Antibiotic resistance in clinically relevant pathogens is a major public health problem worldwide. Since the numbers of antibiotic resistant bacteria are increasing a fast clarification of the resistance is essential for the treatment of affected patients.
Clostridium difficile is the main causative of hospital-acquired diarrhoea worldwide and rifaximin is an antibiotic for the treatment of C. difficile infections (CDI) especially in case of recurring CDI. Reduced rifaximin susceptibility bases on single nucleotide polymorphisms (SNPs) in the rpoB gene which encodes the b-subunit of the RNA polymerase, the main target of rifaximin. The aim of this thesis was the establishment of a new detection method of SNPs in rpoB of clinical C. difficile isolates. In the main study, the screening of 348 C. difficile samples of different PCR ribotypes by conventional PCR (325 bp amplicon) revealed 16 PCR-sequence types (PCR-ST) characterised by 24 detected SNPs. High-resolution melting curve (HRM) analysis established 11 different HRM-curve profiles (HRM-CP) based on 15 of 24 SNPs located in the rpoB hot-spot region (151 bp amplicon). Beside two SNPs associated with reduced rifaximin susceptibility, all previously published SNPs are detectable by this HRM. In another project on the suitability of rifaximin for disc diffusion test in comparison to rifaximin broth microdilution assay 62 phenotypically reduced rifaximin susceptible C. difficile strains were identified. Sequence analysis of the 325 bp rpoB amplicon revealed the presence of non-synonymous SNPs in these strains corroborating the association of SNPs in rpoB with reduced rifaximin susceptibility.
Moreover, this study showed that the rifaximin disc diffusion test is equivalent to the rifaximin broth microdilution assay. ^The third project was dedicated to a RT-PCR for the rapid determination of antibiotic resistance (amikacin, kanamycin and capreomycin) based on SNPs in the 16S rRNA gene in extensive drug resistant Mycobacterium tuberculosis strains. Altogether, this thesis demonstrates that PCR based methods, especially HRM are fast, reliable and cost-effective techniques to determine antibiotic resistance in clinically relevant pathogens. Moreover, fast antibiotic susceptibility testing methods in routine laboratories may have an impact on the treatment of infected patients.