A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification of Ehrlichiaand Borrelia burgdorferi sensu lato. In separate assays the 16S rRNA gene of Ehrlichia species and the 23S-5S rRNA spacer region of B. burgdorferi sensu lato were amplified and labeled by PCR. These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification of Ehrlichia,B. burgdorferi, and Bartonella species in 40 different samples. The application of the assay to DNA extracts from 121 Ixodes ricinus ticks collected from roe deer demonstrated that 45% of these ticks carried EhrlichiaDNA. More than half of these positive ticks carried species with 16S rRNA gene sequences closely related to those of E. phagocytophila and the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identified Ehrlichia-like species. In addition, 13% of the ticks were infected with one or more B. burgdorferigenospecies. In more than 70% of the ticks 16S rRNA gene sequences forBartonella species or other species closely related toBartonella were found. In five of the ticks bothEhrlichia and B. burgdorferi species were detected.

PurposeWe investigated the hypothesis that many total hip arthroplasty revisions that are classified as aseptic are in fact low-grade infections missed with routine diagnostics.MethodsIn 7 Dutch hospitals, 176 consecutive patients with the preoperative diagnosis of aseptic loosening of their total hip arthroplasty were enrolled. During surgery, between 14 and 20 tissue samples were obtained for culture, pathology, and broad-range 16S rRNA PCR with reverse line blot hybridization. Patients were classified as either not being infected, suspected of having infection, or infected according to strict, predefined criteria. Each patient had a follow-up visit after 1 year.Results7 patients were classified as infected, 4 of whom were not identified by routine culture. 15 additional patients were suspected of having infection. 20 of these 22 patients received a cemented prosthesis, fixated with antibiotic-loaded bone cement. All 22 patients received prophylactic systemic antibiotics. 7 of them reported complaints one year after surgery, but only one showed signs of early loosening. However, additional surgery was not performed in any of the patients.InterpretationAlthough the proportions were not as high as previously reported in the literature, between 4% and 13% of patients with the preoperative diagnosis of aseptic loosening were infected. However, as thorough debridement was performed during surgery and prophylactic antibiotics were used, the diagnosis of infection did not have any obvious clinical consequences, as most patients performed well at the 1-year follow-up. Whether this observation has implications for long-term implant survival remains to be seen.

The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes.Based on 16S rRNA gene sequence analysis, viridans streptococci are now separated into six genogroups: the pyogenic, mitis, bovis, salivarius, mutans, and anginosus groups (13). The anginosus group, also designated the Streptococcus anginosus group (SAG), is separated into three distinct species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius (22). Several studies have shown that these species are important human pathogens with the capacity to cause purulent infections and abscesses (3). The three species belonging to the anginosus group are notoriously difficult to identify due to their heterogeneous biochemical and serological characteristics. Furthermore, the clinical spectrum associated with infection with members of the anginosus group also varies considerably. For this reason some scientists have resorted to genotypic rather than phenotypic characterization to differentiate these species (4,8,10). DNA-DNA reassociation data showed that the SAG indeed can be separated into three taxonomically distinct species but that there is considerable heterogeneity within each species (11,22). Analysis of the 16S rRNA gene has been shown to be of value in identifying bacterial species, particularly for fastidious and uncultivable bacteria. Several research groups have used DNA sequencing of the 16S rRNA gene and hybridization with species-specific DNA probes to identify the species of the anginosus group. In previous studies we showed that among the species of the SAG, heterogeneity exists even within the well-conserved 16S rRNA gene (9,10). In this study we show that the 16S rRNA genes of the members of the SAG show mosaic-like structures. These structures suggest that the various species within the SAG may exchange DNA fragments, including parts of the taxonomically important 16S rRNA gene. T ). The other 705 streptococca...

Cats have been shown to provide the only known reservoir of Bartonella henselae, the causative agent of cat scratch disease. To determine the prevalence of Bartonella bacteremia and antibodies in Dutch cats, blood samples from 113 cats from shelters (sheltered cats), 50 pet cats, and 25 specific-pathogen-free (SPF) cats were analyzed. Culture and subsequent PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA intergenic region and 16S rRNA gene PCR-hybridization assays revealed a prevalence of Bartonella bacteremia in 22% of the sheltered cats and showed no bacteremia in the SPF cats. Three spacer RFLP types were found: types A, B, and G, with type B being predominant over types A and G. An important finding was the existence of mixtures of different Bartonella species. Bartonella DNA was detected in 7 of 27 DNA extracts from fleas combed from the sheltered cats (26%). Seropositivity was 50% for sheltered cats and 56% for pet cats, as determined by a B. henselae enzyme-linked immunoassay.

An electronic surveillance network for monitoring antibiotic resistance in The Netherlands has been in operation since 1989. Seven public health laboratories participate and the system covers about 25% of all bacteriological determinations in The Netherlands. This paper reports the results of staphylococci isolated in the period 1989-1995. About 0.3% of the Staphylococcus aureus isolates in the study period were resistant to methicillin. This low percentage may be due to the restrictive use of antibiotics and to strict isolation measures aimed at eradicating methicillin-resistant S. aureus. Low frequencies of resistance among methicillin-resistant S. aureus were found for vancomycin (0%), chloramphenicol (11%), cotrimoxazole (11%), mupirocin (3% low-level resistance) and fusidic acid (7%). Twenty-one percent of the coagulase-negative staphylococci were resistant to methicillin. Low frequencies of resistance among these methicillin-resistant coagulase-negative staphylococci were those to vancomycin (0.4%), nitrofurantoin (2%), doxycycline (20%) and amikacin (20%). Coagulase-negative staphylococci from cerebrospinal fluid, blood and skin were less often resistant to quinolones than isolates from respiratory tract, faeces and urine. A significant increase in resistance of coagulase-negative staphylococci to methicillin, erythromycin, gentamicin and ciprofloxacin was observed in the investigated period but the resistance to doxycycline and co-trimoxazole decreased in the last few years. To confirm the determination of methicillin resistance and coagulase production, a PCR method was developed which detects both the mecA and the coagulase gene. The results of the PCR method correlated well with the methicillin MIC as determined by an agar-dilution method.