Polymorphic GC-rich Repetitive Sequence Research Articles

Fibronectin binding protein and Ca2+ play an access key role to mediate pathogenesis in Mycobacterium tuberculosis: An overview.

The anomalous distribution of adhesive proteins throughout on the cell surface of the Mycobacterium tuberculosis H37 Rv and their contribution in cell surface adhesion and host-pathogen interaction remain elusive. The completion of M. tuberculosis H37 Rv genome sequence analysis gives some interesting information about polymorphic GC-rich repetitive sequence (PGRS) subfamily of M. tuberculosis that encodes fibronectin binding proteins (FnBP), which have been extensively studied, but the function in the pathogenesis of most of these proteins remains unknown and unclear. This review addresses the M. tuberculosis entry mechanism in the host cell. In particular, an effort has been made to focus on several aspects, (a) association of FnBP encodes by PE_PGRS protein family of M. tuberculosis during host-pathogen interactions. (b) Effect of calcium ions in and outside of the host cell is overriding to maintenance of calcium trafficking in phagocytosis. Furthermore, FnBP may be a potential source of antigenic variation that participating in evoking immune response. M. tuberculosis entry mechanism does not have a major influence alone, involvement of calcium ions, perhaps shed light on host-pathogen interaction relationship, and could open up new avenues for development of novel drug by targeting M. tuberculosis FnBP and blockade of selective adhesions could be useful for therapeutics.

Comparative Study of IS 6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems.

Comparison of Restriction Fragment Length Polymorphism with the Polymorphic Guanine-Cytosine-Rich Sequence and Spoligotyping for Differentiation of Mycobacterium tuberculosis Isolates with Five or Fewer Copies of IS 6110

The use of IS6110 as a marker for molecular epidemiological studies is limited when a Mycobacterium tuberculosis isolate has five or fewer copies of IS6110. Restriction fragment length polymorphism analysis with a highly polymorphic GC-rich repetitive sequence located in the plasmid pTBN12 (PGRS RFLP) and spoligotyping (based on the polymorphism of the DR region) are two frequently used secondary typing methods. The aim of this study was to compare the performance of these two methods in a population-based study in San Francisco. We included all patients with culture-positive tuberculosis from 1999 to 2007 with IS6110 RFLP results presenting five or fewer bands. PGRS RFLP and spoligotyping were performed using standardized methods. We determined the concordance between the two methods regarding cluster status and the risk factors for an isolate to be in a cluster with each of the methods. Our data indicate that both methods had similar discriminatory power and that the risk factors associated with clustering by either method were the same. Although the cluster/unique status was concordant in 84% of the isolates, patients were clustered differently depending on the method. Therefore, the methods are not interchangeable, and the same method should be used for longitudinal studies.

IS<I>6110</I> DNA指紋法少コピー数結核菌による精神病院での集団感染事例

We report a case of 4 patients with pulmonary tuberculosis in a mental hospital located in Yamanashi, Japan, between 2000 and 2004 in which 3 isolates of Mycobacterium tuberculosis were analyzed using IS6110 restriction fragment length polymorphism (RFLP). DNA fingerprinting showed them to be identical and the copy number to be two. Additional RFLP using (CGG)5 fingerprinting yielded the same result and a copy number of 15, suggesting a M. tuberculosis outbreak. (CGG)5 analysis thus proved useful in supplementary typing method like polymorphic G-C rich repetitive sequence (PGRS) or spoligotyping.

Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair

BackgroundThe highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability.ResultsComparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of "M. canettii" and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains.ConclusionThis study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.

A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis.

We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35% (95% confidence interval, 28%-41%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide/nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes.

Molecular identification of streptomycin monoresistant Mycobacterium tuberculosis related to multidrug-resistant W strain.

A distinct branch of the Mycobacterium tuberculosis W phylogenetic lineage (W14 group) has been identified and characterized by various genotyping techniques. The W14 group comprises three strain variants: W14, W23, and W26, which accounted for 26 clinical isolates from the New York City metropolitan area. The W14 group shares a unique IS6110 hybridizing banding motif as well as distinct polymorphic GC-rich repetitive sequence and variable number tandem repeat patterns. All W14 group members have high levels of streptomycin resistance. When the streptomycin resistance rpsL target gene was sequenced, all members of this strain family had an identical mutation in codon 43. Patients infected with the W14 group were primarily of non- Hispanic black origin (77%); all were US-born. Including HIV positivity, 84% of the patients had at least one known risk factor for tuberculosis.

