Abstract
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.
Highlights
The emergence of bacterial pathogens that are resistant to clinical antibiotics is an increasing threat to public health
10-fold diluted and 100-fold diluted faecal suspension from 20 patients under the selective digestive decontamination (SDD) regime was inoculated in duplicate on porous aluminium oxide (PAO) chips that were placed on top of Gifu anaerobic agar medium (GAM) and CP agar media that either did or did not contain the SDD antibiotic cocktail
A smaller number of colonies developed on media if the faecal material was inoculated at a higher dilution, if the media contained the SDD cocktail of antibiotics, and if the faecal material was inoculated on CP media
Summary
The emergence of bacterial pathogens that are resistant to clinical antibiotics is an increasing threat to public health. Though novel resistance determinants are typically described once bacteria are obtained in pure culture, resistance genes carried by yet uncultivated gut bacteria appear to be largely uncovered. This perspective is reinforced by the observation that functional metagenomics studies of human gut microbiota consistently yield novel resistance genes [9, 10]. This “black box” of little-studied uncultivated bacteria has been estimated to constitute 40–70% of gut microbes [11, 12]. Even though application of culture-independent methods (e.g. metagenomics [13, 14]) has provided us with useful insights into uncultivated bacteria and their resistance genes, their cultivation will be essential to comprehensively study the antibiotic resistance phenotype, and the potential roles and mechanisms of these bacteria in antibiotic resistance dissemination
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
