Probiotic Product Enhances Susceptibility of Mice to Cryptosporidiosis

Abstract

Cryptosporidiosis, a leading cause of diarrhea among infants, is caused by apicomplexan parasites classified in the genus Cryptosporidium The lack of effective drugs is motivating research to develop alternative treatments. With this aim, the impact of probiotics on the course of cryptosporidiosis was investigated. The native intestinal microbiota of specific pathogen-free immunosuppressed mice was initially depleted with orally administered antibiotics. A commercially available probiotic product intended for human consumption was subsequently added to the drinking water. Mice were infected with Cryptosporidium parvum oocysts. On average, mice treated with the probiotic product developed more severe infections. The probiotics significantly altered the fecal microbiota, but no direct association between ingestion of probiotic bacteria and their abundance in fecal microbiota was observed. These results suggest that probiotics indirectly altered the intestinal microenvironment or the intestinal epithelium in a way that favored proliferation of C. parvumIMPORTANCE The results of our study show that C. parvum responded to changes in the intestinal microenvironment induced by a nutritional supplement. This outcome paves the way for research to identify nutritional interventions aimed at limiting the impact of cryptosporidiosis.

Keywords: Cryptosporidium; cryptosporidiosis; fecal microbiota; gut microbiota; probiotics.

FIG 1

Effect of probiotics on the severity of C. parvum cryptosporidiosis in immunosuppressed mice. The graphs show oocyst counts (on a logarithmic scale), expressed as oocysts per gram of feces, for 3 independent experiments. In experiments 1 and 2, mice were sampled individually. Values represent the means for 4 mice. Error bars show SDs. In experiment 3, each group of 4 mice was sampled together. Error bars for experiment 3 show SDs based on 5 replicate FCM counts for selected samples.

FIG 2

Impact of probiotics on the fecal microbiome of C. parvum-infected mice. PCoA was used to display weighted UniFrac distances between pairs of fecal microbiome samples. Experiment 1 analysis (left) included data for 64 fecal samples collected from individual mice from day 5 of treatment (day 4 p.i.) to day 16 of treatment (day 15 p.i.). For experiment 2, 55 samples from individual mice were analyzed. Each data point represents 1 sample, color coded according to treatment and group as shown in Fig. 1. Matching triangle symbols indicate replicate analyses of the same fecal samples. Data from experiment 3 are not shown because groupwise sample collection resulted in a small number of data points. PC, principal coordinate.

FIG 3

Lack of relatedness of oocyst output to microbiome diversity. Oocyst output, normalized for fecal volume, was plotted against Shannon diversity for 44 samples, collected on 3 days, that were analyzed for both properties in experiment 1 and for 53 samples from experiment 2. Color coding indicates the experimental group, as described in Fig. 1. Samples were collected from individual mice. Due to the small number of data points, data for experiment 3 were not analyzed.

FIG 4

Taxonomy of the fecal microbiome in heavily infected and lightly infected mice, showing that heavy infections were associated with increased abundance of Proteobacteria. For each experiment, bacterial taxa significantly associated with the severity of infection were identified using LEfSe (24), in a comparison of 10 samples with the highest (high) and lowest (low) oocyst concentrations; 20 samples were included for each experiment. The color indicates the phylum and the color intensity the genus or highest taxonomic level (e.g., family or order) identified. Green, Proteobacteria; red, Firmicutes; blue, Actinobacteria.