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Journal of Clinical Microbiology, April 2000, p. 1482-1487, Vol. 38, No. 4
Department of Microbiology, Monash
University,1 and Department of
Infectious Diseases and Clinical Epidemiology, Monash Medical
Centre,7 Clayton, Research and
Development, CSL Limited,3 and
Microbiology Research Unit, Royal Children's
Hospital,4 Parkville, Victorian
Infectious Diseases Reference Laboratory, North
Melbourne,5 and Department of
Pathology, Box Hill Hospital, Box Hill,6
Victoria, Australia, and Institute of Tropical Medicine,
Antwerp, Belgium2
Received 3 November 1999/Accepted 23 January 2000
Two high-copy-number insertion sequences, IS2404 and
IS2606, were recently identified in Mycobacterium
ulcerans and were shown by Southern hybridization to possess
restriction fragment length polymorphism between strains from different
geographic origins. We have designed a simple genotyping method that
captures these differences by PCR amplification of the region between
adjacent copies of IS2404 and IS2606. We have
called this system 2426 PCR. The method is rapid, reproducible,
sensitive, and specific for M. ulcerans, and it has
confirmed previous studies suggesting a clonal population structure of
M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia,
Surinam, Mexico, Japan, China, and several countries in Africa were
easily differentiated based on an array of 4 to 14 PCR products ranging
in size from 200 to 900 bp. Numerical analysis of the banding patterns
suggested a close evolutionary link between M. ulcerans
isolates from Africa and southeast Asia. The application of 2426 PCR to
total DNA, extracted directly from M. ulcerans-infected
tissue specimens without culture, demonstrated the sensitivity and
specificity of this method and confirmed for the first time that both
animal and human isolates from areas of endemicity in southeast
Australia have the same genotype.
Mycobacterium ulcerans is
an environmental mycobacterium with worldwide distribution (Fig.
1) and causes chronic, necrotizing skin
lesions in otherwise healthy humans. The incidence of M. ulcerans disease has been increasing worldwide, particularly
throughout rural west Africa (11, 22). The reasons for this
increase are unknown. Epidemiological and PCR-based evidence suggests
that swamps and slow-flowing water are sources of the organism (1, 6, 8, 17, 25), and recent PCR data from Benin and Ghana have
identified M. ulcerans DNA in aquatic insects
(12). So far M. ulcerans has never been isolated
by culture from any of these environments. It has therefore been
difficult to clearly identify reservoirs or modes of transmission. One
approach to improving our understanding of the ecology of this organism
has been to study the molecular epidemiology of M. ulcerans,
for which two methods have been reported. The first of these identified restriction fragment length polymorphism (RFLP) between isolates by
probing with the polymorphic GC-rich repeat sequence contained in
plasmid pTBN12 (7). This approach distinguished 11 distinct RFLP types among isolates from four countries and
suggested a clonal population structure within a geographic region.
Supporting this conclusion, nucleotide sequence analysis of the 3'
region of the 16S rRNA gene identified three alleles that correlated with the geographic origin of an isolate (13). However, the methods used in these studies were limited by requiring
high-concentration, purified DNA or by offering limited discriminative
capability.
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A Simple PCR Method for Rapid Genotype Analysis of
Mycobacterium ulcerans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (21K):
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FIG. 1.
World map showing regions where the isolates used in the
study were obtained.
PCR genotyping of mycobacteria by targeting insertion sequences (IS) has been well described (10, 16). The recent discovery of two high-copy-number IS elements in M. ulcerans that displayed RFLP between strains (21) suggested that PCR amplification between adjacent copies of these elements (IS2404 and IS2606) may be suitable for genotyping this species.
The aims of this study were twofold. The first was to develop a method that would offer strain discrimination in a simple, reproducible format. Second, using this method, we hoped to improve our understanding of the molecular epidemiology of M. ulcerans by analyzing a selection of isolates from disease foci around the world.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mycobacterial strains, clinical specimens, and culture
conditions.
The origins of the M. ulcerans strains used
in this study are listed in Table 1. The
origins of the other species of mycobacteria that were used and general
culture conditions have been described previously (21).
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DNA preparation.
An approximately 10-µl loopful of cells
was scraped from an egg yolk agar slope and resuspended in 500 µl of
1% Triton X-100. This cell suspension was added to a 2-ml skirted,
screw cap tube containing 200 µl of washed 100-µ-m-diameter glass
beads and 500 µl of chloroform-isoamylalcohol (24:1). The tube was
placed in a Fastprep cell disrupter (Savant Instruments, Holbrook,
N.Y.) at speed setting 6 for 40 s. After cell disruption, the tube
was cooled on ice for 5 min and then centrifuged at 17,000 × g for 5 min. In some instances DNA was also prepared by
resuspending cells in 300 µl of water, heating cells to 100°C for
20 min, and then centrifuging as described above. The aqueous phases
were retained and stored at 
20°C. A 2-µl volume of DNA from
either preparation was used as a template for the PCR.
