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Journal of Clinical Microbiology, June 2004, p. 2558-2565, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2558-2565.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genotypic Variation and Stability of Four Variable-Number Tandem Repeats and Their Suitability for Discriminating Strains of Mycobacterium leprae

Richard Truman,1* Amanda B. Fontes,2 Antonio B. de Miranda,3 Philip Suffys,2 and Thomas Gillis1

Laboratory Research Branch, National Hansen's Disease Program at Louisiana State University, HRSA/BPHC, Baton Rouge, Louisiana 70894,1 Leprosy Laboratory, Department of Tropical Medicine,2 Laboratory of Molecular Biology and Diagnosis of Infectious Diseases, Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Manguinhos 21045-900, Rio de Janeiro, Brazil3

Received 11 December 2003/ Returned for modification 17 February 2004/ Accepted 16 March 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has not been possible to distinguish different strains of Mycobacterium leprae according to their genetic sequence. However, the genome contains several variable-number tandem repeats (VNTR), which have been used effectively in strain typing of other bacteria. To determine their suitability for differentiating M. leprae, we developed PCR systems to amplify 5 different VNTR loci and examined a battery of 12 M. leprae strains derived from patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild armadillos and a sooty mangabey monkey. We found diversity at four VNTR (D = 0.74), but one system (C16G8) failed to yield reproducible results. Alleles for the GAA VNTR varied in length from 10 to 16 copies, those for AT17 varied in length from 10 to 15 copies, those for GTA varied in length from 9 to 12 copies, and those for TA18 varied in length from 13 to 20 copies. Relatively little variation was seen with interspecies transfer of bacilli or during short-term passage of strains in nude mice or armadillos. The TA18 locus was more polymorphic than other VNTR, and genotypic variation was more common after long-term expansion in armadillos. Most strain genotypes remained fairly stable in passage, but strain Thai-53 showed remarkable variability. Statistical cluster analysis segregated strains and passage samples appropriately but did not reveal any particular genotype associable with different regions or hosts of origin. VNTR polymorphisms can be used effectively to discriminate M. leprae strains. Inclusion of additional loci and other elements will likely lead to a robust typing system that can be used in community-based epidemiological studies and select clinical applications.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leprosy is a slow, chronic infection caused by Mycobacterium leprae. It principally affects the peripheral nervous system and involves skin and other tissues. Though M. leprae was one of the earliest bacterial pathogens recognized, the agent is yet to be cultivated on artificial laboratory media, and supplies of leprosy bacilli can only be obtained through propagation in animals. M. leprae has fastidious temperature requirements and few natural hosts. It displays only limited growth when inoculated into the footpads of conventional mice but exhibits uninhibited growth with systemic dissemination in athymic nude mice and armadillos (Dasypus novemcinctus) (24).

Most people incubate the infection for at least 3 to 5 years before developing clinical symptoms, and only a fraction of the people exposed to M. leprae ever develop clinical leprosy. This long latent period, combined with a lack of early diagnostic tests, has confounded our understanding of how the disease is transmitted. Some wild armadillos in the United States are known to harbor M. leprae (23), and there are anecdotal reports of other animals with natural infections. However, humans are considered the principal reservoir. The disease is thought to be spread most effectively through long-term, intimate contact with an infected individual, but the majority of new cases presenting around the world are unable to relate any close association with another person who had leprosy (6).

Leprosy can be treated effectively, and worldwide drug therapy campaigns have reduced the global disease prevalence markedly during the last 30 years. However, the new case detection rate has not declined substantially. A high level of endemicity is still reported in six countries, including India and Brazil, and hyperendemic foci can be found in many areas around the world (27). A better knowledge of how M. leprae is transmitted could benefit our ability to target drug therapy campaigns and lead to improved control strategies.

Genotyping of bacterial pathogens can enhance our ability to identify the source of infection or the time frame of different outbreaks and can facilitate a better understanding of disease transmission. Elegant studies on a closely related pathogen, Mycobacterium tuberculosis, are providing new insights into the international dissemination dynamics of that disease and bringing new clarity about evolutionary changes in the pathogen over wide geographic areas or among reference strains used in research (20, 21). Unfortunately, earlier attempts to differentiate M. leprae strains according to their growth or genotypic characteristics have been largely unsuccessful.

There are no M. leprae strains that exhibit notably different pathogenic or growth characteristics during passage, and most strains have been differentiated only according to their geographic origin or donor. Though in the 1960s Shepard noted the possibility of "fast"- and "slow"-growing M. leprae strains, subsequent studies have shown those observations to be an artifact related to the viability of the individual inocula used and not an inherent trait of the isolates (22). Other than select drug resistance markers, little genotypic variation has been noted. Restriction fragment polymorphism analysis of M. leprae isolates using a combination of different enzymes and probes, as well as sequencing of the internal transcribed spacer region of the 16S-23S rRNA operon, have yielded no polymorphic DNA sequences (4, 5, 26). Some minor structural variation in the polA (7) and rpoT (13) genes have been described, but the value of these elements for differentiating M. leprae appears to be limited.

