1107 7. Bloch AB, Cauthen GM, Onorato IM, et al. Nationwide survey of drug- 11. Dwyer B, Jackson K, Raios K, Sievers A, Wilshire E, Ross B. DNA resistant tuberculosis in the United States. JAMA 1994;271:665 71. restriction fragment analysis to define an extended cluster of tuberculosis 8. Small PM, Hopewell PC, Singh SP, et al. The epidemiology of tuberculosis in homeless men and their associates. J Infect Dis 1993;167:490 4. in San Francisco. A population-based study using conventional and 12. Bay Area Rapid Transit. BART trips for fiscal year 1996. Oakland, CA: molecular methods. N Engl J Med 1994;330:1703 9. BART, 1996. 9. Alland D, Kalkut GE, Moss AR, et al. Transmission of tuberculosis in 13. Caltrans. 1995 traffic volumes. Oakland, CA: Caltrans, 1995. New York City. An analysis by DNA fingerprinting and conventional 14. van Embden JD, Cave MD, Crawford JT, et al. Strain identification of epidemiologic methods. N Engl J Med 1994;330:1710 6. Mycobacterium tuberculosis by DNA fingerprinting: recommendations 10. Daley CL, Small PM, Schecter GF, et al. An outbreak of tuberculosis for a standardized methodology. J Clin Microbiol 1993;31:406 9. with accelerated progression among persons infected with the human 15. Chaves F, Yang Z, el Hajj H, et al. Usefulness of the secondary probe immunodeficiency virus. An analysis using restriction-fragment-length ptbn12 in DNA fingerprinting of Mycobacterium tuberculosis. J Clin polymorphisms. N Engl J Med 1992;326:231 5. Microbiol 1996;34:1118 23. Stability of Mycobacterium tuberculosis DNA Genotypes Robert W. Yeh, Alfredo Ponce de Leon,* Cristina B. Agasino, Judith A. Hahn, Charles L. Daley, Philip C. Hopewell, and Peter M. Small Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford; Department of Epidemiology and Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Francisco To assess genotype stability in Mycobacterium tuberculosis, DNA genotypes were compared in sequential isolates from 49 patients who had sputum cultures separated by at least 90 days that grew M. tuberculosis. By use of IS6110 and the polymorphic GC rich sequence (PGRS) as markers, it was found that paired isolates from 14 (29%) of 49 patients showed changes in their DNA genotypes between isolates (12 in IS6110 genotypes and 2 in PGRS genotypes). Changed IS6110 genotypes were confined to strains with 8 14 bands and were not related to the bacterial drug susceptibility, the patients human immunodeficiency virus serostatus, or adherence to therapy. Although this rate of change complicates the interpretation of molecular epidemiologic studies, it can be exploited to gain additional insight into disease transmission. Furthermore, IS6110-related mutations may be a major source of genetic plasticity in M. tuberculosis and provide insights into the organism s evolution and virulence. Restriction fragment length polymorphism (RFLP) analysis infected with strains of M. tuberculosis that have identical based on the insertion sequence IS6110 has been widely used genotypes ( fingerprints ) are epidemiologically linked, to study the epidemiology of Mycobacterium tuberculosis [1, whereas those with different genotypes are unrelated. This as- 2]. This application is based on the assumption that persons sumption relies on two seemingly paradoxical assertions: that the DNA genotype of a given strain remains constant and that changes in genotypes over time generate considerable genotypic diversity within a population. Received 24 July 1997; revised 29 October 1997. If IS6110-based genotypes change rapidly, it would obscure Presented in part: Molecular Epidemiology and Evolutionary Genetics of epidemiologic links and underestimate transmission. To ac- Pathogenic Microorganisms, Montpellier, France, 26 28 May 1997; Mocommodate this possibility, some studies have considered cases lecular Epidemiology and Control of Tuberculosis meeting, Cordoba, Spain, 15 16 June 1997. with similar but nonidentical patterns as epidemiologically re- Informed consent was obtained from patients or their parents or guardians, lated [2]. On the other hand, if genotypes change too slowly, and human experimentation guidelines of the US Department of Health and then IS6110-based RFLP analysis would link cases that are Human Services and those of Stanford University and the University of Califoronly distantly related, overestimating transmission. For this nia, San Francisco, were followed in the conduct of clinical research. Grant support: NIH (AI-34238, AI-35969). reason, some studies have required that cases match with an Reprints or correspondence: Dr. Peter M. Small, Division of Infectious additional genetic marker to be considered epidemiologically Diseases and Geographic Medicine, S156, Stanford University School of Medilinked [3 5]. The most commonly used alternative marker is cine, 300 Pasteur Dr., Stanford, CA 94305 (peter@molepi.stanford.edu). * Present affiliation: Laboratorio de MicrobiologıB a ClıB nica, Departamento the polymorphic GC rich sequence (PGRS). de InfectologıB a, Instituto Nacional de la Nutrición Salvador Zubirán, Mexico While previous studies have quantified genotype instability City, Mexico. in small numbers of uncharacterized M. tuberculosis isolates The Journal of Infectious Diseases 1998;177:1107 11 [6 8], the bacterial and patient characteristics that affect 1998 by The University of Chicago. All rights reserved. 0022 1899/98/7704 0042$02.00 IS6110 genotypic instability are unknown, and thus the poten-
