Giardia Alters Commensal Microbial Diversity throughout the Murine Gut

Abstract

Giardia lamblia is the most frequently identified protozoan cause of intestinal infection. Over 200 million people are estimated to have acute or chronic giardiasis, with infection rates approaching 90% in areas where Giardia is endemic. Despite its significance in global health, the mechanisms of pathogenesis associated with giardiasis remain unclear, as the parasite neither produces a known toxin nor induces a robust inflammatory response. Giardia colonization and proliferation in the small intestine of the host may, however, disrupt the ecological homeostasis of gastrointestinal commensal microbes and contribute to diarrheal disease associated with giardiasis. To evaluate the impact of Giardia infection on the host microbiota, we used culture-independent methods to quantify shifts in the diversity of commensal microbes throughout the gastrointestinal tract in mice infected with Giardia We discovered that Giardia's colonization of the small intestine causes a systemic dysbiosis of aerobic and anaerobic commensal bacteria. Specifically, Giardia colonization is typified by both expansions in aerobic Proteobacteria and decreases in anaerobic Firmicutes and Melainabacteria in the murine foregut and hindgut. Based on these shifts, we created a quantitative index of murine Giardia-induced microbial dysbiosis. This index increased at all gut regions during the duration of infection, including both the proximal small intestine and the colon. Giardiasis could be an ecological disease, and the observed dysbiosis may be mediated directly via the parasite's unique anaerobic fermentative metabolism or indirectly via parasite induction of gut inflammation. This systemic alteration of murine gut commensal diversity may be the cause or the consequence of inflammatory and metabolic changes throughout the gut. Shifts in the commensal microbiota may explain observed variations in giardiasis between hosts with respect to host pathology, degree of parasite colonization, infection initiation, and eventual clearance.

Keywords: Giardia; microbiome; parasite; pathogenesis.

FIG 1

Sampling scheme for querying murine gut ecological shifts during giardiasis with and without antibiotic treatment. Two cohorts (n = 16) of sibling mice, one not treated with antibiotics and one treated with antibiotics, were used to test the potential effects of Giardia colonization on the microbial diversity of the murine gut. Study animals were infected with Giardia lamblia GS trophozoites (n = 12 animals per treatment group) or a saline vehicle control (uninfected [U]; n = 4 per treatment group) by gavage. Cohorts of infected animals were sacrificed at 3, 7, and 14 days postgavage (n = 4 per day). Gut intestinal tracts for each treatment group and for each day postinoculation were dissected for subsequent diversity analyses (pSI, dSI, CEC, and COL). The total number of reads per cohort (n = 4) are shown for each experimental condition. Luminal and mucosal reads were pooled for most analyses (see Materials and Methods for details). The total reads per animal within each cohort under each experimental condition are summarized in Table S1 in the supplemental material.

FIG 2

Giardia infection significantly alters foregut and hindgut microbial diversity in antibiotic-treated and -naive mice. (A) Comparisons of the fold change in Giardia burden in pSI samples based on QPCR results between antibiotic-treated and nontreated animals are presented for days 3, 7, and 14 post-Giardia gavage. Asterisks indicate significant differences as assessed with Sidek's multiple comparison test. (B to E) Weighted Unifrac comparisons of beta diversity are shown using PCoA plots to show the variance in the diversity of all foregut samples and hindgut samples with and without antibiotic treatment. Samples with a saline vehicle control and samples after 2 weeks of Giardia infection are shown. Significance (P value) and the strength of explained variation (R²) were assessed with the adonis program.

FIG 3

Overall alpha diversity by intestinal site is not significantly changed during giardiasis. (A) Sampling adequacy (alpha diversity) was assessed using rarefaction plots to compare all samples using the Chao1 estimate during the course of giardiasis (uninfected and day 3, day 7, and day 14) with antibiotic treatment (see Fig. S1 for results with no antibiotic treatment). (B to D) Alpha diversity measures of species richness, evenness, and phylogenetic distance, summarizing microbial diversity in regions of the murine gut (pSI, dSI, CEC, and COL) in control, antibiotic-treated, noninfected cohorts (U), and in infected cohorts (days 3, 7, and 14). For these analyses, the OTU tables were rarefied to 3,000 reads/sample and analyzed using QIIME to compare the relative read abundances. Chao 1 estimates of species richness for both antibiotic-treated groups are shown in panel B; increased Chao1 values correspond to increased numbers of unique taxa. Species evenness was also estimated using the Shannon index (C), where a high Shannon value indicated equal distribution between species and a low Shannon value indicated dominance by certain taxa. Lastly, the phylogenetic distance (D) was used to estimate the evolutionary relationships of the community; a higher phylogenetic distance value corresponds to a more diverse community. Error bars correspond to variations between gut samples of each of the four animals in one experimental group.

FIG 4

Significant changes in gut microbiome diversity in the proximal small intestine and colon during giardiasis. At 14 days postinfection, the relative read abundance (as a percentage) of the most abundant bacterial divisions and phyla are shown for the mouse foregut (pSI [A] and dSI [B]) and the mouse hindgut (CEC [C] and COL [D]). Each anatomic section was compared to the cognate uninfected sample.

FIG 5

Significant shifts in beta diversity in both the murine foregut and hindgut during giardiasis. The relative abundance levels of bacterial taxa that were significantly enriched or depleted after 14 days of Giardia infection were plotted by comparing the fold changes in uninfected versus infected animals, using the same anatomical sample. The taxonomy of each species is indicated. Abbreviations: aProt, Alphaproteobacteria; bProt, Betaproteobacteria; gProt, Gammaproteobacteria; BacF, bacilli (Firmicutes); EryF, Erysipelotrichi (Firmicutes); ClosF, Clostridia (Firmicutes). Melainabacteria were categorized as Cyanobacteria-chloroplasts in QIIME. Underlined taxa were used to calculate the murine giardiasis dysbiosis index (see Fig. 7 and also Fig. S7).

FIG 6

Model of Giardia-induced dysbiosis in the proximal small intestine with implications for disease symptoms. Hypothesized metabolic interactions between fermentative Giardia trophozoites with both the host commensal microbiota and host epithelium of the proximal small intestine, the primary site of parasite colonization. Reactive oxygen and nitrogen species (ROS and NOS) are induced by inflammatory responses. Trophozoites ferment sugars and amino acids and can excrete various waste products, depending on the oxygen tension in the surrounding lumen. Primary shifts in diversity of the commensal Rhodocyclaceae and Moraxellaceae and the Clostridiales (as well as Melainabacteria) are indicated and are also summarized in our Giardia microbial dysbiosis index (see Fig. 7).

FIG 7

The murine giardiasis dysbiosis index defines microbial community shifts during the time course of infection in antibiotic-treated animals. The murine giardiasis dysbiosis index was calculated for each sample by dividing the sum of the abundances of the Rhodocylaceae and Moraxellaceae families by the abundance of Clostridiaceae (see Materials and Methods). The average MGDI per body site over the infection time course is presented. (A) The MGDI increased during infection for all body sites analyzed. (B) PCoA revealed the infection time course as an explicit axis. Samples are shaded according to their calculated MGDI, with darker shading intensities representing higher MGDI values.