J. D. A. Van Embden

Genetic Variation and Evolutionary Origin of the Direct Repeat Locus of Mycobacterium tuberculosis Complex Bacteria

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.

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment‐length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30‐bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

A Molecular Epidemiological Approach to Studying the Transmission of Tuberculosis in Amsterdam

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.

Penicillinase-producing Neisseria gonorrhoeae in the Netherlands: epidemiology and genetic and molecular characterization of their plasmids.

Penicillinase-producing Neisseria gonorrhoeae strains were isolated in the Netherlands with increasing frequency during the period of 1976 to 1979. About 3% of the gonococci isolated in the first half of 1979 produced penicillinase. In contrast to the period of 1976 to 1977, most penicillinase-producing N. gonorrhoeae infections during the period of 1978 to 1979 were contracted in the Netherlands. The results of genetic and molecular studies on 80 penicillinase-producing N. gonorrhoeae strains were similar to earlier observations of others: resistance plasmids of only two sizes, 4.5 and 3.3 megadaltons (Md), occurred in penicillinase-producing N. gonorrhoeae strains, and these encoded for the TEM-1 enzyme. The 4.5-Md plasmid could be transferred to Escherichia coli when it coexisted with a plasmid of 24 Md. The latter plasmid was present in the vast majority of the strains carrying the 4.5-Md plasmid. One strain carried a cryptic 7.5-Md plasmid in addition to the commonly found 2.5-Md plasmid. Two penicillinase-producing strains of Haemophilus parainfluenzae isolated were found to carry a 3.3-Md plasmid species which was indistinguishable from the 3.3-Md gonococcal resistance plasmids. No plasmid deoxyribonucleic acid was found in two strains of penicillinase-producing Branhamella catarrhalis, and these strains produced a penicillinase different from the TEM-1 enzyme. Images

Decrease of Drug Resistance in Salmonella in The Netherlands

Since 1974, tetracycline resistance in salmonellae of human and porcine origin has decreased nation-wide in The Netherlands. This decrease has coincided with the ban on incorporation of tetracycline in animal feeds.

Yaws in West Sumatra, Indonesia: Clinical manifestations, serological findings and characterisation of new Treponema isolates by DNA probes

The results of a yaws survey on the island of Sumatra in Indonesia are presented. The prevalence of yaws in the investigated region was found to be very high, a minimum of 300 cases per 100,000 individuals, which indicates that yaws is far from being eradicated and that campaigns for treatment are necessary. Patients suffering from early infectious yaws showed florid skin lesions. Of 101 serum samples from such patients, 100 had a positive reaction in one or more treponemal tests. TheTreponema pallidum haemagglutination assay was found to be the most sensitive test (97 % positive) in detecting antibodies againstTreponema pallidum subsp.pertenue, followed by the fluorescent treponemal antibody absorption test (94 %), the Venereal Disease Research Laboratory test and the TmpA enzyme immunoassay (91 %), and analysis by Western blot usingTreponema pallidum antigens (88 %). Of 42 asymptomatic contacts of yaws patients 32 showed positive reactions in one or more tests, indicating that many people in the investigated region have been infected with treponemes. Eight newTreponema pallidum subsp.pertenue strains were isolated from yaws skin lesions. In vitro amplification of treponemal DNA and hybridisation with specific DNA probes showed that all eight strains were identical withTreponema pallidum subsp.pertenue CDC 2575, with regard to the subsp.pertenue specifictyfl gene.

Heat-Shock Proteins and Mycobacterial Antigens

Most autoimmune diseases in humans are known to be associated with certain HLA alleles. This means that genetic factors contribute to individual susceptibility and that at least some of these factors are encoded within or close to the major histocompatibility complex (MHC). In the case of rheumatological disorders, the association of HLA-B27 with ankylosing spondylitis is well-known and impressive. Of less biological impact, but certainly very significant, is the association of HLA-DR4 and HLA-DR1 with rheumatoid arthritis. Furthermore, evidence has been put forward that the same DR4 allele contributes to the development of arthritis in Lyme borreliosis [42].

Cloning and Expression of the Genes Encoding for the Adhesive Antigens K88 and K99

More than a decade ago, enterotoxigenic E. coli strains were found to be associated with acute diarrhoea in young animals and later such strains were also found to be involved in cases of human diarrhoea. Enterotoxigenic E. coli strains release a heat labile toxin and/or a heat stable toxin which effects the fluid and electrolyte secretion in the intestine by activation of the mucosal enzymes adenyl cyclase and guanyl cyclase, respectively1,2. A number of proteinaceous surface antigens of enterotoxigenic E. coli have been identified, that are involved in the colonization of the gut by facilitating the adherence of the microorganism to the intestinal mucosa. Enterotoxins and several of these colonization factors are encoded by plasmids. The significance of organisms that possess plasmid-mediated pathogenic characteristics is that they constitute a genetic pool from which new lines of pathogenic organisms may arise. To the research worker, they represent genetic material that can be added or removed from organisms, thus permitting the construction of new lines which differ only from the parent microorganism by the presence or the absence of one character. Smith and coworkers exploited this idea to elucidate the pathogenesis of E. coli diarrhoea in animals. They showed in an elegant series of experiments that the antigens K88 and K99 promote colonization of the intestine by implanting K88 and K99 plasmids into non-pathogenic strains of E. coli or alternatively by removal of these plasmids from pathogenic strains and subsequently feeding such modified strains to neonates3,4,5.