Dynamics of co-existing Escherichia colilineages in situ of the infant gut and multiplex phenotypic targeted recovery of previously uncultivated bacteria from the human gut

The work in this thesis explores the dynamic nature of Escherichia coli lineages co-existing in the human intestinal tract. The work is supported via full genomesequencing of co-existing E. coli strains isolated from infants enrolled in the ALLERGYFLORA study. Both sets of isolates examined here were selected due to an observed change in their antibiotic susceptibility profile. Via full genome sequencing, we identified that in both cases a conjugative plasmid harboring antibiotic resistance genes was transferred between co-existing E. coli lineages and is responsible for the change in antibiotic susceptibility. In one case, the transfer occurred in the absenceof antibiotic treatment and the transconjugant remained amongst the gut microbiota for months, providing evidence to the hypothesis that resistance genes are stably maintained once acquired. To our knowledge, this is the first documented case wheretransfer of resistance genes was observed in situ of a non-perturbed gut in the absenceof antibiotic treatment. Additionally, cases of genomic deletions, phage infections andcuring, and plasmid loss were observed, highlighting that strains in the gut are highly dynamic. The detection of these micro-evolutionary events was facilitated throughthe isolation of strains, yielding a high-resolution view into the strain heterogeneity of the human gut. Also included in this thesis is work highlighting that a large proportion of the gutmicrobiota can indeed be cultivated using carefully designed conditions. Antibiotictolerance phenotypes were determined, and this mapping was used to carefully tailor antibiotic combinations to specifically select for previously uncultivated bacteria. Usingthis method, four previously uncultivated species were successfully cultured andgenome sequenced, and two of which had 16S rRNA identities of less than 95% topreviously cultured bacteria. We assessed the genomic coverage and abundance ofthese sequenced isolates in the gut using publicly available metagenomes.