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Journal of Clinical Microbiology, October 2001, p. 3684-3689, Vol. 39, No. 10
Department of Medical Microbiology,
University of Cape Town,1 and Goote
Schuur Hospital,2 Cape Town, South Africa
Received 6 March 2001/Returned for modification 2 May 2001/Accepted 15 July 2001
A 1.6-kb DNA fragment isolated from a
Campylobacter concisus genomic library gave
C. concisus-specific restriction fragment length patterns when it was used as a probe in hybridization studies. All of the strains tested, including type strains and clinical isolates, contained a 0.5-kb HindIII fragment that
hybridized to the probe. DNA sequencing of the 1.6-kb fragment
identified three open reading frames (ORFs). One of the ORFs encodes
the carboxy terminus of GyrB, and the translational products of ORF2 and ORF3 showed similarity to hypothetical proteins,
previously identified in Campylobacter jejuni. DNA-DNA
hybridization studies with a fragment internal to ORF3 showed that this
sequence was responsible for the signal observed with the 0.5-kb
HindIII fragment. A rapid PCR assay was developed
and evaluated. Primers that annealed to the extremities of the 1.6-kb
fragment were used to obtain an amplicon of the correct size from both
reference and clinical strains of C. concisus.
Traditionally
Campylobacter concisus has been recovered exclusively from
and considered part of the bacterial flora of the human oral cavity
(22). This species has been isolated predominantly from
gingival crevices associated with the onset of periodontal disease
(15, 22, 24). Subsequently, strains from outside the oral
cavity have been isolated; however, these were initially misidentified
or not classified (25).
Recent studies have increasingly reported on the isolation of
C. concisus from the stools of patients with diarrhea
(6, 12, 27). This has largely been attributed to an
improvement in culture techniques, where the adoption of the membrane
filter technique has improved isolation rates (13).
Following the introduction of the Cape Town protocol (13),
which utilizes the membrane filter technique in combination with a
hydrogen-enriched microaerobic environment, the prevalence of
C. concisus has increased to 31% of the total
Campylobacter isolates from children at the Red Cross War
Memorial Children's Hospital, Cape Town, South Africa. Of all
the Campylobacter spp., only Campylobacter
jejuni subsp. jejuni is more frequently isolated
from children at this hospital. During a reevaluation of conventional
isolation methods, similar results were obtained in a study in Denmark
(6).
In Cape Town, South Africa, C. concisus displays
clinical and seasonal characteristics similar to those of C. jejuni, an established gastrointestinal pathogen (A. J. Lastovica and M. E. Engel, Abstr. 100th Gen. Meet. Am. Soc.
Microbiol. 2000, abstr. D250, 2000). Monthly isolation frequencies
showed that both C. concisus and C. jejuni were notably higher in the 3-month period towards the end
of summer (February to April). Clinical symptoms of diarrhea, such as
severity and stool consistency, were similar to those associated with
C. jejuni; however, C. concisus was
predominantly isolated from children 1 year old or older, whereas
C. jejuni was mostly found in children younger than 12 months. In addition, C. concisus was more likely to be
associated with children of mixed descent than with indigenous black
children. This observation could not be explained by age, gender
differences, genetic susceptibility, eating habits or geographical
location (Lastovica and Engel, Abstr. 100th Gen. Meet. Am. Soc.
Microbiol.). Although some studies have suggested that C. concisus is not a primary pathogen (6, 27), the role
of C. concisus and its association with diarrhea have yet to be clearly defined.
An understanding of the epidemiology of C. concisus has
been hampered by the lack of a suitable identification tool. Like other
members of the genus, C. concisus is fastidious and is
characterized by low biochemical activity, properties that make the
identification of C. concisus problematic, using
conventional phenotypic techniques (7, 10, 17). DNA-DNA
hybridization, immunotyping and whole-cell protein electrophoresis have
been used to identify C. concisus (11, 23,
25). As these techniques are time-consuming and require
considerable expertise, they are not suitable for large epidemiological
studies. A limited number of molecular approaches for the
identification of C. concisus have been described
(3, 14). A PCR of the 23S ribosomal DNA (rDNA)
(3) did not consistently identify C. concisus (6), probably because of the genotypic heterogeneity within the species (3, 25). A similar
approach is based on the restriction profile of 16S rDNA PCR products
(14). During the course of the study described in this
report, a fragment of C. concisus genomic DNA was
cloned and characterized. DNA-DNA hybridization studies and PCR
assays indicate that this fragment could be used to identify
C. concisus.
Bacterial strains and plasmids.
Reference
Campylobacter strains used in this study included
C. concisus NCTC 11485, C. concisus
NCTC 11684, C. concisus CCUG 13144, C. concisus CCUG 19995, C. curvus NCTC 11649, C. rectus NCTC 11489, C. sputorum
bv. fecalis NCTC 11415, C. coli CCUG 11283, C. lari NCTC 11352, C. upsaliensis
NCTC 12183, C. helveticus NCTC 12470, C. jejuni subsp. doylei NCTC 11487, C. jejuni subsp. jejuni NCTC 11168, C. mucosalis NCTC 11000, and C. fetus
subsp. fetus NCTC 10842. Bacteroides
ureolyticus NCTC 10941 was also used. Local clinical isolates
of C. concisus (n = 104) included in
this study were collected during the period 1992 to 1999. One of the strains was a dental isolate from an adult; the remainder were obtained
from children at the Red Cross War Memorial Children's Hospital. S. L. W. On (Veterinary Research Laboratory, Copenhagen, Denmark)
kindly provided two Danish clinical isolates (SSI 63936 and SSI 11286)
from children with diarrhea. Escherichia coli LK111 (30) was used as a recipient in transformation studies.
The vectors, pEco251 (provided by M. Zabeau, Plant Genetics Systems N. V., Ghent, Belgium) and pUC19 (16) were used in
cloning experiments.
Preparation and analysis of DNA.
Genomic DNA was extracted
using guanidinium thiocyanate (19). Plasmid DNA was
isolated using either the alkaline lysis method (8) or a
Nucleobond kit (Macherey-Nagel). DNA was digested with various
restriction enzymes, and the fragments were separated in horizontal
gels of 0.8 to 1.2% (wt/vol) agarose dissolved in 0.4 M Tris-acetate
and 0.01 M EDTA. Gels were stained with ethidium bromide, and DNA was
visualized by UV transillumination.
Ligations.
Ligations were carried out as described
(20), in 20-µl reaction mixtures containing 1× T4 DNA
ligase buffer and 10 U of T4 DNA ligase (Boehringer Mannheim).
Typically, an approximate ratio of 3 to 1 molar concentrations of
insert and vector were used in ligations, and the reaction mixtures
were incubated at 16°C for 4 to 16 h.
Transformation studies.
Competent E. coli LKIII
cells were prepared as described (4). Ligations were added
to competent cells, and transformants were selected on 2× YT agar
containing appropriate antibiotics.
PCR assays.
Based on the DNA sequence (GenBank accession
number AY026942) of the 1.6-kb BglII-XbaI
fragment (Fig. 1), three pairs of oligonucleotide primers were used to amplify sequences internal to the
three open reading frames (ORFs) identified. pcisus1
(5'-GAGCTTGTGGTAAAGA-3') and pcisus2
(5'-GCGTGGTCTTGGATATAG-3') were used to amplify ORF1. pcisus3 (5'-TTGACGTAGAGAGTGACCTTG-3') and pcisus4
(5'-ATCAGAGCTGATCTCGATAAC-3') were used to amplify ORF2, and
pcisus5 (5'-AGCAGCATCTATATCACGTT-3') and pcisus6
(5'-CCCGTTTGATAGGCGATAG-3') were used to amplify ORF3. PCRs
were carried out with a Perkin-Elmer thermocycler (Gene Amp 2400) in a
final volume of 50 µl containing 50 ng of campylobacter DNA, a 50 pM
concentration of each primer, a 2.5 mM concentration of each
deoxynucleoside triphosphate, and 2.5 U of Taq DNA
polymerase (TaKaRa Biochemicals) in the prescribed buffer. Initial
denaturation (94°C for 120 s) was followed by 30 cycles of
amplification. Each cycle consisted of 94°C for 60 s, 45°C for
60 s, and 72°C for 120 s. A final extension step (72°C
for 10 min) completed the amplification. Aliquots of PCR products were
separated by electrophoresis.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3684-3689.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Identification of
Campylobacter concisus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (6K):
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FIG. 1.
