High throughput cultivation-based screening on porous aluminum oxide chips allows targeted isolation of antibiotic resistant human gut bacteria

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.

Pectin Degradation is an Important Determinant for Alfalfa Silage Fermentation through the Rescheduling of the Bacterial Community

“…Moreover, we still found many unclassified genera bacteria in the PEC and PEE groups. Given the existence of methods to manipulate the inoculants, the sequencing data should be used for further screening and enrichment culture [43]. Manipulating the levels of species or even strains belonging to the Weissella, Leuconostoc, Bacillus, and Aeromonas, as well as other unclassified bacteria that potentially have a close connection to the anaerobic fermentation of alfalfa silage, which may help to improve the quality of alfalfa silage and the development of associated feed industry.…”

Comprehensive genome analyses ofSellimonas intestinalis, a potential biomarker of homeostasis gut recovery

“…In this study, inconsistencies in taxonomic classification were detected at different levels: i) in the allocation of species to families with little phylogenetic relationship, as is the case of Clostridium difficile that had been included within the Clostridiaceae family, but after detailed analysis of the Phylogenetic relationships were classified within the Peptostreptococcaceae family, 26 or ii) in the taxonomic assignment of individuals, as revealed even before this work for S. intestinalis , which in other works had previously been detected as Ruminococcus , but later of the sequencing of its complete genome, it was correctly assigned. 13 These types of findings reveal limitations in the traditional analysis schemes of complete genome data and make clear the need for further studies that lead to clarify the classification of under-studied anaerobic families.…”

“…Additionally, taking into account that this study was aimed at analyzing data from complete genomes of S. intestinalis and no phenotypic tests were performed, it is necessary to carry out further studies that lead to identify the impact of the expression of these VFm and AMRg and their potential role in modulation of the relative abundance of this species under different biotic contexts. Despite this limitation, the identification of these markers could support the hypothesis that some members of the microbiota could fulfill resistance reservoir-function from which bacterial pathogens can acquire resistance is the human gut microbiota, 13 generating interest at the health level. This approach represents the first step conducing to the genomic bases that support S. intestinalis survival under conditions of dysbiosis and subsequent proliferation after the homeostasis reestablishment that could play an important role in maintaining the optimal conditions for host development.…”

“…One of the Lachnospiraceae species recently identified and poorly studied is Sellimonas intestinalis , a Gram positive and obligately anaerobic bacteria, 12 initially considered as part of the gut microbiota fraction that remains uncultivated for its nature extremely oxygen-sensitive ‘EOS’. 13 This limitation could have been the cause of the limited number of studies where S. intestinalis has been detected, being almost all aimed to deciphering the microbiome composition from a shotgun metagenomics approach. 13, 14 In these studies, an increased relative abundance of S. intestinalis was detected in patients which recovered their intestinal homeostasis after suffering dysbiosis caused by chemotherapy treatment against colorectal cancer 15 or therapeutic splenectomy of patients with liver cirrhosis.…”

“…13 This limitation could have been the cause of the limited number of studies where S. intestinalis has been detected, being almost all aimed to deciphering the microbiome composition from a shotgun metagenomics approach. 13, 14 In these studies, an increased relative abundance of S. intestinalis was detected in patients which recovered their intestinal homeostasis after suffering dysbiosis caused by chemotherapy treatment against colorectal cancer 15 or therapeutic splenectomy of patients with liver cirrhosis. 16 These findings suggest the potential of S. intestinalis as a biomarker candidate of gut homeostasis recovery.…”