Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples.

Abstract

To develop a simple and rapid polymerase chain reaction (PCR) method for detecting Oxalobacter formigenes (which degrades oxalate in the gut) in fecal specimens from healthy volunteers and patients with urolithiasis, and to determine whether O. formigenes can be detected in frozen or fresh fecal samples. Whole bacterial DNA was isolated directly from fresh and frozen fecal samples obtained from 30 healthy volunteers free from urolithiasis and from fresh fecal samples obtained from 38 patients with urolithiasis. Genus-specific oligonucleotide sequences were designed, corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase. A PCR-based assay was used on both fresh and frozen fecal samples, and the nucleotide sequences analysed to confirm oxc. A PCR product of 416 bp encoding the oxc gene was detected in 23 (77%) of 30 healthy volunteers free from urolithiasis and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen samples were identical in each subject. The nucleotide sequence analysis showed that the sequence of the amplified product was compatible with that of oxc. O. formigenes can be identified easily and efficiently using this PCR-based detection system. The colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that in healthy volunteers known to be free from urolithiasis. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical to those from fresh samples in each subject, immediate processing of fecal samples may not be necessary to detect O. formigenes in the clinical setting.