Restriction map of DNA fragment from C.
concisus. (A) The shaded block represents pEco251, and the bold
line (5.0 kb) represents the 5.0-kb fragment from C.
concisus cloned in the vector, generating pB3C. (B) Shown
is the 3.0-kb fragment that was cloned into pUC19, generating pNeo.
Preparation of probes. DNA of interest was eluted from agarose gels, purified (21), and labeled using an ECL labeling and detection kit (Amersham International).
DNA-DNA hybridization. Probes were hybridized to genomic DNA restricted with HindIII, separated by agarose gel electrophoresis, and transferred to Hybond-N+ membranes (Amersham International). Prehybridization, hybridization, and the posthybridization washes were carried out according to the protocol supplied with the ECL gene detection system (Amersham International).
DNA sequencing. DNA for sequencing was cloned into pUC19. Sequencing data were generated by automated laser fluorescence (Pharmacia Biotech AB, Uppsala, Sweden) in the Department of Molecular Biology, University of Cape Town. Internal oligonucleotide primers were used where necessary to ensure that both strands were sequenced. DNA sequences were analyzed with DNAMAN (version 4.0; Lynnon BioSoft). BLAST (1) was used to search databases in GenBank for nucleic acid and deduced amino acid sequence similarity with existing sequences. Protein alignments were carried out by the fast alignment method (28), using the Blosum matrix with a k-tuple value of 2 and a gap penalty of 4.
Construction of a DNA library. A DNA library of C. concisus NCTC 11485 was prepared (2). Genomic DNA from C. concisus NCTC 11684 was digested partially with Sau3AI (Boehringer Mannheim). Size fractionation was carried out in a sucrose gradient; the 4- to 5-kb DNA fragments were pooled, recovered by ethanol precipitation, cloned into the BglII site of pEco251, and introduced into competent E. coli LKIII cells.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of C. concisus DNA sequence for identification to species level. To select a recombinant clone that could be used to identify C. concisus, the pEco251 library was screened using DNA-DNA hybridization studies. DNA inserts were restricted from pEco251 with BglII, labeled, and hybridized to HindIII-digested DNA from C. jejuni, C. mucosalis, C. concisus type strains, and clinical isolates from Cape Town (404-96 and 406-96) and Denmark (SSI 11286 and SS1 63936). One of the inserts (5.0 kb), from a recombinant plasmid, designated pB3C, hybridized to four bands (3.0, 1.2, 0.5, and 0.3 kb) in C. concisus NCTC 11485 and to two bands (1.2 and 0.5 kb) in each of the four clinical isolates (data not shown). In addition, signals were obtained with fragments in C. mucosalis and C. jejuni, but the banding patterns were entirely different from those observed in C. concisus. The presence of common bands, which hybridized to the probe in the C. concisus strains, combined with the fact that these bands were not present in C. jejuni and C. mucosalis, suggested that they could be used as identification markers.
