The direct repeat region in Mycobacterium tuberculosis complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking DNA of M. tuberculosis complex strains. We identified 94 different spacer sequences among 26 M. tuberculosis complex strains. No sequence homology was found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare Mycobacterium canettii taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacteriurn tuberculosis in the biochemical tests and in its 16s rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium afiicanurn, M. bovis, and Mycobacteriurn microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".The Mycobactenum tuberculosis complex constitutes a genetically closely related group, and its members, M. tuberculosis, Mycobactenum afncanum, Mycobactenurn bovis, and Mycobacterium microti, may be considered as subspecies of M. tuberculosis (39, 47). The close relatedness between M. tuberculosis complex bacteria has been established by DNA-DNA hybridization (>95%) (l), multiple-locus enzyme electrophoresis (15), and Sequencing of 16s ribosomal RNA and housekeeping genes (15). Furthermore, repetitive DNA elements, such as the insertion sequence IS6110 (43) and the direct repeat (DR) (18), have been found restricted to the M. tuberculosis complex, Nevertheless, the host range and pathogenicity of the M. tuberculosis complex species vary enormously. The natural reservoir of M. tuberculosis and M. afncanum is limited to humans (47) and that of M. microti is limited to voles (48). In contrast, the host range of M. bovis is very broad, and this species causes disease among a wide range of wild and domestic mammals as well as in humans (38).Colonies of primary M. tuberculasis cultures almost invariably have a characterist...

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS,vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element inorf1, of IS1251 in the vanS-vanHintergenic region, and of IS1216V in thevanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.

The rapid increase in genomic sequences provides new opportunities for comparative genomics. In this report, we describe a novel family of repeat sequences that is present in Bacteria and Archaea but not in Eukarya. The repeat loci typically consisted of repetitive stretches of nucleotides with a length of 25 to 37 bp alternated by nonrepetitive DNA spacers of approximately equal size as the repeats. The nucleotide sequences and the size of the repeats were highly conserved within a species, but between species the sequences showed no similarity. Due to their characteristic structure, we have designated this family of repeat loci as SPacers Interspersed Direct Repeats (SPIDR). The SPIDR loci were identified in more than forty different prokaryotic species. Individual species such as Mycobacterium tuberculosis contain one SPIDR locus, while other species such as Methanococcus jannaschii contained up to 20 different loci. The number of repeats in a locus varies greatly from two repeats to several dozens of repeats. The SPIDR loci were flanked by a common 300-500-bp leader sequence, which appeared to be conserved within a species but not between species. The SPIDR locus of M. tuberculosis is extensively used for strain typing. The finding of SPIDR loci in other prokaryotes, including the pathogens Salmonella, Campylobacter, and Pasteurella may extend this surveillance to other species.

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 cultureand ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.

The K88 antigen, a plasmid-specified virulence factor of E. coli involved in porcine neonatal diarrhoea, is often found to be associated with the ability to metabolize raffinose (Raf). Plasmid pRI8801 (51 megadalton) was used to clone the determinants of K88 and Raf with the vector pBR322. K88 was found to be encoded by a 7.7 megadalton HindIII fragment. The expression was highly dependent on the orientation of the HindIII fragment within pBR322. By in vitro generation of deletions, the HindIII fragment was reduced in size to 4.3 megadalton. The expression of K88 by pRI8801 and the recombinant plasmids was studied using an enzyme-linked immunosorbent assay. Raf was found to be located on a 4.0 megadalton SalI fragment. A physical map of pRI8801 was constructed. The K88 antigen and Raf genes are not closely linked but separated by a stretch of DNA of about 20 megadalton.

We conducted a retrospective, population-based study with use of restriction fragment length polymorphism (RFLP) analysis to determine the incidence of and risk factors for clustering of Mycobacterium tuberculosis isolates, indicative of recently transmitted infection, among patients with culture-proven tuberculosis diagnosed between 1 July 1992 and 1 January 1995 in Amsterdam. We found that 214 (47%) of 459 patients were in 53 clusters, probably because of recent transmission of M. tuberculosis among 161 (35%) of these patients. Conventional contact tracing resulted in identification of 5.6% of the 161 patients. Clustering was more frequent among Dutch patients (59.3%) than among foreign ethnic patients (42.1%) (P = .002). The independent risk factor for clustering among Dutch patients was younger age; the independent risk factors among foreign ethnic patients were hard-drug use; alcohol abuse; and country of origin (Surinam or the Netherlands Antilles). These findings suggest the shortcomings of the usual tuberculosis control policies in Amsterdam. We identified several risk factors for clustering, which may guide adjustment of tuberculosis control and contact tracing strategies.

Aims-To detect and diVerentiate Mycobacterium tuberculosis simultaneously by polymerase chain reaction (PCR) in clinical samples prepared for histopathological analysis and for microscopic detection of acid fast bacteria. Methods-ParaYn wax embedded tissue samples and Ziehl-Neelsen (ZN) and auramine stained microscopic preparations from culture positive tuberculosis patients were subjected to DNA extraction and amplification by PCR. PCR was performed with primers specific for direct repeats and the product was detected by hybridisation to a set of 43 diVerent oligonucleotides, a procedure designated as "spoligotyping".