1. Median Total Bacteria by qPCR
THE EFFECT OF RIFAXIMIN ON GUT FLORA
IN NORMAL OUTBRED RATS
Walter Morales, Gene Kim, Mi-Sung Kim, Andres Ardila-Hani, Sharon Kim, Stacy Weitsman,
Christopher Chang, Mark Pimentel
GI Motility Program, Cedars-Sinai Medical Center, Los Angeles, CA
BACKGROUND
Rifaximin, a non-absorbable gut specific antibiotic in the rifamycin class and is currently approved for traveler's diarrhea and hepatic encephalopathy but has also been deemed effective in the treatment of irritable bowel syndrome (IBS). The drug is not absorbed due to its hydrophobic nature and some speculate that rifaximin best works in the small intestine due to the presence of bile.
Two important questions have been raised about this drug by regulators. First, is the effect of rifaximin on Staphylococcal resistance. In a paper now published, a 10 day course of rifaximin does not lead to rifampin resistance rifaximin in outbred rats. In fact, there is less resistance in rifaximin treated rats. The second question is the location and effect of rifaximin on intestinal microflora.
AIM
In this study, we examine the short and long term effects of rifaximin on gut flora in normal outbred Sprague-Dawley rats.
METHOD
Thirty adult, male Sprague-Dawley rats were equally divided into 3 groups:
Group 1 was gavaged daily for 10 days with PBS alone (control).
Group 2 was gavaged daily with 200mg of rifaximin suspended in PBS.
Group 3 included 10 rats that were gavaged for 10 days with rifaximin 200mg daily and then housed for an additional 30 days before euthanasia.
Rats in group 1 and 2 were euthanized at day 10. Prior to and during gavage, rats had daily stool collection for the quantitation of viable Staphylococcal species (PEA agar) and coliforms (MacConkey agar) using serial dilutions. After euthanasia, segments of duodenum, jejunum, ileum, cecum and left colon were ligated to preserve luminal contents. DNA extraction and qPCR of contents was undertaken to determine total luminal bacterial counts in each segment.
RESULTS
At baseline, rats had a median of 2.90x106cfu/ml (range=5.60x x106cfu/ml) combined total stool Staphylococcus spp. After rifaximin, the median total count dropped significantly to 1.20x105cfu/ml (range=0-8.6x105cfu/ml) (p<0.01 by Wilcoxon Rank Sum test for matched pairs) (Figure 1). Interestingly, in the rats followed for 30 additional days off rifaximin, the stool Staphylococcal counts continued to drop to near non-detectable levels.
In the case of stool coliforms, at baseline, rats had a median of 1.86x104 cfu/ml. By day 10 of rifaximin, stool coliform counts dropped to 2.2x103 cfu/mL (P<0.01, Wilcoxon Rank sum test for matched pairs). However, complete recovery of stool coliforms was seen by 3 days after cessation of therapy (Figure 2).
After dissection, coliforms were rare in the duodenum and jejunum as expected in normal animals. However, rifaximin did not affect the large number of coliforms in the colon (Figure 3).
Using qPCR for total bacteria, only the duodenum demonstrated a significant reduction in bacteria (P=0.05). Interestingly, there was a trend for continued decline in bacteria in the duodenum even after cessation of rifaximin (P=0.08) (Figure 4).
Figure 1: Median Staphylococcus spp. counts pre and post treatment
Figure 2: Median stool coliform counts pre and post treatment by culture
P<0.01
P<0.01
Figure 3: Stool coliform counts by segment between rfiaximin treated and controls
Figure 4: Total bacteria in each segment after treatment of rifaximin
P=0.05
Median Total Bacteria by qPCR
Duodenum Jejunum Ileum
CONCLUSIONS
Rifaximin has no durable effect on stool colonic coliform bacteria although durable reductions in stool Staphylococcal species are seen.
Using qPCR, rifaximin has a durable effect on reducing total flora of the duodenum in normal rats.