To refine the probe further, a limited restriction map of pB3C containing the C. concisus insert was determined. Restriction digestion showed that the insert (5.0 kb) contains an internal BglII site 0.4 kb from one of the Sau3AI sites (Fig. 1). Digestion of the insert with BglII and XbaI generated two fragments of 1.6 and 3.0 kb. When the 3.0-kb BglII-XbaI fragment was used to probe the DNA, a strong hybridization signal was obtained with a 3.0-kb band, and weak signals were obtained with 0.5- and 0.3-kb bands in C. concisus NCTC 11485 (data not shown). Hybridization signals, albeit weak, were also obtained with 1.2-kb bands in the local and Danish clinical isolates. No signals were obtained with DNA from the C. jejuni and C. mucosalis type strains. Probing with the 1.6-kb BglII-XbaI fragment resulted in signals with bands of 1.2, 0.5, and 0.3 kb in C. concisus NCTC 11485 (data not shown). In addition, a signal was obtained with a 1.2-kb fragment in the Danish and local clinical isolates; a weak hybridization signal was observed with a 0.5-kb band in one of each of the local and Danish clinical isolates. No hybridization signal was obtained with DNA from C. jejuni, and in C. mucosalis, barely visible signals were detected with fragments (2.7 and 2.2 kb) that had hybridized to the 5.0-kb probe. The fact that similar hybridization profiles were observed in the C. concisus type strain and in the Danish and local clinical isolates suggested that the 1.6-kb sequence and its organization in the genome are conserved in C. concisus. The specificity of the 1.6-kb fragment was investigated, using hybridization assays and an extended panel of Campylobacter type strains and B. ureolyticus, previously considered genotypically similar to campylobacter (26). An expected hybridization banding pattern (1.2, 0.5, and 0.3 kb) was obtained with C. concisus NCTC 11485 (data not shown). In addition, strong signals were obtained with DNA from 2 of the 11 Campylobacter isolates: bands of 2.2 and 1.7 kb in C. curvus and C. sputorum bv. fecalis, respectively, hybridized to the probe. A weak signal was obtained with a 2.6-kb fragment in C. mucosalis, but signals were not detected with DNA from C. rectus, C. coli, C. jejuni subsp. jejuni, C. lari, C. upsaliensis, C. helveticus, C. fetus subsp. fetus, C. jejuni subsp. doylei, and B. ureolyticus. It is important that when the membrane was hybridized with a 16S rDNA (5) probe, signals were obtained with DNA from these species, indicating that DNA had been transferred to the membrane. Based on the above results it was concluded that probing with the 1.6-kb BglII-XbaI fragment generated a species-specific pattern with DNA from C. concisus. To test this further, DNA from 62 clinical isolates, including the dental isolate, and two Swedish reference strains (CCUG 13144 and CCUG 19995), suggested by pulsed-field gel electrophoresis to represent genetically diverse groups (data not shown), was probed with the 1.6-kb fragment. Analysis of the hybridization profiles showed a limited number of restriction fragment length polymorphisms (RFLPs) among the isolates. Five profiles were identified (Table 1). Common to all the profiles was a 0.5-kb fragment that hybridized to the probe, suggesting a genetic marker for the identification of C. concisus.
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Sequence analysis of the 1.6-kb fragment from C. concisus in pNeo. The DNA sequence of the 1.6-kb fragment was determined on both strands (GenBank accession number AY026942). Three major ORFs are contained on the sense strand, while no significant ORFs were identified on the other strand.
Analysis of the sequence upstream of ORF1 did not identify any bacterial transcription signals. This suggested that ORF1 extends upstream of the XbaI site and that the 5' end is contained in the 3.4-kb XbaI-Sau3A fragment (Fig. 1). A comparison of the deduced amino acid sequence of this ORF with protein databases in GenBank showed 76 and 58% similarity to the carboxy termini of GyrB from C. jejuni and Helicobacter pylori, respectively. ORF2 and ORF3 are preceded by ribosome-binding sites upstream of the ATG transcription initiation sites. The sequences (AAGGA) are identical to the C. jejuni consensus sequence (29) but shorter than that proposed previously for C. jejuni (9). Upstream of ORF2 is the sequence TTTAAAAGGT, which shows similarity to the
35
consensus sequence (TTTAAGTNNTT) for C. jejuni promoters (29). Based on the observation that
the spacing between the 35 and 10 regions of C. jejuni promoters varies from 15 to 19 bp (29), two
appropriately spaced putative 10 regions were identified. TTGACCT and CCTAGTT, which are separated from the
putative 35 sequence by 14 and 18 bp, respectively, show similarity
to the 10 consensus sequence, TATAATT, for C. jejuni promoters (29). Two regions, 35
(TTTAAACA) and 10 (TAAGCTA), separated by 18 bp
precede ORF3. The 16 sequence (TTTTTTTG) (29)
was not identified in either of the putative promoters.
