Jan D. A. van Embden

Identification of genes that are associated with DNA repeats in prokaryotes

Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non‐repetitive sequences. To appreciate their characteri‐stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300–500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR‐associated (cas) genes were identified in CRISPR‐containing prokaryotes that were absent from CRISPR‐negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.

Multilocus Sequence Typing Scheme for Enterococcus faecium

ABSTRACT A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.

Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals

In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy.

Molecular Epidemiology of Tuberculosis in the Netherlands: A Nationwide Study from 1993 through 1997

To disclose risk factors for active tuberculosis transmission in the Netherlands, restriction fragment length polymorphism (RFLP) patterns of 78% of the Mycobacterium tuberculosis isolates, from the period 1993-1997, were analyzed. Of the respective 4266 cases, 46% were found in clusters of isolates with identical RFLPs, and 35% were attributed to active transmission. The clustering percentage increased strongly with the number of isolates; taking this into account, fewer cases were clustered than has been reported in other studies. Contact investigations in the five largest clusters of 23-47 patients suggested epidemiological linkage between cases. Of patients identified through contact tracing, 91% were clustered. Demographic risk factors for active transmission of tuberculosis included male sex, urban residence, Dutch and Surinamese nationality, and long-term residence in the Netherlands. Human immunodeficiency virus infection was not an independent risk factor for active transmission. Isoniazid-resistant strains were relatively less frequently clustered, suggesting that these generated fewer secondary cases.

Comparative Genotyping of Campylobacter jejuni by Amplified Fragment Length Polymorphism, Multilocus Sequence Typing, and Short Repeat Sequencing: Strain Diversity, Host Range, and Recombination

ABSTRACT Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.

A Novel Putative Enterococcal Pathogenicity Island Linked to the esp Virulence Gene of Enterococcus faecium and Associated with Epidemicity

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.

Host Specificity of Vancomycin-Resistant Enterococcus faecium

Amplified-fragment length polymorphism (AFLP) analysis was used to investigate the genetic relationships among 255 vancomycin-resistant Enterococcus faecium (VREF) strains isolated from hospitalized patients, nonhospitalized persons, and various animal sources. Four major AFLP genogroups (A-D) were discriminated. The strains of each taxon shared >/=65% of the restriction fragments. Most isolates recovered from nonhospitalized persons (75%) were grouped together with all pig isolates in genogroup A. Most isolates from hospitalized patients (84%), a subset of veal calf isolates (25%), and all isolates from cats and dogs clustered in genogroup C. Most isolates from chickens (97%) and turkeys (86%) were grouped in genogroup B, whereas most veal calf isolates (70%) clustered in genogroup D. Therefore, VREF strains are predominantly host-specific, and strains isolated from hospitalized patients are genetically different from the prevailing VREF strains present in the fecal flora of nonhospitalized persons.

Analysis of Rate of Change of IS6110 RFLP Patterns of Mycobacterium tuberculosis Based on Serial Patient Isolates

The rate of change of IS6110 restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis was determined in serial isolates from 544 patients. In 25 patients (4.6%), the RFLP patterns of the follow-up isolates differed from the initial isolates. Patients with different follow-up strains were less likely to cluster with patients whose strains had indistinguishable RFLP patterns. Changes in RFLP patterns were more common for persons with extrapulmonary disease and for those who had both pulmonary and extrapulmonary isolates. Based on serial isolates spanning for the most part <3 months, the half-life was extrapolated to be 3.2 years (95% confidence interval, 2.1-5.0). The main implication of this study is that the rate of change of IS6110-based RFLP of M. tuberculosis supports the use of IS6110 typing in epidemiologic studies of recent transmission of tuberculosis.

Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers

OBJECTIVE Bacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints. METHODS Samples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species. RESULTS In the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products. CONCLUSION When the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.

Transmission between HIV-infected patients of multidrug-resistant tuberculosis caused by Mycobacterium bovis

Objective:To investigate outbreaks of multidrug-resistant tuberculosis (TB) by using DNA fingerprint databases. Design:Investigation of two outbreaks of multidrug-resistant TB in separate hospitals in Spain by restriction fragment length polymorphism (RFLP) and spoligotyping. Outbreak strains were compared with more than 1500 RFLPs of Mycobacterium tuberculosis complex strains isolated in Spain and 6000 RFLPs from 30 different countries. Methods:Standardized IS6110 DNA fingerprinting and ‘spoligotyping’ was used to type multidrug-resistant isolates belonging to the M. tuberculosis complex amongst the outbreak cases. The DNA types were matched against DNA fingerprint databases in Spain and The Netherlands. Results:The DNA typing analysis indicated that a single multidrug-resistant Mycobacterium bovisstrain was responsible for a nosocomial outbreak in a hospital in Spain involving at least 16 HIV-infected patients with non-treatable to multidrug-resistant TB. Introduction of the fingerprint type of this strain to the international database revealed a single matching strain. This strain was also isolated from an HIV-infected patient in The Netherlands who had died from multidrug-resistant TB. This patient had previously been hospitalized in Spain, where a multidrug-resistant TB nosocomial outbreak involving 20 HIV-infected patients was ongoing. The strains causing this outbreak were also identified as M. boviswith an identical DNA pattern to those strains isolated in the Spanish hospital and the patient in The Netherlands. Conclusions:The use of centralized DNA databases can help to identify rapidly the origin and transmission routes of multidrug-resistant TB across international boundaries and the potential use of such an early warning surveillance system for investigation of nosocomial multidrug-resistant TB outbreaks between HIV-infected patients. To our knowledge this is the first report of transmission of multidrug-resistant M. bovisbetween hospitals.