False Molecular Clusters due to Nonrandom Association of IS 6110 with Mycobacterium tuberculosis

We sought to determine whether nonrandom association of IS6110 with Mycobacterium tuberculosis could result in false-positive clustering in unselected collections of isolates. We typed 196 strains of M. tuberculosis from an unselected community-based study in northern Tanzania by IS6110 and polymorphic GC-rich repetitive-sequence (PGRS) methodologies. The strains were analyzed by Gelcompar computer software. Analysis of 13 out of 25 groups showed that isolates with identical IS6110 and PGRS patterns were likely to be the same strain. Some IS6110 groups containing strains with identical PGRS patterns had similar IS6110 patterns that differed only by movement of the element. Isolates assigned to a single group (i.e., group 11) on the basis of sharing an identical IS6110 fingerprint pattern did not share identical PGRS fingerprint patterns. Six out of the nine bands in these isolates were in hot-spot locations, as previously defined. This indicates that nonrandom association may result in false-positive clustering in unselected community-based studies. Only strains with identical PGRS and IS6110 patterns are likely to be recently transmitted.

Identification of a W variant outbreak of Mycobacterium tuberculosis via population-based molecular epidemiology.

Typing of Mycobacterium tuberculosis could provide a more sensitive means of identifying outbreaks than use of conventional surveillance techniques alone. Variants of the New York City W strain of M tuberculosis were identified in New Jersey. To describe the spread of the W family of M tuberculosis strains in New Jersey identified by molecular typing and surveillance data. Population-based cross-sectional study. All incident culture-positive tuberculosis cases reported in New Jersey from January 1996 to September 1998, for which the W family was defined by insertion sequence (IS) IS6110 DNA fingerprinting, polymorphic GC-rich repetitive sequence (PGRS) typing, spacer oligotyping (spoligotyping), and variable number tandem repeat (VNTR) analysis. Identification and characterization of W family clones supplemented by surveillance data. Isolates from 1207 cases were analyzed, of which 68 isolates (6%) belonged to the W family based on IS6110 and spoligotype hybridization patterns. The IS6110 hybridization patterns or fingerprints revealed that43 patients (designated group A) shared a unique banding motif not present in other W family isolates. Strains collected from the remaining 25 patients (designated group B), while related to W, displayed a variety of IS6110 patterns and did not share this motif. The PGRS and VNTR typing confirmed the division of the W family into groups A and B and again showed group A strains to be closely related and group B strains to be more diverse. The demographic characteristics of individuals from groups A and B were specific and defined. Group A patients were more likely than group B patients to be US born (91 % vs 24%, P<.001), black (76% vs 16%, P<.001), human immunodeficiency virus positive (40% vs 0%, P = .007), and residents of urban northeast New Jersey counties (P<.001). Patients with group B strains were primarily non-US born, of Asian descent, and more dispersed throughout New Jersey. No outbreak had been detected using conventional surveillance alone. The implementation of multiple molecular techniques in conjunction with surveillance data enabled us to identify a previously undetected outbreak in a defined geographical setting. The outbreak isolates comprise members of a distinct branch of the W family phylogenetic lineage. The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment, traditional surveillance outbreak investigations.

DNA typing of Mycobacterium bovis strains from the Castlepoint area of the Wairarapa

Aim. To investigate isolates of Mycobacterium bovis from the Castlepoint area of the Wairarapa using three different methods of DNA typing. Methods. Isolates of M. bovis, obtained from animals in the Castlepoint area between 1982–l998, were characterised by restriction endonuclease analysis. An isolate representing each restriction type was characterised by two newer DNA typing methods based on the polymorphic GC-rich repetitive sequence (PGRS) and spoligotyping. Results. Over 300 isolates were distinguished into 26 restriction types.The 24 available restriction types were differentiated into 11 PGRS types and 7 spoligotypes. The three most common restriction types had the same PGRS type and the same spoligotype. Conclusions. The relatively large number of restriction types found, indicated that restriction endonuclease analysis was well suited for detailed epidemiological studies at Castlepoint. Spoligotyping was less discriminatory than PGRS typing but both methods could be used to group isolates with different restriction types.

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America.

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.

IS6110 transposition and evolutionary scenario of the direct repeat locus in a group of closely related Mycobacterium tuberculosis strains.

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the Mycobacterium tuberculosis genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS6110, and IS1081. These, especially IS6110 and the DR locus, have been widely used as genetic markers to differentiate M. tuberculosis isolates and will continue to be so used, due to the conserved nature of the genome of M. tuberculosis. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of M. tuberculosis. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS6110 transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among M. tuberculosis strains. Our results suggest that IS6110 restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS6110, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS6110 copy in the DR locus was noted, and the mechanisms of this loss are discussed.

Restriction fragment length polymorphism analysis of Mycobacterium bovis isolates from captive and free-ranging animals.