2426 PCR. Reaction conditions used for 2426 PCR were as follows: each PCR mixture (20 µl) contained 1× PCR buffer II (10× PCR buffer II contained 500 mM KCl and 100 mM Tris-HCl [pH 8.3]), 2.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (0.2 mM [each] dATP, dTTP, dCTP, and dGTP), an 0.5 µM concentration of each primer (MU4, 5' ATCGCCGAAGCCTGCCGGAT 3', positions 1119 to 1138, and MU9, 5' TCTTCGTGGTTTTGTGATGGC 3', positions 1306 to 1326), 1 U of Ampli-Taq DNA polymerase (Perkin-Elmer, Melbourne, Australia), and 2 µl of DNA. PCR was performed in an FTS-960 thermal sequencer (Corbett Research, Sydney, Australia) with the following protocol, optimized for this application: 5 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min and 25 cycles (30 cycles for tissue specimens) of 95°C for 20 s, 58°C for 30 s, and 72°C for 40 s, followed by a final extension step at 72°C for 5 min. The PCR products were held at 4°C until analyzed and detected with 7.5% (0.75 mM) native polyacrylamide gel electrophoresis at 200 V for 38 min in a Mini-Protean II apparatus (Bio-Rad) with rapid silver staining. A 100-bp ladder was used as a size marker (Gibco, BRL). The silver stain procedure was adapted from Caetano-Anoelles and Breshoff (Promega Notes 45:13-18, 1994) as follows. After electrophoresis, gels were fixed in 7.5% acetic acid for 6 min, rinsed three times in distilled water, and then stained for 8 min with 50 ml of a 0.075% silver nitrate solution containing 75 µl of formaldehyde. Gels were rinsed twice in distilled water and developed in 50 ml of developing solution, which contained 1.5 g of sodium carbonate, 0.2 µg of sodium thiosulfate, and 75 µl of formaldehyde. The developing reaction was terminated with 7.5% acetic acid.
The banding patterns from all strains were compared visually. A binary matrix was constructed by scoring each pattern for the presence or absence of the 29 PCR products that constituted the total pool of fragments between 200 and 900 bp. It was assumed that comigrating bands were of identical sequence and therefore an indication of genetic relatedness. The patterns were compared by the method of unweighted pair group average linkage and the simple matching coefficient (19). The goodness of fit of the cluster analysis was tested with the cophenetic correlation coefficient, where r values greater than 0.9 suggest a very good fit (18). All analyses were performed with NTSYS, version 1.8, software (Applied Biostatistics Inc., Setauket, N.Y.).| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A test panel of M. ulcerans clinical isolates was
assembled from many of the known M. ulcerans disease foci in
the world (Fig. 1), a collection that represented both temporal and
spatial diversity. All isolates were found to contain IS2404
and IS2606 as determined by PCR, and a selection of these
results is shown in Fig. 2A.
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Several combinations of outward-priming oligonucleotides for
IS2404 and IS2606 were then evaluated in order to
obtain reproducible and discriminatory patterns of bands for different
strains of M. ulcerans. Primers MU4 and MU9 gave the largest
number of clearly resolvable bands when PCR products were separated by
nondenaturing polyacrylamide gel electrophoresis and detected by silver
staining (Fig. 2B). The use of each primer alone in a PCR did not
produce a useful banding pattern. Nine different 2426 PCR genotypes
were observed (Fig. 2B). All isolates were tested at least twice with the same Triton X-100 DNA preparation, and identical profiles were
produced. For the strains from Africa, Queensland, Malaysia, and Papua
New Guinea and for two of the Victorian isolates, DNA was also
extracted by boiling cells in water. The crude DNA preparations from
this method produced banding patterns identical to that obtained from
the detergent-extracted DNA (data not shown). A single pattern for all
seven African isolates was obtained despite the large distances and
long times separating some of these isolates (Table 1; Fig. 1 and
3). The 35 Australian isolates produced
two profiles, representing one genotype from Queensland (northern
Australia) and one from Victoria (southeastern Australia). No variation
was detected among the 32 isolates from Victoria (Fig.
4), but there were two distinct genotypes
among the 3 isolates from Papua New Guinea (PNG I and PNG II). The
Malaysian genotype was nearly identical to PNG II, differing only by
the positions of two bands between 200 and 900 bp. Distinct profiles
were observed for the isolates from Surinam and Mexico; however, the
patterns for isolates from Japan and China were identical.