1108 JID 1998;177 (April) tial impact of sampling biases unclear, and it is not certain that was significantly greater than the frequency of changed PGRS these results can be generalized to population-based studies. patterns (P Å.019). Furthermore, there has been no assessment of the relative stabil- Of the 12 changed IS6110 right-side genotypes, 11 had differences ity of IS6110 and PGRS-based genotypes. To gain a better of only 1 band between the first and second patterns. understanding of genotypic instability of M. tuberculosis, we In some instances, quantitative examination of band intensities genotyped sequential samples from 49 patients from whom M. suggests that there was a mixed population, with only some tuberculosis was repeatedly isolated. bacilli having the new genotype. These included 5 strains that showed an extra band in the second genotype, 4 strains that lost 1 band in the second genotype, and 2 strains in which 1 Materials and Methods band shifted position (figure 1). The genotypes of the isolates from the 12th patient gained 2 bands and had 2 additional Between 1991 and 1995, 1335 cases of active tuberculosis were molecular weight band shifts. The PGRS genotype for this reported in San Francisco. The population identified for the study patient was one of the two PGRS patterns that changed. Of consisted of patients from whom 2 cultures of M. tuberculosis the patients with changes in IS6110 right-sided genotypes, 7 were obtained separated by ú90 days. There were 132 eligible had concomitant changes and 5 had no changes in the left-side patients. Eighty-three of them had only 1 viable isolate available for analysis and thus were excluded from the study. Bacterial genotypes. One additional patient only had changes in the left- isolates from the 49 remaining patients for whom 2 cultures side genotype. were available were examined. To assess the possibility of sampatterns Statistical analyses showed no association between changed pling bias, the 49 subjects included in the study were compared and increasing time interval between which isolates with the 83 excluded patients with respect to the number of bands were taken in the 49 patients (figure 2). All 12 of the strains that hybridized to IS6110, the patients human immunodeficiency with changed IS6110 right-sided patterns could be charactervirus (HIV) serostatus, age, sex, and ethnicity. ized as having an intermediate number of bands (8 14) (figure Each pair of samples for the 49 patients was run on a 20-cm 2). Comparison of groups of isolates with low (1 7 bands), agarose gel side by side to produce the most accurate comparison intermediate (8 14 bands), and high numbers of bands (15 between DNA genotypes. Standard IS6110-based RFLP analysis 21 bands) revealed a statistical association between middle was done with PvuII digestion of genomic DNA and hybridization band number and changed patterns (P Å.026). to bp 631 875, located between the element s PvuII restriction site and the 5 terminus (referred to as the right side), and hybridated with a changed genotype. In addition, no statistical associ- Altered drug susceptibility of the organism was not associization to bp 30 450, located between the PvuII restriction site and the 3 terminus (the left side) [9]. PGRS-based genotyping ations were found between changed patterns and HIV positiv- entailed SmaI digestion and probing for a 32-bp oligonucleotide ity, nonadherence to therapy, or drug resistance of the isolate. (5 -ATCGGCAACGGCGGCAACGGCGGCAACGGCGG-3 ) After matching the RFLP patterns of the 49 patients to the [3]. Visual comparisons between the two RFLP patterns from each existing San Francisco database, 26 of the patients (53%) were patient were done independently by three readers. DNA genotypes found to be involved in clusters between 1991 and 1995. Of of 2 isolates obtained within 5 days of each other from 51 other the 12 patients with changed IS6110 right-side patterns, there patients were analyzed to determine if genotype changes were were four instances in which the initial and final pattern each simply a consequence of repeated sampling. matched a different cluster in San Francisco. Two other patients Two-tailed Fisher s exact tests were done to determine if changwere found to be matched to clusters by only their first RFLP ing DNA genotypes were related to HIV seropositivity, extrapulpatterns. monary tuberculosis disease, drug-resistant isolates, and nonadherence to treatment regimen. Of the 51 patients who had samples separated by 5 days, only 1 pair of isolates had a change in the IS6110 right-side pattern. No changes were seen in PGRS patterns. Results Characteristics of the 49 patients with positive cultures separated Discussion by 90 days were not statistically different from those This study provides a comprehensive population-based anal- of the 83 excluded patients among all variables examined. ysis of the genotype stability of M. tuberculosis. The finding Patient isolates were separated by a range of 90 days to almost that 14 (29%) of 49 patients whose cultures spanned at least 3 years. 