The M. leprae genome contains a number of dysfunctional pseudogenes and has been described as highly degraded. In completing the total genomic sequence, Cole et al. (3) identified several variable-number tandem repeats (VNTR) and other elements that could prove useful for discriminating M. leprae strains (4). Recently, Shin et al. (18) and Matsuoka et al. (14) each reported diversity at one VNTR (TCC) between M. leprae from patients in the Philippines or Japan. However, the discriminatory power of a single VNTR is quite low, and the stability of alleles associated with "pseudogenes" is unclear. In this study, we examined the allelic diversity of M. leprae at five select VNTR using a panel of laboratory strains and clinical isolates obtained from patients and animals from geographically distinct locales, and we assessed the stability of those observed alleles with passage of the strains in nude mice and armadillos.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Identification of the VNTR loci. A PERL script (www.perl.org) was used to search the genome sequence for tandem repetitions of mono-, di-, or trinucleotides. Initially, the search was performed in the M. leprae cosmid collection at National Center for Biotechnology Information, National Institutes of Health, and later with the completed genome sequence at The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/). We also visually inspected the genomic region surrounding the identified patterns for extension to other promising repeats. An arbitrarily defined minimum pattern of 8 mononucleotides and 6 dinucleotide or trinucleotide combinations found 13 VNTR, of which we selected 5 for additional study. Repeats were designated as annotated by the Sanger genome site on the plus DNA strand in the 5-prime-to-3-prime direction and detailed in Table 1.


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  		TABLE 1. Identity and chromosomal location of five M. leprae VNTR and the primer sequences used to amplify them in PCR

 
Mycobacterial isolates, strains, and control tissues. Cultivable mycobacteria obtained from American Type Culture Collection (ATCC) (Manassas, Va.) or recovered from clinical specimens were assembled in a panel to assess specificity of the PCR primers. Identification of each clinical isolate was confirmed by biochemical tests and aided by 16S RNA sequencing, using the RIDOM database (http://www.ridom-rdna.de). Samples of purified DNA or bacterial lysates containing genomic DNA were prepared from liquid cultures of single colonies originating on agar slants. Samples of mouse, armadillo, and human DNA also were included in each testing scheme along with M. leprae DNA as a positive control.

Isolation and propagation of M. leprae. M. leprae was isolated from patient biopsies obtained in different countries and from tissue samples of various wild animals presenting with naturally acquired leprosy. The pedigree of each was recorded, and the M. leprae organisms were propagated in nude mice or armadillos according to the procedure described previously (22). Briefly, after homogenizing the M. leprae-infected tissue in 3 to 5 ml of Hanks' balanced salt solution, aliquots of the supernatant fraction were inoculated into the hind footpads of BALB/c nu/nu mice (50 µl/footpad) or intravenously into armadillos (500 µl). These experimentally induced infections were allowed to advance for different intervals. Typically, the bacteria were transferred between nude mice every 8 months after a 100-fold growth of bacilli in the footpad, while armadillo infections usually resulted in a 10,000-fold expansion after approximately 24 months. Subsequently, bacilli were serially passaged in both nude mice and armadillos. At each passage, aliquots were stored frozen at –80°C, as either a suspension or whole tissue. For armadillos, tissue samples were formalin fixed and embedded in paraffin. In addition, for some of the strains, a coded diagnostic biopsy from the original donor stored in paraffin was available. A battery of such materials was compiled for this study (Table 2). Additional details on the various strains and passage samples are given below.


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  		TABLE 2. VNTR genotype of M. leprae reference strains and passage samplesa

 
Origins of M. leprae strains. M. leprae were isolated from the tissues of multibacillary cases. Patient samples were collected in accordance with an institutionally approved human use protocol and coded to protect individual privacy. Likewise, animal studies were performed in compliance with all applicable standards under an approved protocol. The LWM26 strain was derived from a Philippine biopsy sample (donated by Jerry Walsh, Cebu, Philippines) in 1997 through propagation in nude mice. Bacillary suspensions taken from the third and fourth passage were examined. The NHDP55 strain was isolated in 1996 in nude mice from the skin of a >60-year-old Texas male who had no foreign residence (NFR) history. Bacteria in the diagnostic biopsy and from the fourth nude mouse passage were examined. Strain NHDP10 was isolated in nude mice in 1996 from the skin of a >60-year-old Louisiana NFR male. We examined bacilli from the diagnostic biopsy and two separate nude mice in the fourth passage. Strain NHDP98 was isolated in nude mice in 1990 from the skin of a >25-year-old Mexican-born male patient in Texas. The bacilli had been passed in nude mice along parallel lines (a and b). We examined samples from the diagnostic biopsy and bacilli from both parallel passage lines at the 9th and 11th intervals. Strain SMML was derived from a sooty mangabey monkey skin biopsy donated by R. Gormus (Tulane Regional Primate Center, Covington, La.). The strain was originally isolated in an armadillo in 1994 and transferred to nude mice in 1998. We examined material from the nude mouse passages four and five. The WHO strain was a DNA sample prepared and distributed by M. J. Colston (National Institute for Medical Research, London, United Kingdom). The samples W260, W415, and W508 were clinical biopsies taken from wild Louisiana armadillos with naturally acquired leprosy. The W260 sample was acquired in 1979 in Ascension parish, while samples W415 and W508 were from animals captured in 1983 and 1985, respectively, at locations some 60 miles away from where the W260 sample was recovered. The strain NHDP34 was isolated in an armadillo in 1989 with bacilli from the skin of a >25-year-old female Louisiana NFR case. The strain was passed in another armadillo and then transferred to nude mice. Aliquots from the fourth nude mouse passage were transferred to armadillos Arm80 and Arm72. We examined material from the fourth passage in nude mice and fifth passage in armadillos that had been inoculated with those same bacilli. The strain BR4923 was isolated in nude mice in 1996 from the skin biopsy of a Brazilian patient donated by S. Talhari (University of Amazonas, Manaus, Brazil). The strain was passaged in nude mice three times and transferred to another group of nude mice as well as a group of armadillos (Arm19, Arm21, Arm29, and Arm34) for the fourth passage. We examined samples from passages three and four in nude mice and passage four in armadillos. The strain Thai-53 was originally isolated by M. Matsuoka (National Infectious Disease Institute, Tokyo, Japan) in 1982 with bacilli from a skin leproma of a Thai case and has been carried in continuous passage in nude mice since that time. We examined random-passage samples from 1992 through 1999 carried in nude mice in the National Hansen's Disease program.

