The culture of viable microorganisms from the blood or from cardiac tissue is currently the most important test for diagnosis of IE. This is followed by phenotypic identification methods used for ...taxonomic positioning of isolates. However, in those cases where the invading microorganism is difficult or impossible to culture (including instances of prior antimicrobial treatment), molecular methods provide the best means for detection. Molecular identification methods, either nucleic acid target or signal amplification alone or in combination with sequence analysis can offer a more specific and in some cases a more rapid alternative to the phenotypic methods. We propose revised Duke criteria of IE, including positive identification of an organism by molecular biology methods.
This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may ...either supplement or replace classic culture‐based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture‐based methods still represent the work horse of everyday routine. The number of available FDA‐approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture‐dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy‐to‐perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high‐throughput testing we discuss matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF) of nucleic acids, a technology that has evolved from the post‐genome sequencing era, for high‐throughput sequence variation analysis (1, 2).
1 Institut für Mikrobiologie und Tierseuchen der Freien Universität Berlin, Luisenstr. 56, D-10117 Berlin, Germany
2 Department of Oral Biology, Yonsei University, College of Dentistry, 134 ...Shinchon-Dong, 120--752 Seoul, Korea
3 Institut für Mikrobiologie und Tierseuchen der Freien Universität Berlin, Elektronenmikroskopie, Fabeckstr. 36a, D-14195 Berlin, Germany
4 Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité Humboldt-Universitat zu Berlin, Dorotheenstr. 96, D-10117 Berlin, Germany
Author for correspondence: Ulf B. Göbel. Tel: +49 30 2093 4715. Fax: +49 30 2093 4703. e-mail: ulf.goebel{at}charite.de
ABSTRACT
A novel Treponema species was isolated from an ulcerative lesion of a cow suffering from digital dermatitis (DD), a disease which causes painful ulcerations along the coronary band. Among other anaerobic bacteria, high numbers of spirochaetes have been regularly found in DD lesions. Here data are presented of a spirochaete isolated from a DD ulcer. By chemotaxonomy, protein analysis and comparative 16S rDNA sequence analysis this isolate was classified as a treponeme that differed from all Treponema species described previously. The only isolate, DD5/3 T , for which the name Treponema brennaborense is proposed, is designated the type strain of the novel species. The strain is a small, highly motile spirochaete that has two periplasmc flagella, one flagellum being attached at each cell pole. Strain DD5/31 exhibits -glucosidase and N -acetyl-β-glucosaminidase activity and growth is inhibited by rabbit serum. T. brennaborense was phylogenetically most closely related (89--5% 16S rRNA similarity) to Treponema maltophilum , an oral spirochaete isolated from a periodontitis patient.
Key Words: treponemes digital dermatitis cattle 16S rRNA
The GenBank/EMBL accession number for the 16S rDNA sequence of the novel isolate Treponema brennaborense is Y16568 .
The objective of this study was to visualize borreliae directly in whole-body sections of Ixodes ricinus by fluorescence in situ hybridization (FISH). Borrelia afzelii mono-infected or Borrelia ...burgdorferi sensu stricto (ss)/B. afzelii double-infected nymphs were fixed, embedded in cold polymerizing resin and sectioned. The same sample processing was applied to skin biopsies taken from a Mongolian gerbil after an infectious tick-bite. FISH was carried out using 16S-rRNA-directed, fluorescence-labelled oligonucleotide probes specific for the genus Borrelia and specific within the group of Lyme borreliosis-associated genospecies B. afzelii, B. burgdorferi ss, Borrelia garinii and Borrelia valaisiana. Sensitivity and specificity of the newly designed probes were evaluated using PCR, dot-blot hybridizations and FISH. Despite significant autofluorescence of certain tick tissues (which allowed good histological orientation within the sections), borreliae showing the typical spirochaetal morphotype were clearly visible in five out of six putatively infected ticks. These findings were confirmed by electron microscopy of ticks from the same infected batch as used for FISH. Attempts to produce ticks infected by two different Borrelia genospecies were not successful. FISH on whole-body sections of resin-embedded ticks offers the possibility of visualizing and identifying borreliae within tick tissues. This technique has great potential for the investigation of the transmission of bacteria or other micro-organisms by arthropod vectors. Furthermore, clear visualization of single spirochaetes distributed along subcutaneous fat cell membranes in gerbil skin biopsies suggests that FISH might also be suitable for the detection of borreliae in clinical tissue specimens.
Oral treponemes are related to chronic periodontitis, but the effect of periodontal therapy on the majority of treponemal species is unknown. The aim of this prospective study was to evaluate the ...dynamics in prevalence profiles of treponemes in different habitats of the oral cavity. Thirty-five patients with chronic periodontitis were randomly assigned to mechanical debridement alone (control group) or systemic amoxicillin/metronidazole plus chlorhexidine (test group). Subgingival and mucous membrane plaque samples were taken at baseline, after 10 days, and during supportive periodontal therapy at 3, 6, 9, 12, 18, and 24 months. T. denticola, T. lecithinolyticum, T. maltophilum, T. socranskii, T. vincentii, and treponemal phylotypes I-VII were detected using polymerase chain reaction (PCR) and dot blot analysis. For the majority of the assessed treponemes, a significant intragroup increase in prevalence in the different habitats ( P<0.05) occurred over the study course but, compared to debridement alone, adjunctive antimicrobial therapy resulted in a nonsignificant trend toward lower prevalence in the subgingival habitat. In no case were treponemes eradicated from the oral cavity. After both therapies, possibly new infection with and/or dissemination of Treponema ssp. occurred, which led to treponemes recovering in different habitats and to increased intraoral prevalence. The prescribed adjunctive antimicrobial therapy may limit this increase in the subgingival region.
The major sheath protein-encoding gene (mspA) of the oral spirochete Treponema maltophilum ATCC 51939(T) was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The ...mspA gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an mspA-specific probe showed no cross-reactivity with the msp gene from Treponema denticola.
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