Biology subjects

Wisniewski, J. A.

Publications and source records attributed to Wisniewski, J. A..

4 recordsLinked to original sources

bioRxiv2024

Wild type domestication: loss of intrinsic metabolic traits concealed by culture in rich media

BackgroundBacteria are typically isolated on rich media to maximise isolation success, removing them from their native evolutionary context. This eliminates selection pressures, enabling otherwise deleterious genomic events to accumulate. Here we present a cautionary tale of these quiet mutations which can persist unnoticed in bacterial culture lines. MethodsWe used a combination of microbiological culture (standard and minimal media conditions), whole genome sequencing and metabolic modelling to investigate putative Klebsiella pneumoniae L-histidine auxotrophs. Additionally, we used genome-scale metabolic modelling to predict auxotrophies among completed public genomes (n=2,637). ResultsTwo sub-populations were identified within a K. pneumoniae frozen stock, differing in their ability to grow in the absence of L-histidine. These sub-populations were the same strain, separated by eight single nucleotide variants and an insertion sequence-mediated deletion of the L-histidine biosynthetic operon. The His- sub-population remained undetected for >10 years despite its in inclusion in independent laboratory experiments. Genome-scale metabolic models predicted 0.8% public genomes contained [≥]1 auxotrophy, with purine/pyrimidine biosynthesis and amino acid metabolism most frequently implicated. DiscussionWe provide a definitive example of the role of standard rich media culture conditions in obscuring biologically relevant mutations i.e. nutrient auxotrophies, and estimate the prevalence of such auxotrophies using public genome collections. While the prevalence is low, it is not insignificant given the thousands of K. pneumoniae that are isolated for global surveillance and research studies each year. Our data serve as a pertinent reminder that rich-media culturing can cause unnoticed wild type domestication.

microbiology↗

bioRxiv2024

Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies

Sequencing DNA directly from patient samples enables faster pathogen characterisation compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich for the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in bacterial isolates before comparing enriched and unenriched sequencing of three human faecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56x and 11.3x the number of AMR and MLST reads respectively compared to unenriched sequencing and required approximately one third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterisation outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonised with high-risk strains.

microbiology↗

bioRxiv2023

Predicting Pseudomonas aeruginosa drug resistance using artificial intelligence and clinical MALDI-TOF mass spectra

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used in clinical microbiology laboratories for bacterial identification but its use for prediction of antimicrobial resistance (AMR) remains limited. Here, we used MALDI-TOF MS with artificial intelligence (AI) approaches to successfully predict AMR in Pseudomonas aeruginosa, a priority pathogen with complex AMR mechanisms. The highest performance was achieved for modern {beta}-lactam/{beta}-lactamase inhibitor drugs, namely ceftazidime/avibactam and ceftolozane/tazobactam, with area under the receiver operating characteristic curve (AUROC) of 0.86 and 0.87, respectively. As part of this work, we developed dynamic binning, a feature engineering technique that effectively reduces the high-dimensional feature set and has wide-ranging applicability to MALDI-TOF MS data. Compared to conventional methods, our approach yielded superior performance in 10 of 11 antimicrobials. Moreover, we showcase the efficacy of transfer learning in enhancing the performance for 7 of 11 antimicrobials. By assessing the contribution of features to the models prediction, we identified proteins that may contribute to AMR mechanisms. Our findings demonstrate the potential of combining AI with MALDI-TOF MS as a rapid AMR diagnostic tool for Pseudomonas aeruginosa.

microbiology↗

bioRxiv2023

Genomic dissection of endemic carbapenem resistance: metallo-beta-lactamase gene dissemination through clonal, plasmid and integron transfer pathways

Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades of blaIMP-4 evolution in a hospital using sequence data from 270 clinical and environmental isolates (including 169 completed genomes) and identified extreme gene promiscuity across 7 Gram-negative genera, 68 bacterial strains and 7 distinct plasmid types. An initial multi-species outbreak of conserved IncC plasmids (95 genomes across 37 strains) allowed endemicity to be established through the ability of blaIMP-4 to disseminate in successful strain-genetic setting pairs we termed propagators, in particular Serratia marcescens and Enterobacter hormaechei. From this reservoir, blaIMP-4 persisted through diversification of genetic settings that resulted from transfer of blaIMP-4 plasmids between bacterial hosts and of the integron carrying blaIMP-4 between plasmids. Our findings provide a framework for understanding endemicity and spread of MBLs and may have broader applicability to other carbapenemase-producing organisms.

microbiology↗