Date Published: January 17, 2019
Publisher: Public Library of Science
Author(s): Dennis Versluis, Teresita de J. Bello González, Erwin G. Zoetendal, Mark W. J. van Passel, Hauke Smidt, Zaid Abdo.
The emergence of bacterial pathogens that are resistant to clinical antibiotics poses an increasing risk to human health. An important reservoir from which bacterial pathogens can acquire resistance is the human gut microbiota. However, thus far, a substantial fraction of the gut microbiota remains uncultivated and has been little-studied with respect to its resistance reservoir-function. Here, we aimed to isolate yet uncultivated resistant gut bacteria by a targeted approach. Therefore, faecal samples from 20 intensive care patients who had received the prophylactic antibiotic treatment selective digestive decontamination (SDD), i.e. tobramycin, polymyxin E, amphotericin B and cefotaxime, were inoculated anaerobically on porous aluminium oxide chips placed on top of poor and rich agar media, including media supplemented with the SDD antibiotics. Biomass growing on the chips was analysed by 16S rRNA gene amplicon sequencing, showing large inter-individual differences in bacterial cultivability, and enrichment of a range of taxonomically diverse operational taxonomic units (OTUs). Furthermore, growth of Ruminococcaceae (2 OTUs), Enterobacteriaceae (6 OTUs) and Lachnospiraceae (4 OTUs) was significantly inhibited by the SDD antibiotics. Strains belonging to 16 OTUs were candidates for cultivation to pure culture as they shared ≤95% sequence identity with the closest type strain and had a relative abundance of ≥2%. Six of these OTUs were detected on media containing SDD antibiotics, and as such were prime candidates to be studied regarding antibiotic resistance. One of these six OTUs was obtained in pure culture using targeted isolation. This novel strain was resistant to the antibiotics metrodinazole and imipenem. It was initially classified as member of the Ruminococcaceae, though later it was found to share 99% nucleotide identity with the recently published Sellimonas intestinalis BR72T. In conclusion, we show that high-throughput cultivation-based screening of microbial communities can guide targeted isolation of bacteria that serve as reservoirs of antibiotic resistance.
The emergence of bacterial pathogens that are resistant to clinical antibiotics is an increasing threat to public health. A common route through which pathogens can acquire resistance is by genetic exchange with human-associated bacteria, and especially the gut microbiota [1–3]. Indeed, it has been shown that the commensal gut microbiota harbours diverse resistance genes [4, 5], that these genes are regularly expressed  and that such genes can be acquired by (opportunistic) pathogens . Horizontal gene transfer (HGT) is considered the main mechanism by which resistance genes are disseminated, and it has been shown that HGT events occur exceedingly more often in the gut microbiota than in other environments with complex bacterial communities .
In this study, we investigated the cultivability of anaerobic human faecal bacteria in order to isolate strains that can serve as reservoirs for antibiotic resistance. Therefore, faecal samples from 20 intensive care patients who received SDD therapy were inoculated anoxically on PAO chips placed on top of poor and rich agar media, including media supplemented with the SDD antibiotics. Faecal inocula and associated bacterial growth communities were analysed by 16S rRNA gene amplicon sequencing.