90 days had DNA genotypes that changed is both surprising and Of the 49 patients, 14 (29%) had patterns that changed be- important. Previous studies examining sequential genotypes of tween the first and second isolates by at least one technique. M. tuberculosis isolates from persons with repeatedly positive There were 12 changes in IS6110 right side, 8 in IS6110 left cultures have not found this degree of instability. Cave et al. side, and 2 in PGRS genotypes. In 11 cases, changes in IS6110 [10] examined sequential IS6110 genotypes of M. tuberculosis right-side patterns were not accompanied by changes in PGRS in 18 patients and found that genotypes were identical for 17 patterns. The frequency of changed IS6110 right-side patterns of them. Other studies have examined genotypes in a small
1109 Figure 1. Examples of changed M. tuberculosis DNA genotypes. Lanes contain genomic bacterial DNA from paired isolates separated by 90 days taken from each of 4 patients (A D), digested with PvuII and probed for IS6110 (left) or with SmaI and probed for polymorphic GC rich sequence (PGRS; right). Although all 4 patients showed changed patterns for IS6110, only those from isolates of patient D showed concomitant change in PGRS genotype. that requiring an identical match may in fact underestimate the frequency of epidemiologic links among patients. It is possible that such rapid changes in genotypes could result in a convergence of patterns and inappropriately link unrelated cases. However, this phenomenon has never been observed. In sum, the data presented here suggest that it may be appropriate to include 1-band differences in IS6110 genotype defined clusters. Isolation of bacteria with different IS6110 genotypes could be a consequence of patients being simultaneously infected with bacteria with a diversity of similar but nonidentical pat- terns and that our results are simply an artifact of repeated sampling. Alternatively, it is possible that cultures obtained from different sites in the patient s body could contain different genotypes of bacilli. Both of these possibilities were not sup- ported by the results of this study, in which 98% (50/51) of the patients with isolates spanning õ5 days had identical geno- types, more than half of whom had isolates from different body sites. An additional conclusion of the study is that PGRS has a significantly lower rate of change than IS6110 (2/49 for PGRS vs. 12/49 for IS6110; P Å.019). IS6110 appears to provide number of patients who persistently excreted M. tuberculosis; none found changes in IS6110 genotypes [6 8]. Those studies, however, examined substantially fewer patients from a non population-based sample. The demographic similarity between the study sample and all serial secretors in San Francisco led us to believe that the 29% proportion of genotype instability can be generalized to the population-based molecular epidemiologic studies, such as those conducted in San Francisco. The primary conclusion that can be drawn from these results is that DNA genotypes of M. tuberculosis change at a relatively rapid rate. It is extremely unlikely that our observations were the result of exogenous reinfection with a different strain, since the changes were minor and occurred in a patient population with a relatively low incidence of tuberculosis. The high rate of change suggests that strains with identical DNA genotypes are likely to be epidemiologically linked and supports the use of RFLP analysis for tracking transmission. It has been postulated that IS6110-based RFLP analysis overestimates recent transmission by grouping distantly related strains with genotypes that have remained identical for large amounts time [4]. However, the degree of instability demonstrated here suggests
