentiation of C. pneumoniae isolates by AFLP analysis. Nevertheless, our results
showed that isolates from atherosclerotic lesions are identical to
those from the respiratory tract. These findings are in agreement with
the reported sequences and Southern hybridization analysis data of
these isolates (15, 30).
Three main AFLP fingerprint groups could be recognized within the
species C. trachomatis, related to the host: human, mouse, and swine. This grouping is in agreement with data from other studies
using different approaches (7, 9, 19, 40, 41). Among the
human C. trachomatis isolates, at least 12 different AFLP
fingerprint types could be recognized, despite a high relatedness in
DNA-DNA hybridization of 92 to 100% (6, 9, 19). This observation is in agreement with results obtained with RAPD and RFLP
analyses of genomic DNA (37, 38, 42, 45). However, subgroups
of AFLP fingerprint types did not correlate with serogroups (33), biovar groups (lymphogranuloma venereum or trachoma), or omp1 groups (47). It would be interesting to
study correlations of AFLP fingerprint types with phenotypical or
clinical features.
The C. psittaci isolates showed three AFLP fingerprints
corresponding with their hosts, parakeet, pigeon, and cat, in agreement with previous reports based on other techniques (6, 9, 19, 40, 41,
48).
Our AFLP data support the species status of C. pecorum
(10), in agreement with previous reports (7, 9, 19, 39, 40, 48).
No cutoff SD value exists at which all isolates
are clustered within one of the four currently recognized species.
However, using the criterion of a cutoff SD
value of 80%, all isolates are clustered in seven groups, supporting
the suggestion that at least seven groups within the genus
Chlamydia should be recognized as species: C. pneumoniae, a human C. trachomatis group, a mouse C. trachomatis group, a swine C. trachomatis
group, an avian C. psittaci group, a feline C. psittaci group, and C. pecorum (7, 9, 12,
40). Adding other isolates to the analysis, like C. psittaci abortion and guinea pig inclusion conjunctivitis
isolates, might suggest even more groups. Using previously reported
DNA-DNA hybridization data to infer a phylogram, the C. psittaci and C. trachomatis isolates clustered in two
distinct clusters, in agreement with sequence data (7, 19, 39, 40,
48). However, when using the criterion of 50% or greater DNA-DNA
hybridization relatedness for isolates to be assigned to the same
species (51), six different OTUs could be recognized. This
observation suggests that the currently recognized subgroups of
C. trachomatis and C. psittaci are more distinct
from each other than they appear to be based on sequence data from only
a minor part of the genome. Although these different groups could also
be recognized by AFLP, the clustering pattern of the groups as
calculated from AFLP fingerprints was different from that calculated
from DNA-DNA hybridization data.
Analysis by AFLP has several advantages over other genome-based methods
for typing Chlamydia isolates. In RFLP analysis, many different restriction enzymes and combinations had to be used to
achieve the same level of discrimination (1, 5, 9, 27, 28, 37, 38,
42). RAPD assays are very difficult to standardize compared to
AFLP analysis, since the PCR conditions for RAPD are of low stringency
and therefore prone to variation (50) whereas the PCR
conditions in AFLP analysis are of high stringency. Furthermore, RFLP
analysis of genomic DNA requires large amounts of DNA (1 to 2 µg), or
radioactively labeled DNA, to visualize the generated fragments.
Therefore, the major advantage of the AFLP method is that it requires
only one restriction enzyme combination and much less genomic DNA (<10 ng).
In conclusion, by using AFLP analysis of genomic DNA, differences among
the four currently recognized Chlamydia species, and also
within species, were observed. Furthermore, the cluster analysis of the
AFLP fingerprints provides suggestions for a grouping of chlamydiae
based on the whole genome. In addition, the genome of C. pneumoniae appeared to be highly conserved between isolates of
respiratory and atherosclerotic origin as well.
| |
ACKNOWLEDGMENTS |
We thank Jeroen Stoof, Ankje de Vries, and Geert van Amerongen
for excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Research
Laboratory for Infectious Diseases, National Institute of Public Health
and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. Phone: 31 30 2743595. Fax: 31 30 2744449. E-mail:
Adam.Meijer{at}rivm.nl.
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Abstract
Introduction
