Molecular analysis of faecal and duodenal samples reveals significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel syndrome.

Intestinal microbiota may play a role in the pathophysiology of irritable bowel syndrome (IBS). In this case-control study, mucosa-associated small intestinal and faecal microbiota of IBS patients and healthy subjects were analysed using molecular-based methods. Duodenal mucosal brush and faecal samples were collected from 37 IBS patients and 20 healthy subjects. The bacterial 16S rRNA gene was amplified and analysed using PCR denaturing gradient gel electrophoresis (DGGE). Pooled average DGGE profiles of all IBS patients and all healthy subjects from both sampling sites were generated and fingerprints of both groups were compared. The DGGE band fragments which were confined to one group were further characterized by sequence analysis. Quantitative real-time PCR (q-PCR) was used to quantify the disease-associated microbiota. Averaged DGGE profiles of both groups were identical for 78.2 % in the small intestinal samples and for 86.25 % in the faecal samples. Cloning and sequencing of the specific bands isolated from small intestinal and faecal DGGE patterns of IBS patients showed that 45.8 % of the clones belonged to the genus Pseudomonas, of which Pseudomonas aeruginosa was the predominant species. q-PCR analysis revealed higher levels (P<0.001) of P. aeruginosa in the small intestine of IBS patients (8.3 %±0.950) than in the small intestine of healthy subjects (0.1 %±0.069). P. aeruginosa was also significantly (P<0.001) more abundant (2.34 %±0.31) in faeces of IBS patients than in faeces of healthy subjects (0.003 %±0.0027). This study shows that P. aeruginosa is detected more frequently and at higher levels in IBS patients than in healthy subjects, suggesting its potential role in the pathophysiology of IBS.

Lower Bifidobacteria counts in both duodenal mucosa-associated and fecal microbiota in irritable bowel syndrome patients.

AIM: To determine the composition of both fecal and duodenal mucosa-associated microbiota in irritable bowel syndrome (IBS) patients and healthy subjects using molecular-based techniques. METHODS: Fecal and duodenal mucosa brush samples were obtained from 41 IBS patients and 26 healthy subjects. Fecal samples were analyzed for the composition of the total microbiota using fluorescent in situ hybridization (FISH) and both fecal and duodenal brush samples were analyzed for the composition of bifidobacteria using real-time polymerase chain reaction. RESULTS: The FISH analysis of fecal samples revealed a 2-fold decrease in the level of bifidobacteria (4.2 +/- 1.3 vs 8.3 +/- 1.9, P < 0.01) in IBS patients compared to healthy subjects, whereas no major differences in other bacterial groups were observed. At the species level, Bifidobacterium catenulatum levels were significantly lower (6 +/- 0.6 vs 19 +/- 2.5, P < 0.001) in the IBS patients in both fecal and duodenal brush samples than in healthy subjects. CONCLUSION: Decreased bifidobacteria levels in both fecal and duodenal brush samples of IBS patients compared to healthy subjects indicate a role for microbiotic composition in IBS pathophysiology.

P1A recombinant beta-lactamase prevents emergence of antimicrobial resistance in gut microflora of healthy subjects during intravenous administration of ampicillin.

Ipsat P1A is a recombinant beta-lactamase which degrades antibiotic residue in the gastrointestinal tract. In an open-label, single-center controlled trial, 36 healthy subjects were randomized to receive (i) ampicillin (1 g intravenously [i.v.] every 6 h [q6h]), (ii) oral P1A recombinant beta-lactamase (8.2 mg q6h), or (iii) ampicillin (1 g i.v. q6h) in combination with oral P1A recombinant beta-lactamase (8.2 mg q6h) for 5 days. Fecal samples were collected before treatment, during treatment (days 3 to 5), and at follow-up (day 12). The primary end points were (i) changes in gastrointestinal microflora (determined by temperature gradient gel electrophoresis [TGGE]) and (ii) emergence of bacterial resistance (determined by conventional microbiology and PCR of TEM beta-lactamase genes). Thirty-five subjects completed the study. The mean similarity percentages of TGGE profiles between baseline and each treatment day sample were significantly lower for the ampicillin group than for the group receiving ampicillin plus P1A recombinant beta-lactamase on days 3, 4, and 5 (P < 0.001). Compared with the ampicillin group, subjects receiving ampicillin plus P1A recombinant beta-lactamase had significantly fewer ampicillin-resistant coliforms on days 3, 4, and 5 and at follow-up (P < or = 0.001) and fewer TEM beta-lactamase genes on days 3, 4, and 5 (P < 0.02). P1A recombinant beta-lactamase was safe and well tolerated. In healthy subjects, P1A recombinant beta-lactamase prevents ampicillin-induced alterations in intestinal microflora, emergence of resistance, and the number of TEM genes.

Critical evaluation of diagnosing bacterial overgrowth in the proximal small intestine.

BACKGROUND: Clinical small bowel bacterial overgrowth (SBBO) syndrome can be objectified by bacterial overgrowth tests. As direct culture of jejunal aspirates has disadvantages, noninvasive tests such as breath tests (BTs) are used. Major drawback of lactulose BT might be rapid lactulose transit to the colon. We evaluated diagnosing bacterial overgrowth using experimental and standard BT, and culture and molecular-based methods. STUDY: Bacterial overgrowth was analyzed in 11 controls and 15 SBBO predisposed subjects. During experimental breath testing, an occlusive balloon limited lactulose to the small intestine. Jejunal fluid was analyzed using culture and molecular-based methods. Bacterial overgrowth was diagnosed on the basis of 20 ppm hydrogen or methane increase above baseline within 90 minutes or more than 10 CFU/mL excluding lactobacilli and streptococci and furthermore using all published definitions. RESULTS: Experimental and standard BT showed no changes in timing of hydrogen excretion between controls and SBBO subjects. Using standard BT, 3/11 controls and 8/15 SBBO subjects were bacterial overgrowth positive. Total counts showed no significant differences between controls and SBBO subjects using culture and molecular-based methods. Bacterial overgrowth was diagnosed in 0/9 controls and 4/12 SBBO subjects using culture-based methods. Other definitions used in literature revealed no significant differences between controls and SBBO subjects. CONCLUSIONS: In a small group of subjects, the experimental BT did not improve the ability of lactulose BT to diagnose bacterial overgrowth. Culturing showed less bacterial overgrowth in controls compared with BT. Remarkably, current diagnostic criteria do not seem to be accurate in discriminating between SBBO subjects and controls.