Diarrhoeal events can trigger long-term Clostridium difficile colonization with recurrent blooms

Abstract

Although Clostridium difficile is widely considered an antibiotic- and hospital-associated pathogen, recent evidence indicates that this is an insufficient depiction of the risks and reservoirs. A common thread that links all major risk factors of infection is their association with gastrointestinal disturbances, but this relationship to C. difficile colonization has never been tested directly. Here, we show that disturbances caused by diarrhoeal events trigger susceptibility to C. difficile colonization. Using survey data of the human gut microbiome, we detected C. difficile colonization and blooms in people recovering from food poisoning and Vibrio cholerae infections. Carriers remained colonized for year-long time scales and experienced highly variable patterns of C. difficile abundance, where increased shedding over short periods of 1–2 d interrupted week-long periods in which C. difficile was undetectable. Given that short shedding events were often linked to gastrointestinal disturbances, our results help explain why C. difficile is frequently detected as a co-infecting pathogen in patients with diarrhoea. To directly test the impact of diarrhoea on susceptibility to colonization, we developed a mouse model of variable disturbance intensity, which allowed us to monitor colonization in the absence of disease. As mice exposed to avirulent C. difficile spores ingested increasing quantities of laxatives, more individuals experienced C. difficile blooms. Our results indicate that the likelihood of colonization is highest in the days immediately following acute disturbances, suggesting that this could be an important window during which transmission could be interrupted and the incidence of infection lowered.

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Fig. 1: C. difficile colonization and blooms are associated with recovery from diarrhoeal disturbances.
Fig. 2: C. difficile colonizes patients recovering from V. cholerae infections.
Fig. 3: Azithromycin treatment has no measurable impact on C. difficile colonization and carriage in infants relative to placebo controls.
Fig. 4: Increasing disturbance intensity increases the likelihood and magnitude C. difficile blooms in mice.

Data availability

The publicly available data that we reanalysed here were generated by: David et al.30, European Nucleotide Archive under the accession number ERP006059; Hsaio et al.33, NCBI Short Read Archive (SRA) under the accession number PRJEB6358; Caporaso et al.36, MG-RAST under the accession number MG-RAST:4457768.3-4459735.3; Parker et al.34, SRA under accession number ERP022953; and The NIH Human Microbiome Project37 on NCBI under the accession number PRJNA43017. The amplicon sequencing data from the mouse model of varied disturbance intensity are available on the NCBI SRA under the accession number PRJNA507320. The genome of C. difficile BAA1801 is available on NCBI under the accession number PRJNA562328. All source data necessary to reproduce the figures presented in this manuscript are included in this article and its Supplementary information files. The microscope images used to calculate the cell and spore density are available online at Figshare (https://figshare.com/projects/Source_Data_for_Diarrheal_events_can_trigger_long-term_Clostridium_difficile_colonization_with_recurrent_blooms/72824).

Code availability

All custom computer code necessary to reproduce our results are available on GitHub (https://github.com/polzlab/VanInsberghe_2019_Cdiff_colonization).

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Acknowledgements

This work was supported by a grant from the Center for Microbiome Informatics and Therapeutics to M.F.P. D.V. was partially supported by a fellowship through the Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship-Doctoral. We thank I. Nachamkin (University of Pennsylvania) and N. Pollock (Harvard Medical School) for their comments, which helped improve this study.

Author information

The concept was developed by D.V. and M.F.P. D.V. performed the 16S rRNA amplicon sequence data and C. difficile genomic analyses. D.V., M.F.P. and S.E. designed the mouse model of variable disturbance intensity and D.V. performed the experiment that utilizes this model with the help of B.V. J.A.E. designed the pipeline for analysing the 16S rRNA amplicon sequences from the mouse faecal samples. T.P. and S.E. performed the histological analyses. D.V. and M.F.P. prepared the manuscript.

Correspondence to Martin F. Polz.

