Rapid Detection of Quinolone Resistance Mutations in gyrA of Helicobacter pylori by Real-Time PCR

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the T(m) values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes

Detection of Fusobacterium nucleatum in Patients with Colitis-Associated Colorectal Cancer

Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F.   nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum . The fraction of F. nucleatum -positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself.Open Access funding enabled and organized by Projekt DEAL.Forschungskommission of the Klinikum BayreuthFriedrich-Alexander-Universität Erlangen-Nürnberg (1041

Rapid Detection of Quinolone Resistance Mutations in gyrA of Helicobacter pylori by Real-Time PCR

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the Tm values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes