An Evaluation of Culture Techniques versus 16S Profiling for Investigation of Antibiotic-Mediated Alteration of Microbiota Populations within a Clinically Reflective In Vitro Model of the Human Gut

Next-generation sequencing technologies (e.g. 16S profiling) are increasingly used to investigate complex bacterial communities. They have advantages over classical methods, as a significant proportion of bacteria are ‘non-culturable’. However, they do not distinguish ‘viable’ and ‘non-viable’ populations, which may skew results, particularly following antibiotic exposure. Here we report culture and 16S data from a clinically reflective human gut model, describing changes in the gut microbiota following exposure to multiple antibiotics. A triple-stage chemostat model was inoculated with pooled human faeces from healthy volunteers to establish gut microbiota populations. The model was sequentially exposed to clindamycin (33.9 mg/L, QDS, 7days), vancomycin (125mg/L, QDS, 7days) and fidaxomicin (200 mg/L, BD, 7 days). Specific bacterial populations were enumerated daily on selective agars. Periodically, 16S proling of gut model samples was performed; DNA was extracted on a QIAXtractor, 16S V4 PCR products were sequenced on an Illumina MiSeq, and resulting data were analysed using QIIME. Both culture and 16S profiling demonstrated marked alterations in gut microbiota populations following antibiotic exposure. For many populations, notably bifidobacteria and enterobacteria, changes seen by culture correlated with 16S profiling. However, as culture describes numerical changes in populations, and 16S profiling describes proportional changes, results are not always directly comparable. 16S proling greatly increased microbiome coverage, particularly for clostridia. Population diversity (number of observed species and Shannon index) decreased with sequential antibiotic exposure. Use of culture and molecular methods in tandem can greatly increase understanding of changes occurring in complex microbial populations.