DNA recovery and extraction. DNA was recovered from cultivable mycobacteria, M. leprae suspensions, and fresh and fixed tissue samples. The cultivable agents were grown on 7H11 slants where individual colonies were picked and mixed with deionized water or 10 mM TRIS-HCl (pH 7.4)-1 mM EDTA (TE) buffer prior to boiling for 3 min to liberate their DNA. Cellular debris was clarified by centrifugation, and the DNA-rich supernates were used in PCR. DNA was recovered from M. leprae using the procedure described previously (26). Generally, bacillary suspensions or homogenates were subjected to three freeze-thaw cycles (–80°C/95°C) and treated with protease K at 60°C for 18 h. DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated with 95% ethanol, and resuspended in TE buffer. Any reusable laboratory instruments, such as scissors or glass homogenizers, were treated with a dilute Clorox solution (0.5%) for 30 min and rinsed thoroughly with deionized water before being dried at 200°C overnight to remove residual nucleic acids.

To recover M. leprae DNA from fixed, paraffin-embedded tissues, the blocks were shaved to mechanically remove excess paraffin by using a new, unused blade, and 30- by 5-µm-thick sections were cut from each embedded sample. The sections were mixed with 1 ml of xylene, and the paraffin was extracted at room temperature on a shaker overnight. The tissue sections were next washed with ethanol and collected by centrifugation. After air drying, the pellets were resuspended in TE buffer, along with protease K (15 mg/ml), dissolved in digestion buffer (100 mM TRIS-HCl [pH 7.4], 150 mM NaCl, 10 mM EDTA), and held at 60°C for 18 h. Protease K activity was terminated by heating (95°C for 10 min), and DNA was extracted as described previously (9).

PCR amplification. Primer sequences for regions of interest were derived from the Sanger Center M. leprae sequence and are shown in Table 1. Amplification was performed using both forward and reverse primers annealing to flanking regions of the repeats. PCR was performed in 100-µl volumes consisting of 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxynucleoside triphosphate mix and 2.5 U of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, Calif.). PCR conditions, including annealing temperature and primer concentration, were optimized for each VNTR amplification reaction. PCR conditions for AT17 were 100 pmol of each primer with an annealing temperature of 58°C for 40 cycles; GTA used 50 pmol of each primer with an annealing temperature of 58°C; TA18 used 100 pmol of each primer with an initial denaturation step at 94°C for 5 min, an annealing temperature of 60°C, and a final extension step at 72°C for 7 min. The PCR for GAA was performed as described previously (19). The standard temperatures and cycling conditions for all PCR amplifications described above, except as noted, were an initial step at 94°C for 10 min and then 40 cycles of 94°C for 30 s, the appropriate annealing temperature for 30 s, and 72°C for 30 s. At the end of the cycling period, each reaction was allowed to proceed at 72°C for 10 min. In some instances the BD Advantage 2 PCR enzyme system (Becton-Dickenson, Palo Alto, Calif.) was used as described by the manufacturer to enhance amplicon production and to compare DNA sequences derived from amplicons produced by using either BD Titanium or standard AmpliTaq DNA polymerase as supplied by Applied Biosystems.

