Fosfomycin: Mechanism and Resistance

Silver, Lynn L.

doi:10.1101/cshperspect.a025262

Cited by 241 publications

(194 citation statements)

References 56 publications

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“…Conversely, some genes have negative gene scores in only one or a handful of conditions. For example, consistent with earlier studies we found that overexpression of glpT or uhpT increases susceptibility to phosphomycin 57 . These results also agree with clinical data, which shows that the main cause of phosphomycin resistance in patients is the down-regulation of GlpT via down-regulation of cAMP.…”

Section: Resultssupporting

confidence: 93%

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Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

Mutalik

Novichkov

Price

et al. 2018

Preprint

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35A major challenge in genomics is the knowledge gap between sequence and its 36 encoded function. Gain-of-function methods based on gene overexpression are 37 attractive avenues for phenotype-based functional screens, but are not easily applied in 38high-throughput across many experimental conditions. Here, we present Dual Barcoded 39Shotgun Expression Library Sequencing (Dub-seq), a method that greatly increases the 40 throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression 41 library is cloned between dual random DNA barcodes and the precise breakpoints of 42 DNA fragments are associated to the barcode sequences prior to performing assays. To 43 assess the fitness of individual strains carrying these plasmids, we use DNA barcode 44 sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a 45 demonstration of this approach, we constructed a Dub-seq library with total Escherichia 46 coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental 47 conditions, and identified 813 genes with high-confidence overexpression phenotypes 48 across 4,151 genes assayed. We show that Dub-seq data is reproducible, accurately 49recapitulates known biology, and identifies hundreds of novel gain-of-function 50 phenotypes for E. coli genes, a subset of which we verified with assays of individual 51 strains. Dub-seq provides complementary information to loss-of-function approaches 52 such as transposon site sequencing or CRISPRi and will facilitate rapid and systematic 53 functional characterization of microbial genomes. 54 55 56Importance 57Measuring the phenotypic consequences of overexpressing genes is a classic genetic 58 approach for understanding protein function; for identifying drug targets, antibiotic and 59 metal resistance mechanisms; and for optimizing strains for metabolic engineering. In 60 microorganisms, these gain-of-function assays are typically done using laborious 61 protocols with individually archived strains or in low-throughput following qualitative 62 selection for a phenotype of interest, such as antibiotic resistance. However, many 63 microbial genes are poorly characterized and the importance of a given gene may only 64be apparent under certain conditions. Therefore, more scalable approaches for gain-of-65 function assays are needed. Here, we present Dual Barcoded Shotgun Expression 66Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression 67with DNA barcode sequencing for large-scale interrogation of gene fitness under many 68 experimental conditions at low cost. Dub-seq can be applied to many microorganisms 69and is a valuable new tool for large-scale gene function characterization. 70 71 72 73Advances in DNA sequencing have had a tremendous impact on microbial genomics, 74as thousands of genomes have now been sequenced 1 . However, only a small fraction 75 of these microorganisms have been experimentally studied and as such, our predictions 76 of gene function, metabolic capability, and community function fo...

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Section: Resultssupporting

confidence: 93%

“…These results also agree with clinical data, which shows that the main cause of phosphomycin resistance in patients is the down-regulation of GlpT via down-regulation of cAMP. 57 Accordingly, we also found that overexpression of cpdA (which encodes an enzyme that hydrolyzes cAMP) enhances fitness under phosphomycin stress ( Fig. 4 ).…”

Section: Resultsmentioning

confidence: 59%

Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

Mutalik

Novichkov

Price

et al. 2018

Preprint

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“…Fosfomycin is a derivative of phosphonic acid that blocks an early stage in the synthesis of peptidoglycan, a component of the bacterial cell wall [41]. First discovered in 1969 [42], fosfomycin has recently received renewed interest for MDR Gram-negative bacteria.…”

Section: Contemporary Treatment Strategies For Gram-negative Vapmentioning

confidence: 99%

Resistance Trends and Treatment Options in Gram-Negative Ventilator-Associated Pneumonia

Rhodes

Cruce

O’Donnell

et al. 2018

Curr Infect Dis Rep

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Rapid identification technologies and phenotypic methods, new therapeutic strategies, and novel treatment paradigms have evolved in an attempt to improve treatment outcomes for VAP; however, clinical data supporting alternative treatment strategies and adjunctive therapies remain sparse. Importantly, new classes of antimicrobials, novel virulence factor inhibitors, and beta-lactam/beta-lactamase inhibitor combinations are currently in development. Conscientious stewardship of new and emerging therapeutic agents will be needed to ensure they remain effective well into the future.

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“…All the primers used were designed with Primer3 Input software; their specificity was tested by BLAST alignment against P. aeruginosa genome from Pseudomonas Genome Database (http://www.pseudomonas.com/) and their efficiency was analyzed by Real-time PCR using serial dilutions of cDNA. Differences in the relative amounts of mRNA were determined according to the 2 −ΔΔCT method (41, 42). In all cases, the values of relative mRNA expression were determined as the average of three independent biological replicates each one containing three technical replicates.…”

Section: Methodsmentioning

confidence: 99%

Novel inducers of the expression of multidrug efflux pumps that triggerPseudomonas aeruginosatransient antibiotic resistance

Laborda

Alcalde-Rico

Blanco

et al. 2019

Preprint

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The study of the acquisition of antibiotic resistance (AR) has mainly focused in inherited processes, namely mutations and acquisition of AR genes. However, inducible, non-inheritable AR has received less attention and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about non-antibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tightly regulation of their expression. Their contribution to acquired AR relies in their overexpression. Herein we analyzed potential inducers of the expression of the chromosomally-encoded Pseudomonas aeruginosa clinically-relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able of modifying the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog Phenotype Microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of mexCD-oprJ. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine or atropine are present. This work emphasizes the need of studying compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.

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Fosfomycin: Mechanism and Resistance

Cited by 241 publications

References 56 publications

Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

Resistance Trends and Treatment Options in Gram-Negative Ventilator-Associated Pneumonia

Novel inducers of the expression of multidrug efflux pumps that triggerPseudomonas aeruginosatransient antibiotic resistance

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