Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen.

Background: The potential role of fungal organisms and their co-aggregation with either periodontopathogens or opportunistic pathogens at peri-implantitis sites is unknown. The aim of the present study was to qualitatively/quantitatively analyze and correlate fungal organisms and bacterial species at peri-implantitis sites. Methods: In a total of 29 patients, submucosal/subgingival plaque samples were collected at peri-implantitis and healthy implant sites as well as teeth with a history of periodontitis (controls). A real-time PCR assay was established for the qualification of fungal organisms and a TaqMan assay for the quantification of Porphyromonas gingivalis, Parvimonas micra, Tannerella forsythia, Mycoplasma salivarium, Veillonella parvula, and Staphylococcus aureus. Results: Fungal organisms were more frequently identified at peri-implantitis (31.6%) (i.e., Candida albicans, Candida boidinii, Penicillium spp., Rhodotorula laryngis, Paelicomyces spp., Saccharomycetes, Cladosporium cladosporioides) and healthy implant sites (40% - Candida dubliniensis, C. cladosporioides) than at selected teeth (20% - C. albicans, Fusarium solani). At implant sites, fungal organisms were significantly correlated with P. micra and T. forsythia. Conclusions: Candida spp. and other fungal organisms were frequently identified at peri-implantitis as well as healthy implant sites and co-colonized with P. micra and T. forsythia.

In immunocompromised patients a colonisation with fungi carries the risk to develop serious invasive fungal infection. An early detection is therefore important, but not optimal hitherto. Fortunately, molecular genetic methods have increased the sensitivity of fungal detection and limited the time, until results are available. However, their success depends on an efficient extraction of genomic DNA from the fungal cell in the given diagnostic specimen. To improve the routine DNA preparation method for yeasts and moulds, the impact of bead beating on fungal DNA release was evaluated. PBS, blood and respiratory rinse were spiked with Candida glabrata or Aspergillus fumigatus. DNA was extracted by mechanical bead beating in addition to the different steps of the DNA preparation protocol, which comprised liquid nitrogen treatment, proteinase K digestion and DNA isolation using the EZ1 DNA Tissue Kit and Workstation. In every method variant tested, treatment with liquid nitrogen did not improve the DNA release. Bead beating once followed by proteinase K digestion and EZ1-work-up led to the highest DNA release from fungus, spiked in PBS, and increased the extracted DNA amount of C. glabrata about 100-fold and of A. fumigatus about 10-fold in relation to sole EZ1-work-up. In fungus-spiked respiratory rinse and blood, highest increase in DNA release was measured after triple bead beating with simultaneous proteinase K digestion. Fungal DNA release of C. glabrata increased for >100-fold in respiratory rinse and for >1000-fold in blood and of A. fumigatus for >10-fold in respiratory rinse and about 5- to 10-fold in blood. The data of this study clearly demonstrate that preparation of fungal DNA from human specimens is optimized by introduction of a bead beating step to the conventional DNA-preparation method without the necessity of a liquid nitrogen step.

Rapid diagnosis and appropriate antimicrobial therapy are of major importance to decrease morbidity and mortality in patients with blood stream infections (BSI). Blood culture, the current gold standard for detecting bacteria in blood, requires at least 24–48 hours and has limited sensitivity if obtained during antibiotic treatment of the patient. The aim of this prospective multicenter study was to clinically evaluate the application of a commercial universal 16S/18S rDNA PCR, SepsiTest™ (PCR-ST), directly on whole blood. In total 236 samples from 166 patients with suspected sepsis were included in the study. PCR-ST results were compared to blood culture, the current gold standard for detecting BSI. Because blood cultures can give false-negative results, we performed an additional analysis to interpret the likelihood of bloodstream infection by using an evaluation based on clinical diagnosis, other diagnostic tests and laboratory parameters. Clinical interpretation of results defined the detected organism to be contaminants in 22 of 43 positive blood cultures (51.2 %) and 21 of 47 positive PCR-ST results (44.7 %). Excluding these contaminants resulted in an overall sensitivity and specificity of the PCR-ST of 66.7 and 94.4 % respectively. Of the 36 clinically relevant samples, 11 BSI were detected with both techniques, 15 BSI were detected with PCR-ST only and 10 with blood culture only. Therefore, in this study, SepsiTest™ detected an additional 71 % BSI compared to blood culture alone. More clinically relevant BSI were diagnosed by molecular detection, which might influence patient treatment. An improved SepsiTest™ assay suited for routine use can have additional value to blood culture in diagnosing bacteremia in septic patients.

In the facultative human pathogen Mycoplasma hominis, which belongs to the cell wall-less Mollicutes, the surface-localised substrate-binding domain OppA of the oligopeptide permease was characterised as the main ecto-ATPase.

Mycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100). To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene. With this strategy, we identified a genomic region of about 10.4 kb inM. hominisFBG carrying the P100 gene. Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region. Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized. The organization of the P100 gene andoppBCDFin a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb. Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene. The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system inM. hominis.