Mycobacterium bovis isolates from cattle, captive elk, and free-ranging mule deer and coyotes were examined by restriction fragment length polymorphism (RFLP) analysis. DNA extracted from each isolate was digested with restriction endonucleases AluI and PvuII. DNA probes used for Southern hybridizations were a 37-base oligonucleotide and a 123-base-pair sequence specific for the insertion sequence IS6110 and a plasmid, pTBN12, which contains a polymorphic GC-rich repetitive sequence present in several species of mycobacteria. Generally, M. bovis isolates originating from a single herd of either cattle or captive elk had identical RFLP patterns, whereas isolates from unrelated sources had distinct patterns. The RFLP patterns for M. bovis isolates from free-ranging mule deer and coyotes were identical to patterns observed for isolates from a captive elk herd that was located in the area where the free-ranging animals were found. These results indicate that the captive elk herd may have been the source of M. bovis that infected the free-ranging animals. Results of this study show that RFLP analysis is a useful tool for differentiation of M. bovis isolates and for molecular epidemiology studies to determine possible sources of infection in outbreaks of tuberculosis in animals.

Molecular epidemiology of Mycobacterium tuberculosis strains isolated from patients in a human immunodeficiency virus cohort in Switzerland.

From 1989 to 1995, 46 patients infected with the human immunodeficiency virus were diagnosed with tuberculosis at the University Hospital in Zurich. Using the IS6110 insertion sequence as a genetic marker, restriction fragment length polymorphism analyses were done for 52 Mycobacterium tuberculosis isolates. We have found a large degree of IS6110 polymorphism, ranging from 1 to 16 copies. For isolates from patients from whom multiple isolates had been available, the IS6110 pattern remained virtually stable over a period of up to 4 years, as well as during emerging drug resistance. In none of the cases was a reinfection of a patient with another strain detected. For isolates from 10 patients we detected identical patterns which could be associated with four clusters. In one of these, the strains exhibited a low IS6110 copy number (four bands), and the strains were further analyzed by hybridizing with (i) the polymorphic GC-rich repetitive sequence (PGRS) and (ii) the 36-bp direct-repeat (DR) cluster sequence. One of these isolates had a different pattern with the PGRS as well as with the DR sequence and could therefore be safely excluded from that cluster. These findings point to the importance of applying more than one genetic criterion in the molecular biological study of strain relatedness.

Evaluation of method for secondary DNA typing of Mycobacterium tuberculosis with pTBN12 in epidemiologic study of tuberculosis.

Secondary fingerprinting of Mycobacterium tuberculosis DNA with a probe containing the polymorphic GC-rich repetitive sequence present in pTBN12 has been found to have greater discriminating power than does fingerprinting with the insertion sequence IS6110 for strains carrying few copies of IS6110. To validate the use of pTBN12 fingerprinting in the molecular epidemiology of tuberculosis, M. tuberculosis isolates from 67 patients in five states in the United States and in Spain were fingerprinted with both IS6110 and pTBN12. Epidemiologic links among the 67 patients were evaluated by patient interview and/or review of medical records. The 67 isolates had 5 IS6110 fingerprint patterns with two to five copies of IS6110 and 18 pTBN12 patterns, of which 10 were shared by more than 1 isolate. Epidemiologic links are consistently found among patients whose isolates had identical pTBN12 patterns, whereas no links were found among patients whose isolates had unique pTBN12 patterns. This suggests that pTBN12 fingerprinting is a useful tool to identify epidemiologically linked tuberculosis patients whose isolates have identical IS6110 fingerprints containing fewer than six fragments.

Polymerase chain reaction typing of nonviable Mycobacterium tuberculosis isolates

Repetitive element polymerase chain reaction (PCR) typing was applied to two Mycobacterium tuberculosis isolates for which both viable and nonviable cultures were available. DNA extracted from the nonviable cultures and from fresh subcultures of the viable cultures was amplified with primers directed against the insertion sequence IS6110 and the polymorphic GC-rich repetitive sequence. For both isolates, the nonviable cultures generated PCR-banding patterns identical to those generated by the fresh subcultures.

Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.

Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis

In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS 6110, the direct repeat (DR) sequence flanking IS 6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS 6110 and DR ( DR Pvu II ) and AluI for DR ( DR Alu I ) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS 6110 and DR. IS 6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR Alu I and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.

Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family

To determine whether isolates of Mycobacterium tuberculosis from New York and elsewhere that are resistant to four or more primary antimicrobial agents and responsible for widespread disease in the 1990s represent a newly emerged clone or a heterogeneous array of unrelated organisms. New York City area and selected locations in the United States. M tuberculosis isolates from 1953 patients in New York and multidrug-resistant isolates from six patients from other US communities. Convenience sample of all M tuberculosis strains (M tuberculosis isolates resistant to rifampin, streptomycin, isoniazid, and ethambutol, and sometimes ethionamide, kanamycin, capreomycin, or ciprofloxacin) submitted to the Public Health Research Institute Tuberculosis Center since 1991 and samples submitted to the Centers for Disease Control and Prevention from throughout the United States. The samples submitted were representative of the New York City strains of M tuberculosis. Characterization of resistant M tuberculosis strains studied by IS6110 and polymorphic GC-rich repetitive sequence (PGRS) hybridization patterns, multiplex polymerase chain reaction (PCR) analysis, and automated DNA sequencing of genes containing mutations associated with resistance to rifampin (rpoB), isoniazid (katG and inhA locus), and streptomycin (strA and rrs). Multidrug-resistant M tuberculosis isolates were recovered from 253 New York City patients and had the same or closely allied IS6110 and PGRS patterns, multiplex PCR type, and gene mutations associated with resistance to rifampin, isoniazid, and streptomycin. Isolates with these same molecular characteristics were recovered from patients in Florida and Nevada, health care workers in Atlanta, Ga, and Miami, Fla, and an individual who recently moved from New York City to Denver, Colo, and caused disease or skin test conversion in at least 12 people in a nursing home environment. The results document the molecular origin and spread of progeny of a closely related family of multidrug-resistant M tuberculosis strains that have recently shared a common ancestor and undergone clonal expansion. The multidrug-resistant phenotype in these organisms arose by sequential acquisition of resistance-conferring mutations in several genes, most likely as a consequence of antibiotic selection of randomly occurring mutants in concert with inadequately treated infections. Dissemination of these difficult-to-treat bacteria throughout New York City and to at least four additional US cities has adverse implications for tuberculosis control in the 21st century.

Origin and Interstate Spread of a New York City Multidrug-Resistant Mycobacterium tuberculosis Clone Family

<h3>Objective.</h3> —To determine whether isolates of<i>Mycobacterium tuberculosis</i>from New York and elsewhere that are resistant to four or more primary antimicrobial agents and responsible for widespread disease in the 1990s represent a newly emerged clone or a heterogeneous array of unrelated organisms. <h3>Setting.</h3> —New York City area and selected locations in the United States. <h3>Patients.</h3> —<i>M tuberculosis</i>isolates from 1953 patients in New York and multidrug-resistant isolates from six patients from other US communities. <h3>Design.</h3> —Convenience sample of all<i>M tuberculosis</i>strains (<i>M tuberculosis</i>isolates resistant to rifampin, streptomycin, isoniazid, and ethambutol, and sometimes ethionamide, kanamycin, capreomycin, or ciprofloxacin) submitted to the Public Health Research Institute Tuberculosis Center since 1991 and samples submitted to the Centers for Disease Control and Prevention from throughout the United States. The samples submitted were representative of the New York City strains of<i>M tuberculosis</i>. <h3>Main Outcome Measure.</h3> —Characterization of resistant<i>M tuberculosis</i>strains studied by IS<i>61 10</i>and polymorphic GC-rich repetitive sequence (PGRS) hybridization patterns, multiplex polymerase chain reaction (PCR) analysis, and automated DNA sequencing of genes containing mutations associated with resistance to rifampin (<i>rpoB</i>), isoniazid (<i>katG</i>and<i>inhA</i>locus), and streptomycin (<i>strA</i>and<i>rrs</i>). <h3>Results.</h3> —Multidrung-resistant<i>M tuberculosis</i>isolates were recovered from 253 New York City patients and had the same or closely allied IS<i>6110</i>and PGRS patterns, multiplex PCR type, and gene mutations associated with resistance to rifampin, isoniazid, and streptomycin. Isolates with these same molecular characteristics were recovered from patients in Florida and Nevada, health care workers in Atlanta, Ga, and Miami, Fla, and an individual who recently moved from New York City to Denver, Colo, and caused disease or skin test conversion in at least 12 people in a nursing home environment. <h3>Conclusions.</h3> —The results document the molecular origin and spread of progeny of a closely related family of multidrug-resistant<i>M tuberculosis</i>strains that have recently shared a common ancestor and undergone clonal expansion. The multidrug-resistant phenotype in these organisms arose by sequential acquisition of resistance-conferring mutations in several genes, most likely as a consequence of antibiotic selection of randomly occurring mutants in concert with inadequately treated infections. Dissemination of these difficult-to-treat bacteria throughout New York City and to at least four additional US cities has adverse implications for tuberculosis control in the 21st century. (<i>JAMA</i>. 1996;275:452-457)