For sensitivity testing of each PCR system, a 10-fold serial dilution of M. leprae DNA (10 ng to 1 fg) was added to the PCR mixture and amplified for 40 cycles. Amplification of all systems was performed on an ABI 2700 thermocycler (Applied Biosystems). Specificities of the PCR systems for M. leprae DNA were tested using purified human, mouse, or armadillo DNA, DNA from pathogenic and nonpathogenic mycobacteria, and that from other bacteria frequently found associated with human skin. The bacterial DNA tested included DNA from M. leprae, Mycobacterium aichiense ATCC 27280, Mycobacterium aurum ATCC 2336, Mycobacterium avium ATCC 25291, M. avium (clinical isolate), Mycobacterium bovis BCG ATCC 35736, M. bovis BCG ATCC 35747 and BCG Pasteur, Mycobacterium chelonae NCTC946, M. chelonae ATCC 19977, Mycobacterium flavescens ATCC 14474, Mycobacterium fortuitum ATCC 6841, Mycobacterium gordonae ATCC 14470, Mycobacterium intracellulare ATCC 13950, M. intracellulare 35765, Mycobacterium kansasii 12478, Mycobacterium komossense ATCC 33031, Mycobacterium lepraemurium, Mycobacterium lufu (a gift from F. Portaels, Institute of Tropical Medicine, Antwerp, Belgium), Mycobacterium marinum ATCC 927, Mycobacterium phlei ATCC 11758, Mycobacterium scrofulaceum (clinical isolate), Mycobacterium simiae (clinical isolate), Mycobacterium smegmatis ATCC 14468, M. smegmatis ATCC 19420, M. tuberculosis H37Rv ATCC 27294, Mycobacterium ulcerans (clinical isolate), Mycobacterium vaccae ATCC 15483, Mycobacterium xenopi (gift from the National Institute of Public Health and the Environment, Bilthoven, The Netherlands), Clostridium perfringens ATCC 13124, Stapholococcus epidermidis (clinical isolate), and Streptococcus pyogenes (clinical isolate). To insure that DNA extracts used for specificity testing lacked PCR inhibitors, each extract was tested for reactivity by PCR using primers that anneal to common regions of bacterial 16S ribosomal DNA (rDNA) or, in the case of the human and mouse DNA, primers that recognize common regions of 18S rDNA. Armadillo DNA was tested for the presence of PCR inhibitors by amplifying a segment of the glyceraldehyde 3-phosphate dehydrogenase (gap) gene that has been sequenced and determined to be related to other eukaryotic gap genes (D. L. Williams, National Hansen's Disease Program Laboratory [Baton Rouge, La.], personal communication).

Sequencing of PCR products. To assess quality of the PCR product, a 20-µl sample of each amplification reaction mixture was analyzed on a 3% agarose gel at 100 V for 1 h in buffer and stained with ethidium bromide. Samples of sufficient quality were purified through QIAquick PCR purification columns (QIAGEN, Inc., Ventura, Calif.). To determine the VNTR genotype, each amplicon was sequenced on an ABI 377 automated DNA sequencer (Applied Biosystems) using the ABI PRISM BigDye Terminator v 3.0 sequencing kit (Applied Biosystems). Both DNA strands amplified from forward and reverse primers over each VNTR were sequenced, except for VNTR TA18, for which only the forward sequence gave reproducible sequencing results. To assess reproducibility of the four VNTR PCRs and subsequent DNA sequencing reactions, DNAs from two M. leprae isolates (Br4923 and Thai-53) were extracted and analyzed in triplicate. All DNA sequence reactions produced identical sequence reads across all four VNTR regions specific for each M. leprae isolate.

Statistical analysis. A simple percent distance dissimilarity matrix of the allele values was constructed prior to analysis. Diversities of group clusters were analyzed using unweighted pair group method (UPGMA) cluster routines within the NTSSpc software package (F. J. Rohlf, NTSSpc ed. 2.02; Exeter Software, Stoneybrook, N.Y.). Allelic diversity of individual VNTR was calculated across the entire collection of samples as D (diversity index) = 1 – {Sigma} (allele frequency)2 (19).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VNTR PCR systems. The VNTR selected were distributed around the chromosome and occurred within pseudogenes and intergenic segments (Table 1). Among the regions selected, we included the GAA VNTR, for which polymorphic diversity was reported earlier (18). This sequence is located in an intergenic segment downstream of a putative sugar transporter pseudogene and was described originally as TTC. It is renamed GAA here using the standard 5'-to-3' plus-strand nomenclature. In addition, we selected four other simple VNTR, including a two-segment mononucleotide C16G8 repeat in an intergenic segment, a two-nucleotide AT17 repeat in a conserved hypothetical protein pseudogene, a two-nucleotide TA18 repeat associated within a putative membrane protein pseudogene, and a three-nucleotide GTA repeat located in an intergenic segment. All PCRs were standardized using purified DNA from the WHO strain, and the flanking sequences for each VNTR were confirmed against the standard M. leprae sequence (Sanger). The reverse primer for the TA18 VNTR did not yield reproducible sequencing profiles with most samples, and those sequence results presented are based only on the forward primer sequencing reactions. Sequencing of the C16G8 repeat was not reproducible, perhaps due to the GC-rich structure, and it was omitted from further analysis.

The sensitivities of the different systems were evaluated with a serial dilution of M. leprae DNA. The detection limit of all four systems was in the 10- to 1-pg range. Generally, we found a strong amplification signal using 10 pg and a weak band close to the detection limit with 1 pg of target DNA (data not shown). Specificity tests for all four VNTR systems demonstrated that only M. leprae DNA produced the appropriate amplicon, and all sequencing results were highly reproducible. In addition, tests for PCR inhibitors were negative, as demonstrated by the ability of each bacterial or eukaryotic DNA sample to support the amplification for either 16S or 18S rDNA (data not shown).

Diversity between laboratory culture strains and isolates. We found diversity among our panel of M. leprae strains at each of the VNTR. Between the individual strains, diversity (D) ranged from 0.639 to 0.7919 and averaged 0.74 over the entire panel of strains and passage samples. GTA showed the smallest number of different alleles, and TA18 showed the most. The samples tested were drawn from six different countries and three different host species. The genotypes for individual strains and passage samples are shown in Table 2.

Strains derived from human patients or animals varied markedly and showed no distinguishable genotype for different host species or geographic region. Generally, the human-derived M. leprae strains taken at various passage intervals each showed diverse genotypes (NHDP55, NHDP10, NHDP98, Br4923, and Thai-53). Two human-derived strains were identical (LWM26 and NHDP34). The genotype of each strain tended to remain stable with short-term passage in nude mice. Strains initiated from a single source and passaged in parallel lines or passage intervals (NHDP10 and NHDP98) showed no divergence in VNTR genotype for up to 11 years in nude mice.

