Disruption of the Human Gut Microbiota following Norovirus Infection

<div><p>The gut microbiota, the collection of all bacterial members in the intestinal tract, plays a key role in health. Disruption of the indigenous microbiota by a variety of stressors, including antibiotic therapy and intestinal infections, is associated with multiple health problems. We sought to determine if infection with Norovirus disrupts the gut microbiota. Barcoded pyrosequencing of the 16S rRNA-encoding gene was used to characterize the stool microbiota in Norovirus-infected human patients (n = 38). While the microbiota in most infected patients (n = 31) resembled that seen in uninfected healthy controls, a minority of patients (n = 7) possessed a significantly altered microbiota characterized by reduced relative numbers of Bacteriodetes and a corresponding increase in Proteobacteria. In these patients, the increase in Proteobacteria was due to a single operational taxonomic unit (OTU) of <em>Escherichia coli</em>. We cultured <em>E. coli</em> from Norovirus-infected patients and characterized them using PCR-ribotyping and virulence factor analysis. Multiple ribotypes were encountered, but none possessed typical virulence factors commonly carried by enteropathogenic <em>E. coli</em> strains. Microbiota disruption and elevated Proteobacteria were not significantly correlated to patient age, gender, sampling time following illness onset, or overall gut inflammation. These results demonstrate that some patients have a disrupted microbiota following Norovirus infection, and therefore may be at elevated risk for long-term health complications.</p> </div

Summary of PCR-ribotypes detected in <i>Escherichia coli</i> cultured from several Norovirus patients.

<p>Abbreviations: UG, Norovirus patient with an undisrupted microbiota; DG, Norovirus patient with a disrupted microbiota.</p><p>Individuals with Norovirus infection contained a dominant type of <i>E. coli</i> (80–100% of isolates examined were a single type). Additionally, some PCR ribotypes were shared amongst multiple Norovirus patients, but most types were unique to each patient and each group. Ribotypes were assigned based upon allele combinations seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048224#pone.0048224.s009" target="_blank">Table S5</a>.</p

Comparison of Norovirus and healthy control patient pooled patient data.

<p>Abbreviations: HMP, Human Microbiome Project (healthy control population); DPO, days post-onset (the time between symptom onset and sample collection); n/a, not applicable.</p

Some Norovirus patients have a highly divergent microbiota compared to controls.

<p>Principle coordinates analysis showed the community structure relationship between NoV and HMP patients. This ordination was generated using a Yue and Clayton-based distance matrix representing the relative abundance of OTUs in each community at a 3% OTU definition level. The community of each patient is indicated by a symbol. Arrows depict how the top five most frequently detected OTUs influence the location where each patient is represented. A subset of NoV-infected individuals display a highly divergent community characterized by decreased Bacteriodetes and an increased Proteobacteria. The patients circled represent the disrupted group, which are dominated by Proteobacteria. The majority of the elevated Proteobacteria in these patients was from a single OTU of <i>E. coli</i>.</p

Patients with high Proteobacteria are dominated by high levels of <i>Enterobacteriaceae</i>.

<p>The average family level diversity within all Proteobacteria reads showed that the vast majority of Proteobacteria in disrupted patients was <i>Enterobacteriaceae</i>. Undisrupted and HMP patients had lower overall reads, and showed a higher diversity of reads within Proteobacteria, but lacked a single dominant bacterial family.</p

Patients infected with Norovirus show differential disruption.

<p>A) Dendrogram showing the phylogenetic relationship between samples, as calculated by the Yue and Clayton dissimilarity index at a 3% OTU level was paired with a phylum-level classification of the communities for each individual. B) Comparisons between different groups by a one-way ANOVA test showed that the disrupted patients have significantly less Bacteroidetes and significantly more Proteobacteria compared to the undisrupted and HMP patients. Abbreviations: HMP = Healthy control patient from the Human Microbiome Project.</p

Amplification Ct cut-off.

<p>For each of 71 sputum samples the average SYTO9 Ct value for each of the 11 amplicons is shown. Average Ct are organized according to whether the sputum probe result was wild-type (black -), mutant (black x), or negative. Sputum probe negative results are further stratified into whether the cultured isolate was found to be wild-type (red +), mutant (red -), or to possess an “other” mutation (green X). ROC analysis was performed to examine the optimal Ct whereby SYTO9 to interpret the probe results.</p

Sequencing confirmation of the discordance samples between TAC and phenotypic DST.

<p>Sequencing confirmation of the discordance samples between TAC and phenotypic DST.</p

Correlation between genotypic and phenotypic drug susceptibility test.

<p>Correlation between genotypic and phenotypic drug susceptibility test.</p

Sensitivity of TAC to detect drug resistance on sputum samples.

<p>Sensitivity of TAC to detect drug resistance on sputum samples.</p