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Plasma-sensitive Escherichia coli mutants reveal plasma resistance mechanisms.

ABSTRACT: Non-thermal atmospheric pressure plasmas are investigated as augmenting therapy to combat bacterial infections. The strong antibacterial effects of plasmas are attributed to the complex mixture of reactive species, (V)UV radiation and electric fields. The experience with antibiotics is that upon their introduction as medicines, resistance occurs in pathogens and spreads. To assess the possibility of bacterial resistance developing against plasma, we investigated intrinsic protective mechanisms that allow Escherichia coli to survive plasma stress. We performed a genome-wide screening of single-gene knockout mutants of E. coli and identified 87 mutants that are hypersensitive to the effluent of a microscale atmospheric pressure plasma jet. For selected genes ( cysB, mntH, rep and iscS) we showed in complementation studies that plasma resistance can be restored and increased above wild-type levels upon over-expression. To identify plasma-derived components that the 87 genes confer resistance against, mutants were tested for hypersensitivity against individual stressors (hydrogen peroxide, superoxide, hydroxyl radicals, ozone, HOCl, peroxynitrite, NO•, nitrite, nitrate, HNO3, acid stress, diamide, heat stress and detergents). k-means++ clustering revealed that most genes protect from hydrogen peroxide, superoxide and/or nitric oxide. In conclusion, individual bacterial genes confer resistance against plasma providing insights into the antibacterial mechanisms of plasma.

SUBMITTER: Krewing M

PROVIDER: S-EPMC6451402 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Plasma-sensitive Escherichia coli mutants reveal plasma resistance mechanisms.

Krewing Marco M Jarzina Fabian F Dirks Tim T Schubert Britta B Benedikt Jan J Lackmann Jan-Wilm JW Bandow Julia E JE

Journal of the Royal Society, Interface 20190301 152

Non-thermal atmospheric pressure plasmas are investigated as augmenting therapy to combat bacterial infections. The strong antibacterial effects of plasmas are attributed to the complex mixture of reactive species, (V)UV radiation and electric fields. The experience with antibiotics is that upon their introduction as medicines, resistance occurs in pathogens and spreads. To assess the possibility of bacterial resistance developing against plasma, we investigated intrinsic protective mechanisms t ...[more]

PMID: 30913981

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Project description:BackgroundInterpretative reading of antimicrobial susceptibility test (AST) results allows inferring biochemical resistance mechanisms from resistance phenotypes. For aminoglycosides, however, correlations between resistance pathways inferred on the basis of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and expert rules versus genotypes are generally poor. This study aimed at developing and validating a decision tree based on resistance phenotypes determined by disc diffusion and based on epidemiological cut-offs (ECOFFs) to infer the corresponding resistance mechanisms in Escherichia coli.MethodsPhenotypic antibiotic susceptibility of thirty wild-type and 458 aminoglycoside-resistant E. coli clinical isolates was determined by disc diffusion and the genomes were sequenced. Based on well-defined cut-offs, we developed a phenotype-based algorithm (Aminoglycoside Resistance Mechanism Inference Algorithm - ARMIA) to infer the biochemical mechanisms responsible for the corresponding aminoglycoside resistance phenotypes. The mechanisms inferred from susceptibility to kanamycin, tobramycin and gentamicin were analysed using ARMIA- or EUCAST-based AST interpretation and validated by whole genome sequencing (WGS) of the host bacteria.FindingsARMIA-based inference of resistance mechanisms and WGS data were congruent in 441/458 isolates (96·3%). In contrast, there was a poor correlation between resistance mechanisms inferred using EUCAST CBPs/expert rules and WGS data (418/488, 85·6%). Based on the assumption that resistance mechanisms can result in therapeutic failure, EUCAST produced 63 (12·9%) very major errors (vME), compared to only 2 (0·4%) vME with ARMIA. When used for detection and identification of resistance mechanisms, ARMIA resolved >95% vMEs generated by EUCAST-based AST interpretation.InterpretationThis study demonstrates that ECOFF-based analysis of AST data of only four aminoglycosides provides accurate information on the resistance mechanisms in E. coli. Since aminoglycoside resistance mechanisms, despite having in certain cases a minimal effect on the minimal inhibitory concentration, may compromise the bactericidal activity of aminoglycosides, prompt detection of resistance mechanisms is crucial for therapy. Using ARMIA as an interpretative rule set for editing AST results allows for better predictions of in vivo activity of this drug class.

Project description:Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.

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Plasma-sensitive Escherichia coli mutants reveal plasma resistance mechanisms.

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Plasma-sensitive Escherichia coli mutants reveal plasma resistance mechanisms.

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