The greatest allelic variation was seen in association with transfer of bacilli to armadillos. Armadillo infections require about 2 years to manifest harvestable numbers of bacilli and result in expansion of each inoculum up to 10,000 times. Comparison of inocula prepared in nude mice and the resulting outgrowth in armadillos (Br4923 and NHDP34) show variation among some VNTR with armadillo transfer. The allelic variation among different armadillos was not uniform and appeared to be unrelated to interspecies adaptation of the organisms or inherent environmental pressures within the hosts. Rather, it seemed to correspond with the degree of expansion in the armadillo host and an increased statistical probability for polymorphism. Similarly, some genotypic variation was noted among M. leprae strains in different armadillo tissues (Br4923 and NHDP34). However, no particular tissue type seemed more prone to polymorphism than others. Generally, the TA18 and GTA loci appeared more variable. No allelic variation among passage strains was observed at AT17 or GAA, except with the Thai-53 strain. In an 8-year period of nude mouse passages, Thai-53 showed three complete shifts in genotype, usually involving all the VNTR loci being examined.

The samples W260, W415, and W508 were derived from wild armadillos in Louisiana with naturally acquired leprosy. These samples were collected over a 6-year period from animals at locations separated by at least 60 miles. The VNTR genotypes of two of the samples were identical (Table 2), but the genotype varied at two VNTR loci for one wild armadillo. The genotypes of M. leprae obtained from a naturally infected monkey (SMML) and from M. leprae from wild armadillos also varied. The SMML genotype was identical to the VNTR genotype we determined for the LWM26 and NHDP34 strains, suggesting a likelihood of random associations for some VNTR genotypes.

Amplification from paraffin-embedded samples. Three human biopsies (used to seed strains NHDP55, NHDP10, and NHDP98) previously fixed and embedded in paraffin were extracted and amplified by PCR using primers for AT17, GTA, and GAA. DNA sequence data was obtained for all samples at all three loci, demonstrating the applicability of VNTR typing on archived material (Table 2). VNTR results from the original biopsy containing M. leprae NHDP10 and NHDP98 matched at all three loci compared to subsequent passage of the respective isolate in nude mice. Growth time in nude mice ranged from 54 months for NHDP10 to 135 months for NHDP98. M. leprae obtained from biopsy NHDP55 produced the same VNTR profile for GTA and GAA but showed a minor shift at AT17 from 14 to 13 copies when compared to NHDP55 harvested during its fourth passage (total of 40 months) in nude mice.

The strain Br4923 had been inoculated into four different armadillos, and each had several fresh or paraffin-embedded tissue samples available. Amplification was successful for all tissue types, except lung tissue (Table 2). Among the multiple animals and tissues tested, the VNTR genotypes were identical for all AT17 and GAA loci. Some minor variability between animals was noted for TA18 and GTA. Among the multiple tissue samples examined, most animals showed only a single genotype at all four VNTR loci. However, the lymph node and spleen sample tested from armadillo Arm29 showed a minor variation in copy number at the GTA locus (Table 2).

Genetic relationship of isolates. We examined the genetic relationship of all samples on the panel by using four VNTR loci. UPGMA cluster analysis generated a dendrogram which segregated samples according to their probable genetic similarity and resulted in several logical combinations (Fig. 1). Passage samples of individual strains appropriately clustered together even when allelic variation was noted in some of the passage samples. In this small panel, strains derived from the United States scattered throughout the dendrogram, and no typical regional genotypes could be deduced. Strains derived from wild armadillos in Louisiana tended to cluster with close relationship and appeared proximal to some strains that had been derived from human cases in the region. However, the strain derived from a mangabey monkey appeared to be quite dissimilar from the other animal-derived strains. It was not possible to clearly distinguish human-derived strains from animal-derived strains or deduce any regionally associated genotype within this small panel of samples.



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  		FIG. 1. Dendrogram resulting from UPGMA analysis to demonstrate the discriminatory power of VNTR genotypes in differentiating different M. leprae strains and passage samples in the experimental panel. Analysis is based on four VNTR loci with randomized data input. Coefficient shows similarity of the genotypes. Thai-53 results not included. Samples are listed by strain tested and further identified by suffix. Dx, patient biopsy sample; p, earliest nude mouse passage tested; pl, paraffin-embedded liver sample; ps, paraffin-embedded spleen sample.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found diversity among M. leprae strains in the numbers of short tandemly repeated DNA sequences found at several locations in the genome. The copy number of these sequence repeats varied markedly between M. leprae strains obtained from patients in geographically diverse regions and between strains derived from different animals or from different animal species. These VNTR showed a high degree of allelic diversity within our panel of strains (D > 0.7), but the genotype of most isolates tended to remain stable with interspecies transfer of bacilli between humans and animals and stable over long periods of infection and growth in animals. VNTR genotyping showed strong discriminatory power for differentiating M. leprae strains used in the laboratory, and the technique may have utility for use in community-based epidemiological studies or select clinical applications.

