Ecological conditions leading to the seep of antibiotic resistance genes in the model-type bacterium Escherichia coli

In antibiotic therapy design, conventional wisdom holds that higher antibiotic dosages always leads to the observation of fewer bacterial cells, resulting in a monotonic decay in cell number as a function of increasing antibiotic dose; accordingly, throughout this thesis, we will call this phenomenon a monotone dose-response profile. When we analysed the evolution of antibiotic resistance mediated by the multi-drug efflux pump AcrAB-TolC in Escherichia coli to study if such a monotone dose-response is maintained at all times, our analysis showed that higher dosages can, in fact, lead to higher bacterial loads. This is because selection for drug resistance is mediated by the duplication of the genes, AcrAB-TolC, that encode the aforementioned efflux pump. As explained in detail below, our work highlights the idea that Darwinian selection on additional copies of AcrAB-TolC is a non-linear function of antibiotic dose and that the observed transition from monotone to non-monotone dose-response is a consequence of AcrAB-TolC being strongly selected at very specific dosages. We term this phenomenon an ‘evolutionary hotspot’. Next, we extended the above experimental system to solid media to study how selection on resistance mediated by AcrAB-TolC leads to a ‘spatio-genomic patterning’ effect that we call a ‘bullseye’. Using a bespoke culture device developed as part of this PhD, we show that spatial selection on resistance also depends non-linearly on the distance of the cell from an antibiotic source, and that the non-linearity can be multi-modal as a function of distance, and therefore also of antibiotic dose. This result also contradicts the aforementioned principle that higher antibiotic dosages necessarily lead to fewer bacterial cells. Following on from this, we then studied the ability of microbial competitors for resources to modulate the antibiotic sensitivity of a particular strain of E. coli, namely Tets , using a range of multi-species experiments. We measured the sensitivity to antibiotics of Tets both with, and without, one bacterial or fungal competitor. When that competitor was equally sensitive to the antibiotic, we observed that Tets was less sensitive to it, in part due to an ‘antibiotic sinking’ effect carried out by the competitor strain. However, when the competitor was not sensitive to the antibiotic, Tets was, accordingly, more sensitive than in the absence of competition. In this latter case, the competitor seemed to reduce the growth of Tets by carbon theft as part of a phenomenon known as ‘competitive suppression’. Moreover, this ecological effect is one that synergises with the action of the antibiotic. Finally, we turned to a study of an ecological trade-off motivated by ribosome-binding antibiotics. So, by manipulating the content of ribosomal RNA in the E. coli cell, a large and essential molecule that is bound by antibiotics such as tetracycline or erythromycin, we could subsequently manipulate what is known as a metabolic trade-off between growth rate and growth yield. The latter is the number of cells produced per molecule of carbon found in the extracellular environment of the bacterial population. Using glucose as carbon source we therefore constructed an empirical fitness landscape that shows how the optimum number of ribosomal rRNA operons depends on extracellular glucose concentration. Whilst this study does not relate directly to the presence of an antibiotic, it does show that by altering the number of operons in a manner that is known to affect antibiotic susceptibility, we can also mediate important growth parameters like cell yield, aka efficiency, and growth rate.Engineering and Physical Sciences Research Council (EPSRC

Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the β-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region

Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the β-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region

Hound: A novel tool for automated mapping of genotype to phenotype in bacterial genomes assembled de novo

Increasing evidence suggests that microbial species have a strong within species genetic heterogeneity. This can be problematic for the analysis of prokaryote genomes, which commonly relies on a reference genome to guide the assembly process. Differences between reference and sample genomes will therefore introduce errors in final assembly, jeopardising the detection from structural variations to point mutations—critical for genomic surveillance of antibiotic resistance. Here we present Hound, a pipeline that integrates publicly available tools to assemble prokaryote genomes de novo, detect user-given genes by similarity to report mutations found in the coding sequence, promoter, as well as relative copy number within the assembly. Importantly, Hound can use the query sequence as a guide to merge contigs, and reconstruct genes that were fragmented by the assembler. To showcase Hound, we screened through 5,032 bacterial whole-genome sequences isolated from farmed animals and human infections, using the amino acid sequence encoded by blaTEM-1, to detect and predict resistance to amoxicillin/clavulanate which is driven by over-expression of this gene. We believe this tool can facilitate the analysis of prokaryote species that currently lack a reference genome, and can be scaled either up to build automated systems for genomic surveillance or down to integrate into antibiotic susceptibility point-of-care diagnostics

Capitulación del regimiento suizo de Don Carlos de Reding, para continuar su servicio en el de S. M. C., dispuesta, y arreglada por el Loable Canton de Schwitz, por medio de sus Ilustres Diputados, y Apoderados Don Carlos Domingo Yutz, Banderet, y Landamano, Regente; y Don Nazaro Ignacio de Ceberg Landamano, en la forma, y con las Condiciones, que explican los Articulos siguientes

Texto fechado en Lucerna a 5 de octubre de 1757 incluyéndose al final el texto de la aprobación fechada en Madrid a 5 de noviembre de 1757 y refrendada por Sebastián de EslavaSign.: A1