{"title":"Engineering of a monitorable expression system to characterize β-lactamase genes in Enterobacteriaceae","authors":"Anna Schumann , Ahmed Gaballa , Martin Wiedmann","doi":"10.1016/j.mimet.2025.107120","DOIUrl":null,"url":null,"abstract":"<div><div>Bacteria are becoming progressively more resistant to available antimicrobials. The increased ease and availability of genome sequencing has made it possible to identify putative, novel antimicrobial resistance (AMR) genes bioinformatically. However, no standardized system is available to phenotypically characterize the ability of novel AMR genes in <em>Enterobacteriaceae</em> to confer resistance and impact bacterial physiology and pathogenicity in relation to expression levels. We previously used plasmid pBAD24, which allows for arabinose-inducible expression of heterologous genes, and <em>Escherichia coli</em> Top10 to characterize mobile colistin resistance genes. Based on the pBAD24 backbone, we constructed a new plasmid (pBAD25) that carries a kanamycin resistance gene (instead of an ampicillin resistance gene). We show that our expression system allows for the characterization of five different <em>bla</em><sub><em>OXA</em></sub> genes, which differ in their ability to confer susceptibility to β-lactams, detected protein levels, and impact on bacterial growth. We characterized <em>bla</em><sub><em>OXA-48b</em></sub>, a close relative of <em>bla</em><sub><em>OXA-48</em></sub>, previously uncharacterized in <em>E. coli</em>, to be phenotypically similar to <em>bla</em><sub><em>OXA-48,</em></sub> and <em>bla</em><sub><em>OXA-549</em></sub>, a previously uncharacterized gene of the <em>bla</em><sub><em>OXA-548</em></sub> family, as encoding a β-lactamase that is detected intra- but not extracellularly, has moderate growth defects, and decreases susceptibility to carbapenems and ampicillin. Additionally, we found that, in <em>bla</em><sub><em>OXA</em></sub> expressing strains, (i) levels of intracellular proteins and bacterial growth negatively correlate and (ii) susceptibility to 2nd and 3rd generation cephalosporins and susceptibility to different carbapenems positively correlate. Our results demonstrate that the expression of AMR genes, specifically <em>bla</em><sub><em>OXA</em></sub> genes, through pBAD25 allows for easy characterization of putative, novel AMR genes.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107120"},"PeriodicalIF":1.7000,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of microbiological methods","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0167701225000363","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Bacteria are becoming progressively more resistant to available antimicrobials. The increased ease and availability of genome sequencing has made it possible to identify putative, novel antimicrobial resistance (AMR) genes bioinformatically. However, no standardized system is available to phenotypically characterize the ability of novel AMR genes in Enterobacteriaceae to confer resistance and impact bacterial physiology and pathogenicity in relation to expression levels. We previously used plasmid pBAD24, which allows for arabinose-inducible expression of heterologous genes, and Escherichia coli Top10 to characterize mobile colistin resistance genes. Based on the pBAD24 backbone, we constructed a new plasmid (pBAD25) that carries a kanamycin resistance gene (instead of an ampicillin resistance gene). We show that our expression system allows for the characterization of five different blaOXA genes, which differ in their ability to confer susceptibility to β-lactams, detected protein levels, and impact on bacterial growth. We characterized blaOXA-48b, a close relative of blaOXA-48, previously uncharacterized in E. coli, to be phenotypically similar to blaOXA-48, and blaOXA-549, a previously uncharacterized gene of the blaOXA-548 family, as encoding a β-lactamase that is detected intra- but not extracellularly, has moderate growth defects, and decreases susceptibility to carbapenems and ampicillin. Additionally, we found that, in blaOXA expressing strains, (i) levels of intracellular proteins and bacterial growth negatively correlate and (ii) susceptibility to 2nd and 3rd generation cephalosporins and susceptibility to different carbapenems positively correlate. Our results demonstrate that the expression of AMR genes, specifically blaOXA genes, through pBAD25 allows for easy characterization of putative, novel AMR genes.
期刊介绍:
The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach.
All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.