Genotyping of M. leprae presents several unique challenges. The organism cannot be cultivated on laboratory media and must be maintained with passage in animals. Even among highly susceptible animal hosts, multiplication of M. leprae is quite slow and requires months to years to manifest a suitable bacterial mass. With such cumbersome maintenance requirements, there are relatively few recognized M. leprae strains, and we know very little about the adaptive traits or potential genetic alterations of M. leprae during long-term culture. In addition, M. leprae samples derived directly from patient biopsies can have many limitations. They are often of poor quality, the bacilli typically show a low percent viability, they are frequently contaminated with other cultivable agents, and the donors may have an unknown treatment history. Furthermore, M. leprae obtained through propagation in animals or derived from patient biopsies cannot be selected as a single colony and may not represent a single clone of bacteria. Nonetheless, our study of allelic polymorphisms in the M. leprae genome found diversity at all four of the VNTR sites tested and relatively good stability of most genotypes following subsequent passage in laboratory animals.

Only limited genetic polymorphism has been noted previously among M. leprae, and it has not been possible to discriminate different isolates by genotype. Recently, Cole et al. (4) presented an in silico analysis of the M. leprae genome (3) and showed that about 2% of its chromosome consists of repetitive sequences. Such elements can be subject to changes in size and sequence through both random and evolutionary events and have been exploited as strain typing tools for other genetically homogeneous pathogens. A particular class of short repeats, VNTR, have been successfully used for genotyping Neisseria gonorrhoeae (15), Helicobacter pylori (11), Haemophilus influenzae (25), Bacillus anthracis (9), Yersinia pestis (1), and M. tuberculosis (20). Recently, polymorphism among M. leprae was reported at the triplet TTC repeat (18). However, the degree of diversity was extremely high. With 12 different TTC alleles found among only 34 patient isolates in a single Philippine city, the potential epidemiological utility of tracking this polymorphism was unclear (18).

VNTR can be found in intergenic and nonintergenic regions of genomic DNA and their specific function is unknown. Aside from their coding potential, VNTR may act as molecular switches in some microorganisms and may be involved in regulating transcription or translation (16, 20). Our in silico analysis of the M. leprae genome found 13 different simple VNTR sequences spread throughout the chromosome. Like others before us, we found binucleotide and trinucleotide repeats, as well as more complex elements; but no tetranucleotide repeats were revealed (4). Most earlier reports on repetitive sequence variability in M. leprae or other pathogens with highly conserved genomes have considered small bi- or trinucleotide repeats (9, 13, 14, 18). Therefore, we concentrated our study on analysis of four such loci, including the GAA (TTC) VNTR, for which polymorphism had been previously reported (18). Our observations for polymorphism at GAA are consistent with the earlier report and include three new GAA alleles (11-, 12-, and 16-repeat lengths). We also found diversity at three additional VNTR loci elsewhere on the chromosome and showed that VNTR genotype can effectively discriminate different M. leprae strains.

The M. leprae genome contains a number of degraded gene segments that do not appear to be capable of expressing functional proteins. The degree of conservation or control that an organism may exercise over these so-called pseudogenes is unknown, but several M. leprae VNTR are located on the chromosome in probable association with them or their nearby intergenic spaces (3, 4). Therefore, we examined the stability of the VNTR genotype after isolation of the bacilli from their diagnostic biopsy and subsequent serial passage in laboratory animals. Interspecies transfer and short-term passage of the different strains did not markedly influence the various alleles determined for most VNTR. However, the TA18 repeat associated with a putative membrane protein pseudogene showed marked variability among different armadillos inoculated with the same passage preparation. Least variation was with AT17, which is located within a conserved hypothetical protein pseudogene, and the GAA VNTR, which is found in an intergenic segment of the chromosome, suggesting that VNTR stability within noncoding regions is similar. Extended analysis of polymorphic DNA in other regions of the M. leprae chromosome, including authentic open reading frames, may help define evolutionary trends in M. leprae, as well as establishing appropriate markers for a robust strain typing system.

The most prominent allelic variation seen was with transfer of bacilli to armadillos. These experimental infections require approximately 2 years to manifest, and they result in a 10,000-fold expansion in the numbers of bacilli. A similar, and perhaps markedly larger, degree of expansion is likely to occur in human cases of lepromatous leprosy. Passaging M. leprae in nude mice usually results in only 100-fold increases in bacterial mass and does not seem to favor VNTR variation. A similar appearance of genotypic stability has been noted for variability in the IS6110 element of M. tuberculosis during short-term animal culture. Though later found to be highly polymorphic in humans, initial studies with IS6110 found no polymorphic variation during short-term culture of M. tuberculosis strains in guinea pigs. Therefore, animal models or experimental panels constructed to assess genotypic stability of bacterial pathogens should likely include long periods of infection and multiple passage intervals (8). Armadillos appear to be ideal hosts for this kind of study with M. leprae, and they will likely become a useful adjunct to field studies for these evaluations.

The relative stability of these VNTR alleles among most M. leprae strains appears to be similar to, to slightly less stable than, the VNTR markers studied in other mycobacteria. VNTR within the mycobacterial interspersed repetitive unit of BCG have been observed to remain unchanged over a 30-year period with hundreds of passages on laboratory media (21). Similarly, others have noted that mycobacterial interspersed repetitive unit VNTR among M. tuberculosis strains remain stable in serial passage over a 6-year period in the laboratory or in vivo for at least 18 months during outbreaks (12, 17). However, one particular M. leprae strain that we studied (Thai-53) showed a remarkable amount of variation in VNTR genotype even over relatively short periods.

