Biology subjects

Hubbard, A. T. M.

Publications and source records attributed to Hubbard, A. T. M..

6 recordsLinked to original sources

bioRxiv2026

In vitro evolution of uropathogenic Escherichia coli to fosfomycin resistance in a 3D cultured human bladder microtissue model

In vitro studies of antimicrobial resistance (AMR) using laboratory growth media produce important, fundamental information. However, their inability to more closely replicate the in vivo environment limits the translational potential of this work. Here, we used a 3D cultured microtissue model which reflects the human bladder microenvironment to select for resistance to fosfomycin in two uropathogenic strains of Escherichia coli, UTI-34 and UTI-59. To assess the clinical relevance of the mutations produced, we screened the observed mutations in the fosfomycin-selected variants against a curated dataset of 14,163 E. coli genomes isolated from urine. The four independent fosfomycin-selected variants of UTI-34 contained diverse mutations, while the mutations in the five independent fosfomycin-selected variants of UTI-59 were more constrained. All variants contained mutations in glpT, uhpT, uhpA and uhpC, which are commonly linked to fosfomycin-resistance in clinical isolates of E. coli. Screening of the mutations against the 14,163 E. coli genomes from urine confirmed that four of these mutations were found as exact matches in the dataset, while other mutation types were confirmed at a regional and gene level. These mutations did not result in any collateral susceptibility or resistance to other antibiotics recommended for the treatment of urinary tract infections. The use of a human 3D microtissue model, which closely replicates the urothelial microenvironment to study AMR during urinary tract infection treatment, could improve the clinical relevance of in vitro AMR studies. This has the potential to provide a better understanding of how AMR is acquired and expressed, and inform new strategies to combat AMR.

microbiology↗

bioRxiv2023

The effect of antibiotic selection on collateral effects and evolvability of uropathogenic Escherichia coli

Trimethoprim is recommended as a first-line treatment of urinary tract infections (UTIs) in the UK. In 2018, 31.4% of Escherichia coli isolated from UTIs in England were trimethoprim resistant, leading to overreliance on other first and second-line antibiotics. Here, we assessed whether prior selection with trimethoprim results in collateral effects to other antibiotics recommended for the treatment of UTIs. As collateral effects, we considered changes in susceptibility, mutation-selection window and population establishment probability. We selected 10 trimethoprim-resistant derivatives from three clinical isolates of uropathogenic Escherichia coli. We found that mutations conferring trimethoprim resistance did not have any collateral effects to fosfomycin. In contrast, resistance to trimethoprim resulted in decreased susceptibility (collateral resistance) to nitrofurantoin, below the clinical breakpoint, and narrowed the mutation-selection window thereby reducing the maximum concentration for selection of nitrofurantoin resistance mutations. Our analyses demonstrate that multiple collateral responses should be accounted for when predicting and optimising antibiotic use, limiting future AMR emergence.

microbiology↗

bioRxiv2020

Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae

Escherichia coli and other Enterobacteriaceae are highly diverse species with open pangenomes1,2, where genes move intra- and inter-species via horizontal gene transfer3. These species can cause clinical infections4,5 as well as persist environmentally6,7. Environmental populations have been suggested as important reservoirs of antimicrobial resistance (AMR) genes. However, as most analyses focus on clinical isolates8,9, the pangenome dynamics of natural populations remain understudied, particularly the role of plasmids. Here, we reconstructed near-complete genomes for 828 Enterobacteriaceae, including 553 Escherichia spp. and 275 non-Escherichia species with 2,293 circularised plasmids in total, collected from nineteen locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30km radius at three timepoints over the course of a year. We find different dynamics for the chromosomal and plasmid-borne components of the pangenome, showing that plasmids have a higher burden of both AMR genes and insertion sequences, and AMR plasmids show evidence of being under stronger selective pressure. Focusing on E. coli, we observe that plasmid dynamics are more strongly dominated by niche and local geography, rather than phylogeny or season. Our results highlight the diversity of the AMR reservoir in these species and niches, and the importance of local strategies for controlling the emergence and spread of AMR.

microbiology↗

bioRxiv2020

A novel hemA mutation is responsible for small colony variant phenotype in Escherichia coli.

We identified a small colony variant (SCV) of a clinical isolate of Escherichia coli from Malawi following sequential in vitro selection in sub-inhibitory concentrations of amoxicillin-clavulanic acid and gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase responsible for the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed this phenotype was stable over multiple generations and the SNP in hemA resulted in a significant fitness cost to the isolate which persisted even in the presence of hemin. As hemA is not found in mammalian cells, and disruption of the gene results in impaired biofilm formation and a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by biofilm-producing E. coli.

microbiology↗

bioRxiv2020

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1B

A novel phenotype of Escherichia coli and Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3rd generation cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the {beta}-lactamase blaTEM, however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a {beta}-lactamase but lacked mutations within {beta}-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS26-flanked composite transposon containing several antibiotic resistance genes, including blaTEM-1B, which was designated Tn6762. These resistance genes are also found to be present on a translocatable unit which had excised from Tn6762 in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn6762 following exposure to TZP and capture the TU in a plasmid containing a copy of IS26. Subsequent amplification of the TU, and by extension blaTEM-1B, leads to {beta}-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value = <0.0001), we found that the TZP-resistant isolate was as fit as the susceptible ancestor. This mechanism of gene amplification, and the subsequent hyperproduction, of blaTEM-1B is an important consideration when using genomic data to predict resistance/susceptibility to TZP.

microbiology↗

bioRxiv2019

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

BackgroundShotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth ([~]200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ResPipe.\n\nResultsTaxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve <1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.\n\nConclusionsSequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

microbiology↗