Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 2 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrugresistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.

Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene. This gene has been shown to be part of a 150-kb putative pathogenicity island. A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients. In the present study we analyzed the polymorphism in the esp gene of E. faecium, and we investigated the association of esp with neighboring chromosomal genes. The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N-and C-terminal domains of the Esp protein as well as differences in the number of repeats. DNA sequencing of chromosomal regions flanking the esp gene of E. faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance. These flanking regions were invariably associated with the presence or absence of the esp gene in E. faecium, indicating that esp in E. faecium is part of a distinct genetic element. Because of the presence of virulence genes in this gene cluster, the lower G؉C content relative to that of the genome, and the presence of esp in E. faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E. faecium. Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E. faecalis.Enterococci are common inhabitants of the gastrointestinal tracts of humans and animals, and although they have been recognized as pathogens able to cause endocarditis, they were generally considered second-rate pathogens. Recent estimates, however, indicate that enterococci are now among the leading causes of nosocomial infections (57). Of all enterococcal species, Enterococcus faecalis accounted for the most infections in humans (26). However, during the past decade, the incidence of bloodstream infections caused by Enterococcus faecium increased, an increase which has been linked to the emergence of antibiotic resistance in this species (26,40).Little is known about virulence determinants in E. faecium (20). Recently, however, three potential virulence genes, esp, hyl, and acm, have been described for E. faecium. They were all found more frequently in clinical isolates than in fecal isolates or nonhuman isolates (13,41,44,65).Of these three putative virulence genes, only the esp gene is also found in E. faecalis (51). The Esp protein in E. faecalis is expressed as a large surface-exposed protein with a molecular mass of approximately 202 kDa. In E. faecalis, Esp is thought to be an adhesin contributing to colonization of urinary tract epithelial cells and biofilm formation (50, 59). Although detailed experimental evidence is not yet available, the higher prevalence of the E. faecium esp gene in clinical isolates suggests a role of Esp in the pathogenesis of E. faeci...

A total of 188 Bordetella strains were characterized by the electrophoretic mobilities of 15 metabolic enzymes and the distribution and variation in positions and copy numbers of three insertion sequences (IS). The presence or absence of IS elements within certain lineages was congruent with estimates of overall genetic relationships as revealed by multilocus enzyme electrophoresis. Bordetella pertussis and ovine B. parapertussis each formed separate clusters, while human B. parapertussis was most closely related to IS1001-containing B. bronchiseptica isolates. The results of the analysis provide support for the hypothesis that the population structure of Bordetella is predominantly clonal, with relatively little effective horizontal gene flow. Only a few examples of putative recombinational exchange of an IS element were detected. Based on the results of this study, we tried to reconstruct the evolutionary history of different host-adapted lineages.

SuSm and N-Tc, have been characterized. Both were of relatively small size (5 x 106 to 6 x 106 daltons) and present in multiple copies within their respective bacterial hosts. N-SuSm possessed a guanine plus cytosine content of 55%, whereas N-Tc was 49% guanine plus cytosine. Although these plasmids were inherently nontransmissible they could be mobilized by a large variety of transfer agents including Ent, Hly, and K88. The fih transfer factors tested were far more likely (about 200x) to mobilize these nonconjugative plasmids than were the fi+ transfer factors tested. Although the mobilization phenomenon was not found to be associated with a detectable level of direct stable recombinational union between N-SuSm or N-Tc with a transfer factor, we were able to demonstrate a low level of recombination between these replicons and a transfer factor by P1-mediated transduction. The isolation of recombinants between transfer factors and nonconjugative plasmids presumably represents one means by which unitary molecular types of R-plasmids arise and by which existing R-plasmids may acquire new resistance determinants.

In a recent work, we have shown that mycobacterial heat-shock proteins (hsp) of 65-kDa (GroEL-type) and 70-kDa (DnaK-type) acted as carrier molecules in mice, previously primed with Mycobacterium tuberculosis var. bovis (bacillus Calmette-Guérin, BCG), for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)40. Anti-peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial hsp, was given in the absence of any adjuvants (Lussow et al., Eur. J. Immunol. 1991. 87:2960). In this report, we show that mice immunized with peptides or oligosaccharides conjugated to the 70-kDa hsp produced high titers of IgG antibodies in the absence of any previous priming with BCG. The anti-peptide antibody response persisted for at least 1 year. This adjuvant-free carrier effect of the 70-kDa hsp was T cell dependent, since no anti-peptide nor anti-70-kDa IgG antibodies were induced in athymic nu/nu mice. Previous immunization of mice with the 65-kDa or 70-kDa hsp did not have any negative effect on the induction of anti-peptide IgG antibodies after immunization with hsp-peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65-kDa hsp could substitute for BCG in providing an effective priming for the induction of anti-(NANP) antibodies. Finally, both the 65-kDa and 70-kDa hsp acted as carrier molecules for the induction of IgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants. These findings strongly suggest that the use of hsp as carriers in conjugated constructs for the induction of anti-peptide and anti-oligosaccharide antibodies could be of value in the design of new vaccines for eventual use in humans.

The amino acid sequences of the 65-kilodalton antigens of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis BCG display greater than 95% homology. 1932

Tuberculosis and leprosy are chronic infectious diseases that presently affect more than 65 million people (1, 2). Apparently the majority of individuals exposed to or infected with pathogenic mycobacteria develops effective immunity that is exclusively based on the cellular immune response, and recent evidence suggests that T helper/inducer (T4) lymphocytes are of particular importance for protection (3-6). In previous studies whole bacteria or crude bacterial extracts have been used for stimulation of heterogeneous T cell populations. Thus, the bacterial proteins recognized by mycobacteria-reactive T lymphocytes are completely unknown. Recently, a 64 kD protein antigen of Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been cloned in Escherichia coli (7), and T cell clones (TC) with reactivity to mycobacterial antigens have been established (8). It has therefore become possible to characterize defined mycobacterial antigens by their potential to stimulate distinct TC. We have used this approach to characterize the reactivity against a recombinant M. bovis BCG antigen (antigen A) of human TC that were derived from a leprosy patient by in vitro stimulation with M. leprae and from three normal PPD-reactive individuals by stimulation with BCG. This is of particular interest for vaccine development, and for the design of better-defined and more specific diagnostic skin test reagents.

DNA fingerprinting techniques were used to type 273 isolates ofMycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.