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To investigate primer specificity 24 other species of mycobacteria were
tested by 2426 PCR. Some PCR products from other mycobacteria were
observed, but these were generally faint, and none produced profiles
similar to any of the patterns obtained from the different M. ulcerans strains (Fig. 5).
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A dendrogram was constructed to display the relatedness between strains
based on banding pattern similarity (Fig.
6). The clustering of the southeast Asian
genotypes and the African genotype is suggestive of an evolutionary
link among these strains; however, the PNG I genotype was an
interesting exception to this cluster. The genotypes representing the
other regions appeared less related to each other, displaying 30 to
60% similarity (r = 0.91) (Fig. 6).
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The detection sensitivity of 2426 PCR was tested with a dilution series
of purified M. ulcerans genomic DNA from an African isolate.
This demonstrated a detection limit of 100 genomes (Fig. 7).
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The relatively low concentration of cells required to produce a profile
and the specificity of the target sequences to M. ulcerans
suggested that this method may be useful for genotyping directly from
clinical samples. To investigate this, DNA extracted from 50 patient
specimens was subjected to 2426 PCR. These samples included swabs and
formalin-fixed and paraffin-embedded tissue specimens that had been
screened previously by IS2404 PCR for M. ulcerans
diagnosis (unpublished data). Of these 50 samples, 40 were
IS2404 positive and the remainder were IS2404
negative. All 50 samples were then tested by 2426 PCR. An example of
some profiles obtained is shown in Fig.
8. A recognizable genotype was obtained
for 20 of the 40 IS2404-positive specimens. All samples that
were IS2404 negative were also 2426 PCR negative. The
specificity of the 2426 PCR was 100% (calculated as the number of true
negatives divided by the number of true negatives plus false
positives), and the sensitivity was 66.7% (calculated as the number of
true positives divided by the number of true positives plus false
negatives). From the 20 recognizable profiles, 13 were identified as
the Victorian genotype and 7 as the Queensland genotype. These profiles
also correctly predicted the geographic origin of the specimen.
Analysis of tissue samples from an M. ulcerans-infected
alpaca (Quechua alpako) and possum (Trichosurus
vulpecula) clearly demonstrated that these animals were infected
with the same genotype as the human cases from these areas (Fig. 8).
Both these animals inhabited regions in southeast Australia where
M. ulcerans disease is known to be endemic.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In mycobacteria, genetic diversity is driven by the activity of mobile DNA such as IS rather than nucleotide sequence drift (20). Thus IS elements are potentially ideal targets for indexing rapidly evolving change in mycobacterial populations (10, 26). M. ulcerans has at least two high-copy-number IS elements, IS2404 and IS2606, that demonstrate RFLP between strains (21) and that appear to be present in isolates of diverse geographic origin (Fig. 2A) (4). However, M. ulcerans is a slow-growing organism, and extraction of a sufficient quantity and quality of DNA to perform a Southern analysis is a time-consuming and impractical process for routine analysis.
The PCR typing method we have developed is a simple and robust tool for quickly determining the origin of an isolate, requiring a minimum of 100 genomic equivalents of DNA (Fig. 7). The high detection sensitivity and specificity permitted typing directly from patient specimens without requiring culture. Other mycobacteria tested by 2426 PCR did produce some bands, but none reproduced any of the profiles seen with M. ulcerans (Fig. 5). The sensitivity of 2426 PCR for genotyping directly from tissue specimens was, however, found to be somewhat low (66.7%). This was most likely due to the low numbers of target cells in some samples; however, DNA extracted from swabs or tissue specimens served equally well as a template. The detection sensitivity of 2426 PCR in this application may be improved by ensuring that specimens are taken from areas of ulceration likely to contain large numbers of bacilli.
Analysis by 2426 PCR of IS2404-positive swab and tissue specimens, obtained from animals and humans in areas of southeast Australia where M. ulcerans disease is endemic identified the same genotype in all specimens, suggesting that animals are infected from the same environmental source as humans (Fig. 8). Genotyping directly from tissue specimens will be particularly useful for studying the epidemiology of M. ulcerans, as culture of this organism is slow and relatively insensitive (4).
To investigate the discriminating capability of 2426 PCR, 55 M. ulcerans isolates were tested (Table 1). The combination of primers MU4 and MU9 produced nine distinct profiles that correlated absolutely with the geographic source of the isolates, i.e., the origin of an isolate could be unambiguously assigned based on its banding pattern. This result is somewhat surprising as other mycobacteria, such as Mycobacterium avium, exhibit considerable IS diversity even within a geographic region when analyzed by a similar genotyping technique (26). The lack of genotype variation in M. ulcerans within a region may be a reflection of its slow generation time and perhaps a low population density within its environmental niche. PCR-based incidence data, gathered during a disease outbreak, suggested a very low abundance of bacteria in the environment (17).