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Extended data

Extended Data Fig. 1 Representative histology of caecum, ileocecal junction, ileum and ascending colon from mice fed varying amounts of polyethylene glycol 3350 (PEG335) while being exposed to avirulent C. difficile spores.

All 40 mice from all treatment groups have comparable intestinal histology and show no evidence of typhlitis, enteritis or colitis. Tissues were collected ten days after treatment. Hematoxylin and Eosin. Bars= 100 m.

Extended Data Fig. 2 Increasing disturbance intensity increases the likelihood and magnitude C. difficile blooms in mice.

Four groups of ten C57BL/6J Mice were single housed in cages with grated bottoms. Non-toxinogenic C. difficile spores were suspended in drinking water at 104 spores/ml for one day prior, during, and two days following PEG3350 laxative treatment. Daily faecal samples were characterized using 16S rRNA PCR amplicon sequencing. The top panels summarize the family-level phylogenetic structure of the faecal microbial community over time, where different shades of the same colour represent different families in the same phyla; Firmicutes and Bacteroidetes families are depicted in green and blue, respectively. Lower panels show the relative abundance of C. difficile over time. Source data

Extended Data Fig. 3 C. difficile blooms occur the day after treatment when cell density and stool consistency have returned to pre-treatment levels.

(a) The cell density of faecal samples was measured by direct microscopic counts of diluted samples stained with the fluorescent dye fluorescein isothiocyanate (FITC), which covalently binds the amine groups of proteins. Cell density was calculated using 25 images of random fields of the fixed and stained stool samples; center values represent the average cell density and error bars express the 95% confidence intervals of the densities observed for each sample. (b) To confirm the abundance of C. difficile measured using V4 16S rRNA amplicon sequencing (solid line), the abundance was also measured using a qPCR assay that specifically amplifies the rplP gene of C. difficile. (c) Representative stool samples from mice before, during, and after treatment are shown to illustrate how loose the stools of mice fed 150 mg/ml PEG3350 were at the end of treatment, but returned to normal by the following day. All ten mice in the highest PEG3350 treatment group experienced the same pattern of changes in stool consistency over time shown here. Source data

Extended Data Fig. 4 Isolation of C. difficile colonies and genotype-specific colony PCR confirms that the inoculating strain is responsible for the blooms observed in mice treated with laxatives.

Faecal samples from the two mice that experienced the highest magnitude increase in C. difficile measured via 16S rRNA amplicon sequencing were anaerobically homogenized and two dilutions were plated on cycloserine-cefoxitin fructose agar (CCFA) in duplicate. (A) Only one colony morphology was detected from all samples; ground class texture and diffuse edges are consistent with C. difficile. (B) Colony forming unit (CFU) counts roughly reflect the abundance of C. difficile measured via 16S rRNA sequencing. (C) PCR confirms that all isolates isolated on CCFA plates are the same genotype as the inoculating strain (ATCC BAA1801). The C. difficile specific PCR assay amplifies the rplP locus present in all C. difficile strains, while the assay specific to the BAA1801 genotypic cluster amplifies a gene that is present in BAA1801 and its 19 closest sequenced relatives (differentiated by only 270 SNPs, genome-wide) but no other C. difficile lineage. C. difficile strain ATCC-9689 is a toxinogenic control that can be successfully amplified using the rplP PCR assay but not the assay specific to the BAA1801 genotypic cluster. All 72 isolated colonies from all plated dilutions that grew on the CCFA plates were genotyped with both the rplP and BAA1801 genotypic cluster specific PCR assays once. Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Summary of 16S rRNA amplicon sequencing data necessary to reproduce the analysis of the mouse model of variable disturbance intensity.

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VanInsberghe, D., Elsherbini, J.A., Varian, B. et al. Diarrhoeal events can trigger long-term Clostridium difficile colonization with recurrent blooms. Nat Microbiol (2020). https://doi.org/10.1038/s41564-020-0668-2

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