During an 8-year span of nude mouse passages, the Thai-53 strain showed three complete shifts in VNTR genotype. Thai-53 is one of the oldest known M. leprae strains, since it was isolated more than 20 years ago and has been carried in continuous passage in nude mice since then. Although we could hypothesize that VNTR sequence changes might be more frequent with adaptation and long-term passage of M. leprae in mice, the genotype shifting that we observed occurred simultaneously across almost all the VNTR loci. Therefore, we find it more likely that this isolate contained a mixed population of bacilli expressing different alleles. A similar phenomenon is seen when genotyping clinical samples of other difficult-to-grow organisms, such as P. carinii, and the problem associated with mixed bacterial populations is a complication largely avoided when the bacterial population under study originates from a single colony or clone (2, 10). The possibility of infections occurring with mixed bacterial populations expressing multiple alleles, or of rapid genotype shifting among some M. leprae strains, highlights a potential pitfall for using the VNTR genotype to assess relapse or reinfection in individual patients, and this problem merits additional attention.

Cluster analysis of these VNTR alleles suggested a number of logical associations among strains in serial passage. The ability to link infections through bacterial genotype raises an intriguing possibility, that of using VNTR in community-based transmission studies. However, some VNTR appear to be hypervariable and may not be useful in either laboratory or field studies. Others can be flanked or composed of sequences that are problematic for reliable amplification. Certainly, little is yet known about regulation in the M. leprae genome or the influence of pseudogenes on VNTR mutation rates. Additional, well-controlled studies with select sets of patients or model animal groups will be needed to describe the nature and frequency of these polymorphisms.

The high degree of allelic diversity among M. leprae VNTR imparts good discriminatory power for differentiating laboratory strains. This application alone suggests that genotyping is likely to become an effective tool to help monitor for drift and evolutionary change within the M. leprae strains used in research. More advanced clinical and epidemiological applications will require a better understanding of the biological diversity of M. leprae in different environments and populations. In particular, confirmation of the concordance of VNTR genotyping for associating regional types of M. leprae or specific incident clusters of infection is needed. Successful application of these techniques in the community could lead to development of a robust typing system for M. leprae. Such development would clearly benefit our understanding of how this agent is transmitted and could ultimately lead to improved methods to effectively interrupt its transmission and reduce the spread of leprosy.


   ACKNOWLEDGMENTS
 
This study was supported in part by funds from the National Institute for Allergy and Infectious Disease, contract number Y1-AI-2646-01, and PAPES III of Fiocruz, the CNPq, FAPERJ, and the Damien Foundation.

We express our appreciation to Naoko Robbins, Roena Domingue, and Mike Deshotels of the NHDP for their expert technical assistance in this project.


