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Establishment of a method to rapidly assay bacterial persister metabolism.

ABSTRACT: Bacterial persisters exhibit an extraordinary tolerance to antibiotics that is dependent on their metabolic state. Although persister metabolism promises to be a rich source of antipersister strategies, there is relatively little known about the metabolism of these rare and transient phenotypic variants. To address this knowledge gap, we explored the use of several techniques, and we found that only one measured persister metabolism. This assay was based on the phenomenon of metabolite-enabled aminoglycoside killing of persisters, and we used it to characterize the metabolic heterogeneity of different persister populations. From these investigations, we determined that glycerol and glucose are the most ubiquitously used carbon sources by various types of Escherichia coli persisters, suggesting that these metabolites might prove beneficial to deliver in conjunction with aminoglycosides for the treatment of chronic and recurrent infections. In addition, we demonstrated that the persister metabolic assay developed here is amenable to high-throughput screening with the use of phenotype arrays.

SUBMITTER: Orman MA

PROVIDER: S-EPMC3754326 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Establishment of a method to rapidly assay bacterial persister metabolism.

Orman Mehmet A MA Brynildsen Mark P MP

Antimicrobial agents and chemotherapy 20130701 9

Bacterial persisters exhibit an extraordinary tolerance to antibiotics that is dependent on their metabolic state. Although persister metabolism promises to be a rich source of antipersister strategies, there is relatively little known about the metabolism of these rare and transient phenotypic variants. To address this knowledge gap, we explored the use of several techniques, and we found that only one measured persister metabolism. This assay was based on the phenomenon of metabolite-enabled a ...[more]

PMID: 23817376

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Project description:Persisters represent a small subpopulation of cells within a bacterial culture that are tolerant to killing by antibiotics. Persisters have been linked to recalcitrant infections caused by numerous bacterial pathogens, including Pseudomonas aeruginosa A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation for P. aeruginosa are poorly understood. We generated a high-density transposon insertion library of P. aeruginosa PAO1 and determined the relative frequency of each insertion following fluoroquinolone treatment using transposon sequencing (Tn-seq). Of the 4,411 disrupted genes included in the screen, 137 had a ≥10-fold impact on survival. The gene disruption that resulted in the lowest survival rate was disruption of carB, which codes for the large subunit of carbamoyl phosphate synthetase (CPSase). CPSase is a metabolic enzyme that is involved in pyrimidine and arginine synthesis. Disruption of carB resulted in survival rates that were reduced by up to 2,500-fold following antibiotic treatment, and this phenotype was abolished by the addition of uracil, highlighting the importance of de novo pyrimidine biosynthesis for persister formation. Disruption of carB resulted in intracellular ATP accumulation, and lowering ATP levels using arsenate restored the antibiotic tolerance profile of the mutant to levels similar to those seen with the wild type. A decrease in ATP would lead to reduced antibiotic target activity and increased survival.IMPORTANCE Antibiotic treatment of P. aeruginosa residing in the lung of cystic fibrosis patients is ineffective. Treatment failure is attributed in part to antibiotic-tolerant phenotypic variants known as persister cells. Understanding how these cells emerge will likely inform future therapeutic strategies. In the current study, we identified carB, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in P. aeruginosa Disruption of carB resulted in a metabolic perturbation that increased cellular ATP and reduced persister formation. Conversely, lowering ATP in the mutant restored antibiotic tolerance. Our data support the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.

Project description:Background. As whole genome sequence data from bacterial isolates becomes cheaper to generate, computational methods are needed to correlate sequence data with biological observations. Here we present the large-scale BLAST score ratio (LS-BSR) pipeline, which rapidly compares the genetic content of hundreds to thousands of bacterial genomes, and returns a matrix that describes the relatedness of all coding sequences (CDSs) in all genomes surveyed. This matrix can be easily parsed in order to identify genetic relationships between bacterial genomes. Although pipelines have been published that group peptides by sequence similarity, no other software performs the rapid, large-scale, full-genome comparative analyses carried out by LS-BSR. Results. To demonstrate the utility of the method, the LS-BSR pipeline was tested on 96 Escherichia coli and Shigella genomes; the pipeline ran in 163 min using 16 processors, which is a greater than 7-fold speedup compared to using a single processor. The BSR values for each CDS, which indicate a relative level of relatedness, were then mapped to each genome on an independent core genome single nucleotide polymorphism (SNP) based phylogeny. Comparisons were then used to identify clade specific CDS markers and validate the LS-BSR pipeline based on molecular markers that delineate between classical E. coli pathogenic variant (pathovar) designations. Scalability tests demonstrated that the LS-BSR pipeline can process 1,000 E. coli genomes in 27-57 h, depending upon the alignment method, using 16 processors. Conclusions. LS-BSR is an open-source, parallel implementation of the BSR algorithm, enabling rapid comparison of the genetic content of large numbers of genomes. The results of the pipeline can be used to identify specific markers between user-defined phylogenetic groups, and to identify the loss and/or acquisition of genetic information between bacterial isolates. Taxa-specific genetic markers can then be translated into clinical diagnostics, or can be used to identify broadly conserved putative therapeutic candidates.

Dataset Information

Establishment of a method to rapidly assay bacterial persister metabolism.

Publications

Establishment of a method to rapidly assay bacterial persister metabolism.

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