Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease

Abstract

dRecurrent Clostridium difficile infection (CDI) is of particular concern among health care-associated infections. The role of the microbiota in disease recovery is apparent given the success of fecal microbiota transplantation (FMT) for recurrent CDI. Here, we present a murine model of CDI relapse to further define the microbiota recovery following FMT. Cefoperazone-treated mice were infected with C. difficile 630 spores and treated with vancomycin after development of clinical disease. Vancomycin treatment suppressed both C. difficile colonization and cytotoxin titers. However, C. difficile counts increased within 7 days of completing treatment, accompanied by relapse of clinical signs. The administration of FMT immediately after vancomycin cleared C. difficile and decreased cytotoxicity within 1 week. The effects of FMT on the gut microbiota community were detectable in recipients 1-day posttransplant. Conversely, mice not treated with FMT remained persistently colonized with high levels of C. difficile, and the gut microbiota in these mice persisted at low diversity. These results suggest that full recovery of colonization resistance against C. difficile requires the restoration of a specific community structure. R ecently, the Centers for Disease Control identified Clostridium difficile as an urgent nosocomial threat. In particular, recurrent C. difficile infection (CDI) is a major concern (1). Up to 30% of patients experience recurrent symptoms following cessation of antibiotic therapy, and subsequent recurrences are more likely after a primary recurrence incidence (2, 3). A healthy, diverse gastrointestinal microbial community, termed the gut microbiota, is important in resistance against the development of CDI. The normal gut microbiota provides colonization resistance (the ability to resist pathogen colonization) against C. difficile (4). In both human studies and animal models, perturbation of the indigenous gut microbiota by antibiotic administration has been correlated to a susceptible community structure, ultimately leading to CDI (5-8). Although it is clear that antibiotic use is correlated with the majority of CDI cases (9), the standard therapy for CDI is the administration of an antibiotic regimen with activity against C. difficile (typically vancomycin or metronidazole). Paradoxically, this may further perturb the intestinal microbial community (10, 11). Therefore, it is important to understand the impact of antibiotic treatment for CDI on recovery of the gut microbiota and colonization resistance.In a murine model of persistent CDI, we have previously shown that the use of sequential antibiotics can delay recovery of bacterial diversity (11). Previous studies have suggested that patients who develop recurrence have a less diverse community than patients who have stable recovery (12). The idea that restoration of the gut microbiota is necessary to prevent or mitigate recurrence is supported by an ϳ90% success rate of fecal microbiota transplantation (FMT) (13), and significant recovery of diver...

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“…DNA was isolated from fecal and cecal samples using a PowerMag™ Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) on an epMotion® 5075 TMX automated liquid handling system (Eppendorf, Hamburg, Germany) according to manufacturer instructions. The V4 region of the 16S rRNA gene was amplified and sequenced as described previously [47]. PCR reactions used 1 μl undiluted DNA, 1 μl of DNA diluted 1:10, 5 μl of DNA diluted 1:50, 1 μl of DNA diluted 1:50, or 1 μl of DNA diluted 1:100.…”

“…PCR reactions used 1 μl undiluted DNA, 1 μl of DNA diluted 1:10, 5 μl of DNA diluted 1:50, 1 μl of DNA diluted 1:50, or 1 μl of DNA diluted 1:100. Sequencing was performed on an Illumina® MiSeq sequencer (Illumina, Inc., San Diego, CA), as described previously[47]. 16S rRNA gene sequences were processed and analyzed using the mothur software package version 1.34.4, 1.35.1 and 1.36.1 according to the publicly available MiSeq SOP [48,49].…”

The gut microbiome composition associates with bipolar disorder and illness severity

“…DNA was isolated with a PowerMag Microbiome RNA/DNA Isolation Kit (Mo Bio Laboratories, Inc.) using an epMotion 5075 liquid handling system. The V4 region of the bacterial 16S rRNA gene was amplified using a dual-indexing sequencing strategy and sequenced on the Illumina MiSeq platform as described previously (18). Negative control wells including all PCR reagents with water replacing sample template DNA were included across the plates and showed no visible bands upon gel analysis.…”

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“…[21]. The V4 amplicon pool was prepared and sequenced with a MiSeq (Illumina) as described previously [22]. …”