   FOOTNOTES
 
* Corresponding author. Mailing address: NHDP at LSU, P.O. Box 25072, Baton Rouge, LA 70894. Phone: (225) 578-9848. Fax: (225) 578-9860. E-mail: rtruma1{at}lsu.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Adair, D. M., P. L. Worsham, K. K. Hill, A. M. Klevytska, P. J. Jackson, A. M. Friedlander, and P. Keim. 2000. Diversity in a variable-number tandem repeat from Yersinia pestis. J. Clin. Microbiol. 38:1516-1519.[Abstract/Free Full Text]
  2. Beard, C. B., J. L. Carter, S. P. Keely, L. Huang, N. J. Pieniazek, I. N. Moura, J. M. Roberts, A. W. Hightower, M. S. Bens, A. R. Freeman, S. Lee, J. R. Stringer, J. S. Duchin, C. del Rio, D. Rimland, R. P. Baughman, D. A. Levy, V. J. Dietz, P. Simon, and T. R. Navin. 2000. Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission. Emerg. Infect. Dis. 6:265-272.[Medline]
  3. Cole, S. T., K. Eiglmeier, J. Parkhill, K. D. James, N. R. Thomson, P. R. Wheeler, N. Honore, T. Garnier, C. Churcher, D. Harris, K. Mungall, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. M. Davies, K. Devlin, S. Duthoy, T. Feltwell, A. Fraser, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, C. Lacroix, J. Maclean, S. Moule, L. Murphy, K. Oliver, M. A. Quail, M. A. Rajandream, K. M. Rutherford, S. Rutter, K. Seeger, S. Simon, M. Simmonds, J. Skelton, R. Squares, S. Squares, K. Stevens, K. Taylor, S. Whitehead, J. R. Woodward, and B. G. Barrell. 2001. Massive gene decay in the leprosy bacillus. Nature 409:1007-1011.[CrossRef][Medline]
  4. Cole, S. T., P. Supply, and N. Honore. 2001. Repetitive sequences in Mycobacterium leprae and their impact on genome plasticity. Lepr. Rev. 72:449-461.[Medline]
  5. de Wit, M. Y., and P. R. Klatser. 1994. Mycobacterium leprae isolates from different sources have identical sequences of the spacer region between the 16S and 23S ribosomal RNA genes. Microbiology 140:1983-1987.[Abstract]
  6. Fine, P. E. M. 1982. Leprosy: the epidemiology of a slow bacterium. Epidemiol. Rev. 4:161-187.[Free Full Text]
  7. Fsihi, H., and S. T. Cole. 1995. The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP-dependent transport systems and a novel polA locus associated with genomic variability. Mol. Microbiol. 16:909-919.[CrossRef][Medline]
  8. Hermans, P. W., D. van Soolingen, J. W. Dale, A. R. Schuitema, R. A. McAdam, D. Catty, and J. D. van Embden. 1990. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J. Clin. Microbiol. 28:2051-2058.[Abstract/Free Full Text]
  9. Keim, P., L. B. Price, A. M. Klevytska, K. L. Smith, J. M. Schupp, R. Okinaka, P. J. Jackson, and M. E. Hugh-Jones. 2000. Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J. Bacteriol. 182:2928-2936.[Abstract/Free Full Text]
  10. Latouche, S., E. Ortona, E. Mazars, P. Margutti, E. Tamburrini, A. Siracusano, K. Guyot, M. Nigou, and P. Roux. 1997. Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions. J. Clin. Microbiol. 35:383-387.[Abstract]
  11. Marshall, D. G., D. C. Coleman, D. J. Sullivan, H. Xia, C. A. O'Morain, and C. J. Smyth. 1996. Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences. J. Appl. Bacteriol. 81:509-517.[Medline]
  12. Mathema, B., P. J. Bifani, J. Driscoll, L. Steinlein, N. Kurepina, S. L. Moghazeh, E. Shashkina, S. A. Marras, S. Campbell, B. Mangura, K. Shilkret, J. T. Crawford, R. Frothingham, and B. N. Kreiswirth. 2002. Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster. J. Infect. Dis. 185:641-649.[CrossRef][Medline]
  13. Matsuoka, M., S. Maeda, M. Kai, N. Nakata, G. T. Chae, T. P. Gillis, K. Kobayashi, S. Izumi, and Y. Kashiwabara. 2000. Mycobacterium leprae typing by genomic diversity and global distribution of genotypes. Int. J. Lepr. Other Mycobact. Dis. 68:121-128.[Medline]
  14. Matsuoka, M., L. Zhang, T. Budiawan, K. Saeki, and S. Izumi. 2004. Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission. J. Clin. Microbiol. 42:741-745.[Abstract/Free Full Text]
  15. Poh, C. L., V. Ramachandran, and J. W. Tapsall. 1996. Genetic diversity of Neisseria gonorrhoeae IB-2 and IB-6 isolates revealed by whole-cell repetitive element sequence-based PCR. J. Clin. Microbiol. 34:292-295.[Abstract]
  16. Renders, N., L. Licciardello, C. IJsseldijk, M. Sijmons, L. van Alphen, H. Verbrugh, and A. van Belkum. 1999. Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonization of cystic fibrosis patients. FEMS Microbiol. Lett. 173:95-102.[CrossRef][Medline]
  17. Savine, E., R. M. Warren, G. D. van der Spuy, N. Beyers, P. D. van Helden, C. Locht, and P. Supply. 2002. Stability of variable-number tandem repeats of mycobacterial interspersed repetitive units from 12 loci in serial isolates of Mycobacterium tuberculosis. J. Clin. Microbiol. 40:4561-4566.[Abstract/Free Full Text]
  18. Shin, Y. C., H. Lee, H. Lee, G. P. Walsh, J. D. Kim, and S. N. Cho. 2000. Variable numbers of TTC repeats in Mycobacterium leprae DNA from leprosy patients and use in strain differentiation. J. Clin. Microbiol. 38:4535-4538.[Abstract/Free Full Text]
  19. Smith, K. L., V. DeVos, H. Bryden, L. B. Price, M. E. Hugh-Jones, and P. Keim. 2000. Bacillus anthracis diversity in Kruger National Park. J. Clin. Microbiol. 38:3780-3784.[Abstract/Free Full Text]
  20. Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht. 2001. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.[Abstract/Free Full Text]
  21. Supply, P., E. Mazars, S. Lesjean, V. Vincent, B. Gicquel, and C. Locht. 2000. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36:762-771.[CrossRef][Medline]
  22. Truman, R. W., and J. L. Krahenbuhl. 2001. Viable M. leprae as a research reagent. Int. J. Lepr. Other Mycobact. Dis. 69:1-12.[Medline]
  23. Truman, R. W., J. A. Kumaresan, C. M. McDonough, and C. K. Job. 1991. Seasonal and spatial trends in the detectability of leprosy in nine-banded armadillos. Epidemiol. Infect. 106:549-560.[Medline]
  24. Truman, R. W., and R. M. Sanchez. 1993. Armadillos: models for leprosy. Lab. Anim. 22:28-32.
  25. van Belkum, A., S. Scherer, W. van Leeuwen, D. Willemse, L. van Alphen, and H. Verbrugh. 1997. Variable number of tandem repeats in clinical strains of Haemophilus influenzae. Infect. Immun. 65:5017-5027.[Abstract]
  26. Williams, D. L., T. P. Gillis, and F. Portaels. 1990. Geographically distinct isolates of Mycobacterium leprae exhibit no genotypic diversity by restriction fragment-length polymorphism analysis. Mol. Microbiol. 4:1653-1659.[CrossRef][Medline]
  27. World Health Organization. 2000. Leprosy global situation. Wkly. Epidemiol. Rec. 28:226-231.

Journal of Clinical Microbiology, June 2004, p. 2558-2565, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2558-2565.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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