Efficacy of oral ramoplanin for inhibition of intestinal colonization by vancomycin-resistant enterococci in mice

Abstract

Ramoplanin is a glycolipodepsipeptide antibiotic with activity against gram-positive bacteria that is in clinical trials for prevention of vancomycin-resistant Enterococcus (VRE) bloodstream infections and treatment of Clostridium difficile diarrhea. Orally administered ramoplanin suppresses VRE intestinal colonization, but recurrences after discontinuation of treatment have frequently been observed. We used a mouse model to examine the efficacy of ramoplanin for inhibition of VRE colonization and evaluated the etiology of recurrences of colonization. Eight days of treatment with ramoplanin (100 microg/ml) in drinking water suppressed VRE to undetectable levels, but 100% of mice developed recurrent colonization; a higher dose of 500 microg/ml in water was associated with recurrent colonization in 50% of mice. Two of eight (25%) mice treated with the 100-microg/ml dose of ramoplanin had low levels of VRE in their cecal tissues on day 8 despite undetectable levels in stool and cecal contents. Mice that received prior ramoplanin treatment did not develop VRE overgrowth when challenged with 10(7) CFU of oral VRE 1, 2, or 4 days later. In communal cages, rapid cross-transmission and overgrowth of VRE was observed among clindamycin-treated mice; ramoplanin treatment effectively suppressed VRE overgrowth in such communal cages. Ramoplanin treatment promoted increased density of indigenous Enterobacteriaceae and overgrowth of an exogenously administered Klebsiella pneumoniae isolate. These results demonstrate the efficacy of ramoplanin for inhibition of VRE colonization and suggest that some recurrences occur due to reexpansion of organisms that persist within the lining of the colon. Ramoplanin treatment may be associated with overgrowth of gram-negative bacilli.

FIG. 1.

Efficacy of oral ramoplanin treatment for decolonization of VRE stool colonization in mice. High-density VRE colonization was established in all mice by administering orogastric VRE on day −8 in conjunction with subcutaneous clindamycin from days −10 to 0. Oral ramoplanin in drinking water (100 [□] or 500 [◊] μg/ml) was given from days 0 to 8 (solid line). Control mice received regular drinking water (•). Error bars represent SE.

FIG. 2.

Mean densities (error bars, SE) of members of the indigenous stool microflora before, during, and after oral ramoplanin treatment from days 0 to 7 (solid line). Symbols: ⧫, total anaerobes; □, Bacteroides; ▴, Enterobacteriaceae; ×, lactobacilli; ○, enterococci.

FIG. 3.

DGGE patterns derived from stool samples before and during antibiotic treatment. Lanes 1, a control pattern containing PCR products obtained from strains of E. coli, Fusobacterium nucleatum, Bacteroides thetaiotaomicron, and Bacteroides uniformis. Lanes 2 and 3, untreated control mice at baseline; lanes 4 and 5, the same control mice 7 days later; lanes 6 and 7, ramoplanin-treated mice at baseline; lanes 8 and 9, the same mice on day 7 of ramoplanin treatment; lanes 10 and 11, clindamycin-treated mice at baseline; lanes 12 and 13, the same mice on day 7 of clindamycin treatment.

FIG. 4.

Mean densities (error bars, SE) of VRE for groups of mice in communal cages. A mouse with high-density VRE stool colonization (∼7 log₁₀ CFU/g) was placed in a cage along with four mice with no previous exposure to VRE. All mice were treated with subcutaneous clindamycin (1.4 mg) once daily from day 0 to day 5. The control cage received regular drinking water (•), and the experimental cage received ramoplanin in drinking water (□) from days 0 to 9 (solid line). On day 9 all mice were placed in individual cages and ramoplanin was discontinued.

FIG. 5.

Effect of oral ramoplanin administration on the establishment of intestinal colonization with ESBL-producing K. pneumoniae (ESBL-KP) (A) or C. glabrata (B) in mice. Mice received 10⁶ CFU of ESBL-KP or C. glabrata by orogastric inoculation on day 0 and either regular drinking water (controls [solid symbols]) or oral ramoplanin in drinking water (open symbols) from days −2 to 4 (solid line). Error bars represent SE.