Association Between Clostridium innocuum and Antibiotic-Associated Diarrhea in Adults and Children: A Cross-sectional Study and Comparative Genomics Analysis

Abstract

Background: A recent study from Taiwan suggested that Clostridium innocuum may be an unrecognized cause of antibiotic-associated diarrhea (AAD) and clinically indistinguishable from Clostridioides difficile infection. Our objective was to compare C. innocuum prevalence and strain between those with AAD and asymptomatic controls.

Methods: In this cross-sectional study, we collected stool from 200 individuals with AAD and 100 asymptomatic controls. We evaluated the association between AAD and C. innocuum in stool using anaerobic culture and quantitative polymerase chain reaction (qPCR). To identify strain-specific associations with AAD, we performed whole-genome sequencing of C. innocuum isolates using Illumina MiSeq and constructed comparative genomics analyses.

Results: C. innocuum was isolated from stool of 126/300 (42%) subjects and more frequently from asymptomatic controls than AAD subjects (50/100 [50%] vs 76/200 [38%], respectively; P = .047). C. innocuum isolation frequency was not associated with AAD in either the adult or pediatric subgroups. C. innocuum and C. difficile were frequently co-prevalent in individuals with and without diarrhea. There were no phylogenetic differences or accessory genome associations between C. innocuum isolates from AAD subjects and asymptomatic controls.

Conclusions: C. innocuum was frequently isolated and at a greater frequency in asymptomatic controls than those with AAD. We did not identify strain lineages or accessory genomic elements associated with AAD. These data highlight that differentiating C. innocuum-associated diarrhea from asymptomatic colonization, and differentiating diarrhea caused by C. difficile from C. innocuum, are clinical microbiology challenges that require additional investigation to identify host-specific factors and/or biomarkers that distinguish these conditions.

Keywords: Clostridioides difficile; Clostridium innocuum; antibiotic-associated diarrhea; comparative genomics; whole-genome sequencing.

Figure 1.

Flowchart of subjects included in this study. ^atcdB PCR negative, toxin EIA negative; ^btcdB PCR positive, toxin EIA negative. Abbreviations: EIA, enzyme immunoassay; PCR, polymerase chain reaction.

Figure 2.

Clostridium innocuum culture positivity rates and Ct values from subjects with AAD and asymptomatic controls. A, B, Percentage of CI culture positivity rates for a cohort of subjects with (A) diarrhea (Di) or asymptomatic controls (As), and (B) subjects with diarrhea who are either C. difficile colonized (Di-CDC) or C. difficile negative (Di-CDN). C, D, Box plots of CI Ct values as determined by qPCR of (C) As and Di subjects and (D) As, Di-CDC, and Di-CDN subjects. Boxes show the median and 25th and 75th percentiles. The whiskers represent the maximum and minimum data values. *P < .05 (or Bonferroni-corrected P < .25 for pairwise comparisons of multiple groups in panels B and D); **P < .01; ***P < .001. Abbreviations: AAD, antibiotic-associated diarrhea; CI, Clostridium innocuum; Ct, cycle threshold; ns, not significant; qPCR, quantitative polymerase chain reaction.

Figure 3.

Cladogram of C. innocuum isolates stratified by the presence of AAD showing 119 successfully sequenced isolates from adults (Ad) and children (Ch), 71 with AAD (Di), with and without C. difficile colonization (CDC and CDN, respectively), and 50 asymptomatic controls (As). Isolates from those with AAD are indicated by the black bars. The 2 reference strains, LC-CI [8] and CI-ATCC [7], are indicated by the gray bar bars. Abbreviations: AAD, antibiotic-associated diarrhea; ATCC, American Type Culture Collection; CI,Clostridium innocuum.