Succession in the Gut Microbiome following Antibiotic and Antibody Therapies for <em>Clostridium difficile</em>

<div><p>Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to <em>Clostridium difficile</em>. <em>C. difficile</em> infection is typically lethal in hamsters, but protection can be conferred with neutralizing antibodies against the A and B toxins. We compare treatment with neutralizing monoclonal antibodies (mAb) to treatment with vancomycin, which prolongs the lives of animals but ultimately fails to protect them from death. We carried out longitudinal deep sequencing analysis and found distinctive waves of succession associated with each form of treatment. Clindamycin sensitization prior to infection was associated with the temporary suppression of the previously dominant <em>Bacteroidales</em> and the fungus <em>Saccinobaculus</em> in favor of <em>Proteobacteria</em>. In mAb-treated animals, <em>C. difficile</em> proliferated before joining <em>Proteobacteria</em> in giving way to re-expanding <em>Bacteroidales</em> and the fungus <em>Wickerhamomyces</em>. However, the <em>Bacteroidales</em> lineages returning by day 7 were different from those that were present initially, and they persisted for the duration of the experiment. Animals treated with vancomycin showed a different set of late-stage lineages that were dominated by <em>Proteobacteria</em> as well as increased disparity between the tissue-associated and luminal cecal communities. The control animals showed no change in their gut microbiota. These data thus suggest different patterns of ecological succession following antibiotic treatment and <em>C. difficile</em> infection.</p> </div

PCoA analysis displaying the community structure through clindamycin treatment and recovery.

<p>The x-axis indicates time, the y-axis indicates the value of the first principal coordinate, a summary of the UniFrac matrix of distances between bacterial communities. The color code for the different groups is shown to the lower right. Each point represents the community of one animal on one day and the large colored dot on each day is the mean. Red dots- untreated control group; green triangles- UIg; teal squares- IIg; purple plus signs- IV. Vertical bar- range of two standard deviations from the mean.</p

Protection of CD4-IgG2-treated rhesus macaques in a high-dose SIVmac239 challenge experiment.

<p>To maintain serum concentrations, CD4-IgG2 (or control human polyclonal IgG) was administered subcutaneously over a two-week period by an ALZET osmotic pump. Animals were challenged intrarectally with a single high dose inoculum (3–5×10³ TCID₅₀) of SIVmac239 3-days after initiation of CD4-IgG2 administration. (<b>A</b>) Viral loads for animals treated with 200 mg of control polyclonal human IgG as a function of time following SIVmac239 challenge. All control animals became infected. (<b>B</b>) Viral loads for animals administered 20 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Three out of 6 animals were fully protected and one infected animal showed delayed primary viremia. Due to a technical problem with the ALZET osmotic pump, one of the protected animals (98045) did not receive the full dose of 20 mg but this animal did not become infected. (<b>C</b>) Viral loads for animals administered 200 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Five out of 7 animals were protected and showed no sign of infection at any time point. The minimum detection level was 125 SIV RNA copies/ml with a 95% confidence level. Open symbol indicates protected animal, closed symbol indicates infected animal.</p

Plasma concentration of CD4-IgG2 in treated animals.

<p>Animals administered 200 mg of CD4-IgG2 showed plasma concentrations in the range of 500 to 1400 ng/ml at the time of challenge. No apparent correlation between plasma concentration and protection was observed. The CD4-IgG2 concentration at the time of challenge in animals administered 20 mg was 100 ng/ml for 1 animal and below the limit of detection (8 ng/ml) for 2 animals. Serum samples from the remaining 3 animals administered 20 mg were unavailable for this analysis.</p

Taxonomic heatmap of 18S OTUs demonstrating microeukaryote succession in clindamycin-treated hamsters.

<p>Taxonomic heatmap comparing the three untreated control hamsters to five of the UIg individuals. The color code for the heat map is as in previous figures (more intense blue indicates a higher proportion).</p

<i>C. difficile</i>-specific Taqman qPCR analysis of hamster feces and ceca.

<p>(A) A custom qPCR specific for <i>C. difficile</i> DNA was consistently negative for all tested samples from the untreated control group of hamsters. <i>C. difficile</i> DNA in the UIg group rises above the limit of detection by day 7 and remains elevated. The infected hamsters have a detectable spike by day 3 despite having no detectable <i>C. difficile</i> initially. The mAb-treated groups have near-complete survival to day 40 despite having a significant pathogen DNA burden as determined by PCR analysis. Vancomycin-treated animals have a lower spike by day 3 that falls below the limit of detection for most of the subsequent samples. (B) <i>C. difficile</i> 16S RNA copies were detected in luminal and cecal samples. Red bar- group mean 16S copies/ng DNA; gray dots- individual mean 16S copies/ng DNA. Limit of detection is 27 copies per reaction, or ca. 7.9×10³<i>C. difficile</i> genome equivalents per gram of dry weight feces assuming perfect recovery.</p

Anti-human CD4 response in animals treated with CD4-IgG2.

<p>Animal sera were tested in a human CD4-specific ELISA to detect macaque antibody responses against CD4-IgG2. Serum samples were tested up to 23 days post-viral challenge and no responses were detected before day 15, indicating that the animal protection outcome was independent of a response against human CD4. Serum samples from 3 animals administered 20 mg CD4-IgG2 were unavailable for this analysis.</p

Experimental design and pathology.

<p>(A) Hamsters were divided into seven groups, corresponding to one untreated control group (n = 3) and two clindamycin-treated arms with and without <i>C. difficile</i> challenge. Within each arm, hamsters were divided into three treatment groups- none, vancomycin, and antitoxin monoclonal antibody (n = 10 per treatment group). (B) Timeline of interventions. Clindamycin, <i>C. difficile</i>, vancomycin (green bar), and mAb (blue indicator) were given to the designated groups on the days indicated. (C) Gross pathology of Golden Syrian hamsters. Arrow indicates the cecum. [Left] Untreated control (UCtrl) animal on day 40; [Right] Infected, no treatment (I0) animal on day 2. The cecum of the infected, untreated hamster is enlarged, hyperemic, inflamed, and partially necrotic.</p

Natural history of infection includes weight loss and high mortality rate.

<p>(A) Mean change in percent weight per group over the course of the experiment. Deceased hamsters were dropped from the plot. Red dot- mean weight as percent of initial; gray dot- individual weight as percent of initial; red bar- range within 2 standard deviations of the mean; gray dashed line- original weight. (B) Kaplan-Meier survival curve. Untreated control (UCtrl) hamsters survived the duration of the study. Both infected and uninfected untreated animals showed 100% mortality within six days. One animal died in both vancomycin-treated groups on day 2, while the rest of the animals survived between 11 and 16 days post infection, approximately one week after cessation of treatment. Two animals died in the uninfected, mAb-treated group, and one animal died in the infected, mAb-treated group about two weeks post infection. One more hamster from the infected, mAb-treated group died shortly before the end of the experiment and the rest survived until they were euthanized on day 40.</p

Taxonomic heatmap of fecal and cecal bacterial communities demonstrating large-scale community shift and multiple waves of succession following clindamycin administration.

<p>Heatmap showing relative abundance of taxa as a percent of total 16S rRNA tag reads. Each column represents the read-aggregated mean community for that treatment group and day, each row represents a taxon. The uninfected, clindamycin-naïve group is mostly stable and homogenous for the entirety of the experiment, while clindamycin induces a large-scale perturbation in the microbiota. The numbers of animals and the treatments are shown at the top (white plus (“+”) signs indicate dates of administration) and sample origin at the bottom. Proportion contributed by each taxon is shown by the color scale at the top right.</p