A lack of IS mobility as a cause of the limited genotype diversity is possible but unlikely, as the different 2426 PCR genotypes are indicative of ongoing transposition events or IS-associated genome rearrangements. Furthermore, these events appear to have occurred relatively recently, considering (i) the absence of IS2404 and IS2606 from the closely related M. marinum and (ii) the very high nucleotide sequence similarity between the 16S rRNA and groEL genes of M. ulcerans and M. marinum (13-15, 23), which is indicative of the recent evolutionary divergence of these species. The inferred close genetic relationship among the five genotypes obtained from the southeast Asian isolates (Fig. 6) suggests that M. ulcerans is introduced into an area and then is isolated or sequestered, undergoing occasional transposition or genome rearrangement events to produce a unique genotype in a particular region. However, rapid dispersal over large distances also seems possible considering that only one genotype was observed among the isolates from five countries throughout central and west Africa (Fig. 3). This analysis suggests that the spread of disease across Africa has occurred after its spread throughout southeast Asia and Australia if the rate of genome rearrangements displayed by 2426 PCR is assumed to be the same for all strains.
The 2426 PCR method is highly reproducible. The DNA from all isolates was tested at least twice, and each set of tests produced identical profiles. In addition, 2426 PCR performed with DNA extracted from cells in crude form by boiling produced profiles identical to those produced by PCR performed with purified DNA. The reproducibility of this method is further reinforced by the ability to produce the same pattern from DNA over a 5-log-unit dilution range of template DNA (Fig. 7) and by the fact that the same pattern was obtained for the 32 Victorian isolates and 7 African isolates (Fig. 4).
The findings of previous genotype studies of M. ulcerans by Southern hybridization with the pTBN12 probe (7) and nucleotide sequence analysis of the 16SrRNA gene (13) generally concurred with the results from 2426 PCR analysis, where identical genotypes were detected within a region. However the pTBN12 probe method also identified some additional RFLP among African isolates. The apparent increased discriminatory capability of the pTBN12 probe is countered by the length of time taken to perform a Southern hybridization (3 to 4 days) compared with that taken by 2426 PCR (4 to 6 h) and the ability of 2426 PCR to obtain a genotype directly from a clinical specimen.
Interestingly the pTBN12 probe offered no additional discrimination among Victorian isolates, thus supporting the suggestion of clonality of M. ulcerans within a region. Analysis of additional African isolates with the pTBN12 probe and 2426 PCR is warranted to more thoroughly examine evidence for clonality between endemic foci and to better compare the capabilities of each method.
It is of interest that two genotypes were detected in Papua New Guinea (Fig. 1B). The PNG II pattern appears closely related those of the Malaysian and Australian strains. By comparison, the PNG I pattern is quite distinct and appears, at least by this method, less related to those of the other southeast Asian strains than to those of the African isolates (Fig. 6). Unfortunately further details regarding the origin of this strain were unavailable but, it is likely to represent an endemic focus distant to the PNG II isolates. We are currently sequencing multiple gene loci from M. ulcerans to better establish the genetic relatedness between all strains.
It has been proposed that the global distribution of M. ulcerans may be linked to the breakup of the supercontinents at the end of the Jurassic period (5). However, the data presented in this study, previous 16S rRNA sequence comparisons of M. ulcerans and M. marinum, and the discovery of M. ulcerans in Japan and China (3, 24) argue against this hypothesis and suggest a far more recent mechanism of dispersal. More-extensive nucleotide sequence analyses of M. ulcerans strains are now required to investigate intraspecies and interspecies evolution and to further our understanding of the molecular epidemiology of M. ulcerans.
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ACKNOWLEDGMENTS |
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We thank David Dawson for the provision of mycobacterial isolates.
This work was supported in part by funding from the Government of Victoria through the Department of Human Services.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Monash University, Wellington Rd., Clayton 3168, Australia. Phone: 61 3 9905 4809. Fax: 61 3 9905 4811. E-mail: tim.stinear{at}med.monash.edu.au.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, April 2000, p. 1482-1487, Vol. 38, No. 4
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Marsollier, L., Stinear, T., Aubry, J., Saint Andre, J. P., Robert, R., Legras, P., Manceau, A.-L., Audrain, C., Bourdon, S., Kouakou, H., Carbonnelle, B.
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