1110 JID 1998;177 (April) Figure 2. Changes in IS6110-based DNA genotypes as function of time interval between isolates (A) and number of IS6110 hybridizing bands (B). Solid regions of bars represent patterns that changed; open regions represent unchanged patterns. these strains have copies in relatively stable positions in the genome and thus have not spawned progeny with greater numbers of copies. If specific isolates have intrinsically high (or low) rates of mutations, it may be difficult to deduce general rules governing genotype stability. It is also plausible that increasing numbers of IS6110 impose a burden on the strain and that transposition in strains that already have a high number of copies frequently results in nonviable organisms. An understanding of the dynamics of IS6110 transposition in M. tuberculosis may provide insight into the mechanism by which this organism generates genomic vari- ability. For DNA genotypes to change, a mutation or transposition must occur in the genome of at least one mycobacterial cell, and that cell must progressively outcompete the remaining un- changed cells within the host. It is interesting to postulate that the 12 new IS6110 patterns reflected the emergence of more fit strains within the patients. Tracking these strains as they are transmitted between patients in San Francisco over coming years to see if they differ in fitness may provide valuable information concerning the virulence of M. tuberculosis. greater resolution in detecting genotypic differences between strains of M. tuberculosis and, thus, should be considered a more stringent test for epidemiologic links between cases. It is important to note that PGRS and IS6110 instability are largely independent of one another, explaining previous findings that IS6110 clusters can sometimes be subdivided by PGRS. The data also support the potential for the generation of phylogenetic trees describing degrees of relatedness among strains of M. tuberculosis on the basis of similarities in RFLP patterns. If 1-band differences can be thought of as related, it follows that 2-band differences can be considered related, albeit slightly less so, and so on. Phylogenetic trees may provide valuable information about the evolution of M. tuberculosis within a population and the migration of strains between distinct geographic regions [11]. From a microbiologic standpoint, it is interesting that all of the changed patterns occurred in strains with an intermediate number of bands (8 14). Lack of transposition in strains with only a few copies of IS6110 may be a consequence of there simply being fewer opportunities for transposition. Alternatively, it may be that
1111 Acknowledgments 4. Warren R, Richardson M, Sampson S, et al. Genotyping of Mycobacterium tuberculosis with additional markers enhances accuracy in epidemiologic studies. J Clin Microbiol 1996;34:2219 24. We are indebted to the personnel of the San Francisco Depart- 5. Ross BC, Raios K, Jackson K, Bwyer B. Molecular cloning of highly ment of Public Health Division of Tuberculosis Control for contin- repeated DNA element from Mycobacterium tuberculosis and its use ued support in molecular epidemiologic studies; to H. Salamon, as an epidemiological tool. J Clin Microbiol 1992;30:942 6. M. Javanillo, M. Farid-Moamar for assistance in the implementapolymorphisme de longueur des fragments de restriction (RFLP) de 6. Lemaitre N, Sougakoff W, Truffot C, Grosset J, Jarlier V. Analyse du tion of the study design and analysis of the data; and to M. Feldman souches de Mycobacterium tuberculosis isolees de malades ayant fait and his group for consultation on issues pertaining to population plusieurs episodes de tuberculose. Pathol Biol (Paris) 1996;44:452 5. biology. 7. Strässle A, Putnik J, Weber R, Fehr-Merhof A, Wüst J, Pfyffer GE. Molecular epidemiology of Mycobacterium tuberculosis strains isolated from patients in a human immunodeficiency virus cohort in Switzerland. J Clin Microbiol 1997;35:374 8. 8. van Soolingen D, de Haas PE, Hermans PW, Groenen PM, van Embden References JD. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculo- 1. Small PM, Hopewell PC, Singh SP, et al. The epidemiology of tuberculosis sis. J Clin Microbiol 1993;31:1987 95. in San Francisco. A population-based study using conventional and 9. van Embden JD, Cave MD, Crawford JT, et al. Strain identification of molecular methods. N Engl J Med 1994;330:1703 9. Mycobacterium tuberculosis by DNA fingerprinting: recommendations 2. Yang ZH, de Haas PE, van Soolingen D, van Embden JD, Andersen AB. for a standardized methodology. J Clin Microbiol 1993;31:406 9. Restriction fragment length polymorphism analysis of Mycobacterium 10. Cave MD, Eisenach KD, Templeton G, et al. Stability of DNA genotype tuberculosis strains isolated from Greenland during 1992: evidence of pattern produced with IS6110 in strains of Mycobacterium tuberculosis. tuberculosis transmission between Greenland and Denmark. J Clin Mi- J Clin Microbiol 1994;32:262 6. crobiol 1994;32:3018 25. 11. Hermans PWM, Messad F, Guesbrexabher H, et al. Analysis of the popula- 3. Chaves F, Yang Z, el Hajj H, et al. Usefulness of the secondary probe tion structure of Mycobacterium tuberculosis in Ethiopia, Tunisia, and ptbn12 in DNA fingerprinting of Mycobacterium tuberculosis. J Clin the Netherlands: usefulness of DNA typing for global tuberculosis epi- Microbiol 1996;34:1118 23. demiology. J Infect Dis 1995;171:1504 13.