Generation of Rho Zero Cells: Visualization and Quantification of the mtDNA Depletion Process.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen. In contrary, cells devoid of endogenous mitochondrial genomes (ρ° cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ° cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

Effect of sub-MIC concentrations of metronidazole, vancomycin, clindamycin and linezolid on toxin gene transcription and production in Clostridium difficile.

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5x MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

Antimicrobial phenotypes and molecular basis in clinical strains of Clostridium difficile.

Clostridium difficile remains the leading cause of nosocomial-acquired diarrhea. This study investigated antimicrobial susceptibility patterns of C. difficile over a 3-year period. Three hundred seventeen C. difficile isolates recovered between 2002 and 2004 were analyzed for their susceptibility to erythromycin (ERY), clindamycin (CLI), moxifloxacin (MXF), doxycycline (DOX), vancomycin (VAN), and metronidazole (MTR) by Etest. The molecular basis for resistance was investigated using polymerase chain reaction (PCR) and DNA sequencing. PCR ribotyping was used to differentiate strains. All strains were susceptible to VAN and MTR. Resistance rates to ERY/CLI, MXF, and DOX increased during the study period. Eighty-four (26.5%) strains exhibited resistance against ERY/CLI, MXF, and DOX. Prevalence of resistance genes was as follows: ermB, 83; ermQ, 0; ermFS, 1; tetM, 84; tetP, 0; tetO, 2; and gyrA mutation, 76. These results indicate an increasing trend in the prevalence of combined resistance against macrolide-lincosamide-streptogramin B antibiotics, fluoroquinolones, and tetracycline in C. difficile. The lack of understanding of antibiotic resistance mechanisms in C. difficile and the increased resistant strains warrants further investigations.

In vitro activity of OPT-80 against Clostridium difficile.

Clostridium difficile remains the major cause of nosocomial diarrhea. Reports on impaired susceptibility of C. difficile to metronidazole and vancomycin and frequent relapses of patients after therapy necessitate the search for new substances. With this study, the activity of OPT-80, a new macrocycle, against 207 C. difficile strains and against other obligately anaerobic bacteria was tested. OPT-80 showed high in vitro activity against all C. difficile strains tested.