Influence of rifaximin treatment on the susceptibility of intestinal Gram-negative flora and enterococci

Authors


H. L. DuPont, 6720 Bertner Ave, MC 1-164, Houston, TX 77030, USA
E-mail: hdupont@sleh.com

Abstract

The development of rifaximin- and rifampicin-resistant intestinal coliforms was studied in 27 subjects receiving rifaximin for 3 days by plating stool samples on media containing rifaximin 200 mg/L or rifampicin 64 mg/L before treatment (day 0), after treatment was completed (day 3), and after a further 2 days (day 5). The susceptibility of enterococci grown on day 0 and day 3 was also studied in 71 subjects. Significant increases in antimicrobial-resistant coliform flora were not seen in either the rifaximin-treated or the placebo-treated subjects. Enterococci recovered pre- and post-treatment showed similar susceptibilities. Rifaximin did not select for significant resistance in the Gram-negative and Gram-positive intestinal flora during therapy.

The aim of this study was to determine whether rifaximin, used for treatment of acute travellers' diarrhoea, encouraged the development of rifaximin- and rifampicin-resistant Gram-negative and Gram-positive faecal flora. College students from the USA who suffered from diarrhoea in Guadalajara, Mexico during the summer of 2000 were included in a placebo-controlled trial evaluating rifaximin treatment [1]. Stool samples were collected from subjects receiving rifaximin 200 mg three times daily (600 mg/day) for 3 days, rifaximin 400 mg three times daily (1200 mg/day) for 3 days, or a placebo for 3 days. Samples were collected immediately before therapy (day 0), immediately after therapy (day 3), and after a further 2 days (day 5), and were investigated for resistant flora as described previously [2].

Ten-fold dilutions (to 10−5) of samples from 27 patients were plated (100 µL) on to Mueller–Hinton agar (Becton Dickinson, Sparks, MA, USA) containing rifaximin (Salix Pharmaceuticals, Raleigh, NC, USA) 200 mg/L or rifampicin (Sigma, St Louis, MO, USA) 64 mg/L. Rifaximin 200 mg/L was selected because this concentration is eight-fold higher than the median MIC90 for enteric pathogens, and is at the upper range of susceptibility for any of the enteric bacterial pathogens studied [3]. Rifampicin 64 mg/L was selected because this is the upper range of susceptibility for Gram-positive and Gram-negative bacteria [4,5]. Following incubation overnight at 37°C, one colony of a Gram-negative bacterium isolated on rifaximin- or rifampicin-containing agar from each of three stool samples (days 0, 3 and 5)/patient (three colonies/patient) was identified with the API 20E system (bioMérieux, Marcy L'Etoile, France). MICs of rifaximin were determined [3] in the Houston laboratory for one colony of Gram-negative bacteria/stool sample growing on either rifaximin or rifampicin.

Enterococci were recovered from the day 0 and day 3 samples taken from 71 patients by plating on to Streptococcus faecalis medium (Bacto SF Medium; Difco Laboratories, Detroit, MI, USA) and Bacto Bile Esculin Agar (Difco), and incubating at 45.5°C for 72 h. Putative colonies of enterococci were transferred to storage media stabs (BHI agar; Difco) and transported to Houston for further testing. Colonies isolated from both pre- and post-treatment samples were tested for susceptibility to rifaximin [3]. Differences in numbers of rifaximin- or rifampicin-resistant coliforms, and differences in post-treatment vs. pre-treatment susceptibilities of enterococci, were compared between groups with Student's t-test.

The numbers of rifaximin- and rifampicin-resistant coliforms seen in the pre-treatment stool sample and following treatment with rifaximin or placebo are shown in Fig. 1. Low numbers of rifaximin- and rifampicin-resistant coliforms were isolated from the initial stool sample in all groups before treatment. Baseline pre-treatment numbers of antimicrobial-resistant coliforms were higher for the groups randomised to receive 1200 mg/day and 600 mg/day, compared with the group randomised to receive a placebo, but these differences were not significant (p 0.0683; Wilcoxon Mann–Whitney test). Significant increases in the numbers of rifaximin- or rifampicin-resistant coliforms were not found in the day 3 and day 5 post-treatment samples, compared with the pre-treatment baseline samples for the three groups. Similar values were seen for the same subjects when stool samples were plated on to rifampicin-containing medium.

Figure 1.

Numbers of faecal rifaximin- and rifampicin-resistant coliforms (GNB) isolated pre-treatment (day 0), immediately post-treatment (day 3) and 2 days post-treatment (day 5) from patients treated for 3 days with either rifaximin 1200 mg/day, rifaximin 600 mg/day or placebo.

Twenty-seven coliforms growing on rifaximin-containing agar were identified biochemically as Escherichia coli. These isolates did not show increased resistance when pre-treatment stool isolates were compared with post-treatment isolates in the three groups. The MIC90 for coliforms growing on rifaximin- or rifampicin-containing media from pre-treatment (day 0) and post-treatment (days 3 and 5) stools was 64 mg/L for subjects randomised to receive either of the two doses of rifaximin (range 1–128 mg/L) or for subjects in the placebo group (range 2–128 mg/L).

Enterococci were isolated from pre-treatment and post-treatment samples at similar frequencies (10/24 vs. 20/24 for the 1200 mg/day group; 9/23 vs. 18/23 for the 600 mg/day group; 8/24 vs. 17/24 for the placebo group). In total, enterococci were recovered from both pre- and post-treatment stool samples for 27 (38%) of 71 patients. MICs of enterococci were ≤ 64 mg/L for rifaximin and ≤ 16 mg/L for rifampicin. No significant changes in susceptibility were seen between the pre-treatment and post-treatment samples.

Rifaximin is a poorly absorbed rifamycin derivative showing bactericidal activity against a broad range of enteric pathogens [3,6]. Rifaximin is available in several countries in Europe, Asia and Latin America for the treatment of bacterial diarrhoea [7]. It has also been used for the treatment of small-bowel bacterial overgrowth syndrome [8], hepatic encephalopathy [9], and diverticular disease of the colon, as well as for preventing infectious complications in colorectal surgery [7]. A paediatric suspension of this drug has been developed for children with bacterial diarrhoea [10].

Although the development of resistance is a major concern when rifamycins are used for therapy, rifaximin did not select for significant resistance among gut flora when given for 3 days. It is unlikely that rifaximin would stimulate the development of rifampicin-resistant Mycobacterium tuberculosis, since the drug remains largely in the gut during short-term use for the treatment of diarrhoea. Extra-intestinal tissues infected with M. tuberculosis should not be exposed to significant concentrations of the drug, and growth of M. tuberculosis on media containing varying concentrations of rifaximin does not lead to the selection of rifampicin-resistant strains [11].

In summary, rifaximin appears to be a suitable drug for the management of travellers' diarrhoea [12] and other non-systemic, non-dysenteric enteric bacterial infections. This drug has now been licensed for use in the USA.

Acknowledgements

This work was supported by a grant from Salix Pharmaceuticals and Public Health Service grant DK56338, which funds the Texas Gulf Coast Digestive Diseases Center.

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