Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases

Abstract

Aim: To investigate the presence of Helicobacter species by nested PCR of 16S rRNA genes followed by the presence of Helicobacter pylori (H pylori) 16S rRNA, ureA, cagA genes in bile obtained at endoscopic retrograde cholangio-pancreatography (ERCP) from 60 Indian subjects.

Methods: Sixty bile samples were obtained from patients diagnosed with various hepato-biliary diseases and control subjects at ERCP. PCR analysis was carried out using primers for Helicobacter genus 16S rRNA gene and H pylori (16S rRNA, ureA and cagA) genes. Gastric H pylori status was also assessed from biopsies obtained at endoscopy from patients with various hepato-biliary diseases and controls. The control group mainly consisted of subjects with gastric disorders. Sequencing analysis was performed to confirm that PCR products with 16S rRNA and cagA primers were derived from H pylori. RESULTS No Helicobacters were grown in culture from the bile samples. Helicobacter DNA was detected in bile of 96.7% and 6.6% of groups I and II respectively. Ten from group I were positive for 16S rRNA and ureA and 9 were positive for cagA gene. In contrast of the 2 from the control, 1 amplified with 16S rRNA, ureA and cagA primers used. The sequences of the 16S rRNA genes and cagA were 99% similar to Helicobacter pylori.

Conclusion: Helicobacters are associated with the pathogenesis of various hepato-biliary disorders.

Figure 1

A schematic representation of the PCR products of the Helicobacter spp DNA. A: Gel image showing first amplification products of 16S rRNA PCR with Helicobacter genus specific primers at 1 300 bp. Lanes 1 to 6 represent Helicobacter DNA isolated from bile samples, ‘P’ represents positive control andLanes C, N and M represents negative control, reaction negative control and 1 Kb molecular weight marker. B: Second amplification products of 16S rRNA PCR with Helicobacter genus specific primers at 480 bp. Lanes 1 to 5 represent bile DNA, ‘P’ represents positive control. Lanes N and M represent reaction negative control and 100 bp molecular weight marker ladder respectively. C: Gel picture showing 16S rRNA amplification products specific to H pylori of bile samples at 534 bp. Lanes 1, 2, 3 and 5 represent bile DNA samples while Lanes M and P represent 100 bp ladder and positive control respectively. D: ureA amplification products at 411 bp. Lanes 1 to 3 represent bile DNA, Lane ‘P’ represents positive control and Lanes C, N and M represent negative control, reaction negative control and 100bp molecular weight ladder respectively. E: Gel picture illustrating the cagA amplification products at 349 bp. Lanes 1, 2 and 3 represent bile DNA while Lanes M, P, N and C represent 100bp molecular weight ladder, positive and reaction negative control, negative control respectively.