Laboratory of Microbiology, Wageningen University & Research, Wageningen, the Netherlands.
Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, the Netherlands.
PLoS One. 2019 Jan 17;14(1):e0210970. doi: 10.1371/journal.pone.0210970. eCollection 2019.
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.
细菌病原体对临床抗生素产生耐药性，对人类健康构成的威胁日益增加。人类肠道微生物群是细菌病原体获得耐药性的重要储存库。然而，迄今为止，大部分肠道微生物群仍未被培养，其耐药储存功能也很少被研究。在这里，我们旨在通过靶向方法分离尚未培养的耐药肠道细菌。因此，我们从接受选择性消化道去污染（SDD）预防性抗生素治疗的 20 名重症监护患者的粪便样本中，通过厌氧接种在多孔氧化铝芯片上，这些芯片放在贫瘠和丰富的琼脂培养基上，包括补充了 SDD 抗生素的培养基。通过 16S rRNA 基因扩增子测序分析芯片上的生物量，显示出个体间细菌可培养性的巨大差异，并富集了一系列分类多样的操作分类单位（OTU）。此外， Ruminococcaceae（2 个 OTU）、肠杆菌科（6 个 OTU）和 Lachnospiraceae（4 个 OTU）的生长受到 SDD 抗生素的显著抑制。属于 16 个 OTU 的菌株被认为是纯培养的候选菌株，因为它们与最接近的模式菌株的序列同一性≤95%，且相对丰度≥2%。其中 6 个 OTU 检测到存在于含有 SDD 抗生素的培养基中，因此是研究抗生素耐药性的主要候选菌株。其中一个 OTU 是通过靶向分离获得纯培养的。该新型菌株对抗生素甲硝唑和亚胺培南具有耐药性。它最初被分类为 Ruminococcaceae 的成员，但后来发现它与最近发表的 Sellimonas intestinalis BR72T 共享 99%的核苷酸同一性。总之，我们表明，高通量培养为基础的微生物群落筛选可以指导靶向分离作为抗生素耐药性储存库的细菌。