The translational product of ORF2 showed 70% similarity to a conserved
hypothetical protein of 127 amino acids in C. jejuni (CDS Cj 1724c), while that of ORF3 showed 70% similarity with the
NH2-terminal of a conserved hypothetical protein
(408 amino acids) in C. jejuni (CDS Cj 0015c).
DNA-DNA hybridization studies with sequences internal to ORF1,
ORF2, and ORF3 as probes.
Internal portions of each ORF were
amplified, labeled, and hybridized to DNA from Campylobacter
spp. When the fragment internal to ORF1 (gyrB) was used to
probe the DNA, a strong signal was obtained with the 1.2-kb band in
C. concisus (Fig. 2A,
lane 1). In addition, signals were obtained with a 2.2-kb fragment in
C. curvus (Fig. 2A, lane 3), a 1.7-kb fragment in
C. sputorum bv. fecalis (Fig. 2A, lane 5) and a 0.5-kb
fragment in C. helveticus (Fig. 2A, lane 10). Weak
hybridization signals were obtained with DNA fragments in C
mucosalis, C. rectus, C. coli,
C. upsaliensis, B. ureolyticus, and
C. jejuni subsp. doylei (Fig. 2A, lanes 2, 4, 6, 9, 11, and 12). No signals were obtained with the DNA from C. jejuni subsp. jejuni, C. lari, and
C. fetus subsp. fetus.
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PCR assay for the identification of C.
concisus.
As hybridization studies are time consuming and
not the preferred approach in a diagnostic laboratory, a PCR assay
based on the DNA sequence of the 1.6-kb fragment was developed and
evaluated. Using pcisus1 and pcisus6, which anneal to DNA sequences in
ORF1 and ORF3, respectively, a product of the expected size (1.5 kb) was obtained from C. concisus NCTC 11485 (Fig.
3). No PCR products were obtained from
C. jejuni, C. lari, C. upsaliensis, C. helveticus, B. ureolyticus, C. sputorum bv. fecalis,
C. curvus, C. mucosalis, C. rectus
and super-family member Helicobacter pylori. Amplicons were not obtained with DNA from unrelated bacteria
Acinetobacter baumannii, Mycobacterium
bovis BCG, E. coli, and Salmonella sp. It is noteworthy that, although hybridization signals were
obtained with the DNA from C. curvus and C. sputorum bv. fecalis when it was probed with the 1.6-kb fragment,
no PCR products were obtained from these species. It must also be noted
that in a PCR assay using universal primers for 16S rDNA
(5), a product of the appropriate size was obtained from
all of the species tested, indicating the suitability of the DNA for
PCR and the absence of inhibitors in the reaction mixture.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Hybridization studies and PCR assays carried out in this study indicate that a 1.6-kb BglII-XbaI fragment, which was cloned from C. concisus NCTC 11485, could be used to identify this species. When this fragment was used to probe DNA from Campylobacter spp., a C. concisus-specific HindIII hybridization profile was generated. Strong hybridization signals were obtained with the DNA from only two other species, C. sputorum bv. fecalis and C. curvus, suggesting that these species are more closely related to C. concisus than are the other species, a finding that is supported by phylogenetic rRNA studies (26). A weak signal was obtained with the DNA from C. mucosalis, but signals were not obtained with DNA from C. rectus, C. coli, C. jejuni, C. lari, C. upsaliensis, C. helveticus, B. ureolyticus, C. jejuni subsp. doylei, and C. fetus subsp. fetus.
