Date Published: April 15, 2019
Publisher: Public Library of Science
Author(s): Lena M. Biehl, Debora Garzetti, Fedja Farowski, Diana Ring, Martin B. Koeppel, Holger Rohde, Philippe Schafhausen, Bärbel Stecher, Maria J. G. T. Vehreschild, Alexander V. Alekseyenko.
Large-scale clinical studies investigating associations between intestinal microbiota signatures and human diseases usually rely on stool samples. However, the timing of repeated stool sample collection cannot be predefined in longitudinal settings. Rectal swabs, being straightforward to obtain, have the potential to overcome this drawback. Therefore, we assessed the usability of rectal swabs for microbiome sampling in a cohort of hematological and oncological patients.
We used a pipeline for intestinal microbiota analysis from deep rectal swabs which was established and validated with test samples and negative controls. Consecutively, a cohort of patients from hematology and oncology wards was established and weekly deep rectal swabs taken during their admissions and re-admissions.
Validation of our newly developed pipeline for intestinal microbiota analysis from rectal swabs revealed consistent and reproducible results. Over a period of nine months, 418 rectal swabs were collected longitudinally from 41 patients. Adherence to the intended sampling protocol was 97%. After DNA extraction, sequencing, read pre-processing and filtering of chimeric sequences, 405 of 418 samples (96.9%) were eligible for further analyses. Follow-up samples and those taken under current antibiotic exposure showed a significant decrease in alpha diversity as compared to baseline samples. Microbial domination occurred most frequently by Enterococcaceae (99 samples, 24.4%) on family level and Enterococcus (90 samples, 22.2%) on genus level. Furthermore, we noticed a high abundance of potential skin commensals in 99 samples (24.4%).
Deep rectal swabs were shown to be reliable for microbiome sampling and analysis, with practical advantages related to high sampling adherence, easy timing, transport and storage. The relatively high abundance of putative skin commensals in this patient cohort may be of potential interest and should be further investigated. Generally, previous findings on alpha diversity dynamics obtained from stool samples were confirmed.
The intestinal tract harbors a complex microbial community which plays a central role in human health. Disruption of the gut microbiota (or dysbiosis) is associated with pathological intestinal conditions such as obesity and malnutrition, metabolic diseases such as diabetes, and chronic inflammatory diseases such as inflammatory bowel diseases [1–4]. The recent increase in clinical microbiome studies has been facilitated by advances in high throughput sequencing technologies, which offer rapid and comprehensive culture-independent techniques, mostly relying on DNA sequencing of the 16S ribosomal RNA gene .
In this analysis, we present an intestinal microbiome analysis from a longitudinal cohort study utilizing deep rectal swabs for intestinal microbiota profiling. Previous studies have already compared rectal swabs with samples from different origins [6–9]. We validated the pipeline for DNA extraction and 16S rRNA gene sequencing on rectal swabs and stools obtained from seven healthy volunteers and one patient, confirming reproducibility of the results. Between swab- and stool-derived microbiota profiles we found some small differences, which may partly be due to dissimilarities in the sampled body sites (rectal mucosal wall versus stool), as previously reported . Since it was not our aim to prove equality of these two sampling techniques, only this small number of comparative specimen from volunteers or patients were collected. Furthermore, the relatively low number of contaminant reads found in sterile swabs and negative controls is within the range of previously reported contamination of laboratory consumables , and is therefore expected not to confound the taxonomic composition of the patient samples.