Five RFLP profiles were observed in the C. concisus strains (n = 106) probed with the 1.6-kb fragment. All of the strains contained a 0.5-kb HindIII fragment that hybridized to the probe. The profiles obtained were consistent in both local and Danish clinical isolates. In addition, profiles indicative of C. concisus were observed in the two Swedish reference strains (CCUG 13144 and CCUG 19995), belonging to two separate DNA-DNA hybridization homology groups (25). These data suggest that the observed hybridization pattern is not confined to strains from a specific geographic region. Moreover, the stability of the profile is suggested by the fact that the strains were collected over a period of 7 years (1992 to 1999).
An analysis of the DNA sequencing data of the 1.6-kb fragment identified three ORFs. ORF1 contains the 3' end of gyrB. It is assumed that the 5' end of this gene is contained in the 3.4-kb XbaI-Sau3A portion of the 5.0-kb insert in pB3C (Fig. 1). As gyrB is contiguous with gyrA in C. jejuni it is probable that the latter gene is also contained in the 3.4-kb XbaI-Sau3A portion. The strong signals obtained with DNA from C. curvus, C. sputorum bv. fecalis, and C. helveticus when probed with a portion of ORF1 (Fig. 2) suggest that the 3' ends of the gyrB genes in these species are closely related to their counterpart in C. concisus.
The fragment internal to ORF2 hybridized to DNA from C. concisus and C. curvus. As ORF2 contains a HindIII site, two fragments (1.2 and 0.3 kb) in C. concisus hybridized to the probe. On the other hand, only one band (2.2 kb) in the DNA from C. curvus hybridized to the probe. Since ORF1 also hybridized to a fragment of 2.2 kb in this strain, it is likely that as in C. concisus, ORF1 and ORF2 are contiguous in C. curvus.
Hybridization studies using a portion of ORF3 showed that this sequence was responsible for the signal obtained with the 0.5-kb HindIII fragment in all of the C. concisus strains investigated. The translational product of this ORF showed 70% similarity with the amino terminus of a hypothetical protein (408 amino acids) in C. jejuni (CDS Cj 0015c)
A rapid PCR assay for the identification of C. concisus was evaluated, using primers that annealed to the extremities of the 1.6-kb fragment. The assay was specific for C. concisus, and PCR products were not obtained from any of the other Campylobacter spp. tested. Amplicons were obtained from local (87 of 89) and Danish (n = 2) clinical isolates. Importantly, amplicons were also obtained from the two Swedish reference strains shown to be genetically diverse (25). It was not necessary to obtain a restriction enzyme profile of the amplicons, making the method rapid and suitable for epidemiological studies. It was not the purpose of this study to evaluate the use of the PCR assay to detect C. concisus directly from stools. In other studies, PCR assays have not always detected Campylobacter spp. in stools, suggesting the presence of inhibitors in the specimen (18).
The two clinical isolates from which PCR products were not obtained were investigated further. Hybridization studies, using the 1.6-kb fragment as a probe, supported the results of the PCR assay (data not shown). Using the 16S rDNA method (14), the resulting DdeI restriction profiles of the amplicons obtained from these two strains were different from the Campylobacter profiles described, including that of C. concisus. These data suggest that the two strains might have been misidentified as C. concisus and underscore the necessity for an unequivocal identification tool.
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ACKNOWLEDGMENTS |
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We thank Harold Zappe for his help in the preparation of the C. concisus DNA library.
M. I. Matsheka and this study were funded by the Rockefeller and Carnegie Foundations through the University Science, Humanities, and Engineering Partnerships in Africa (USHEPiA) program. M. I. Matsheka thanks the University of Botswana for financial assistance received during the course of his studies. A. J. Lastovica thanks the South African Medical Research Council for financial assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, Medical School, UCT, Anzio Rd., Observatory 7925, Cape Town, South Africa. Phone: (021) 4066378. Fax: (021) 4488153. E-mail: gelisha{at}curie.uct.ac.za.
Present address: Department of Biological Sciences, University of
Botswana, Gaborone, Botswana.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, October 2001, p. 3684-3689, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3684-3689.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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