pH-Mediated Potentiation of Aminoglycosides Kills Bacterial Persisters and Eradicates In Vivo Biofilms

Lebeaux, David; Chauhan, Ashwini; Létoffé, Sylvie; Fischer, Frédéric; Reuse, Hilde De; Beloin, Christophe; Ghigo, Jean‐Marc

doi:10.1093/infdis/jiu286

Cited by 112 publications

(94 citation statements)

References 41 publications

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“…In fact, persistence can be regarded as a metabolic program, where shutdown of metabolic processes participates in entry into this quasidormant state, metabolic activity during stasis maintains viability, and reactivation of metabolism is required for reawakening and growth after the conclusion of antibiotic treatment. The importance of metabolism is further highlighted by the discovery of antipersister strategies which depend on metabolic stimulation in persisters (9,(35)(36)(37)(38). These findings advocate for the need for greater understanding of persister metabolism.…”

Section: Discussionmentioning

confidence: 99%

Impacts of Global Transcriptional Regulators on Persister Metabolism

Mok

Orman

Brynildsen

2015

Antimicrob Agents Chemother

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Bacterial persisters are phenotypic variants with an extraordinary capacity to tolerate antibiotics, and they are hypothesized to be a main cause of chronic and relapsing infections. Recent evidence has suggested that the metabolism of persisters can be targeted to develop therapeutic countermeasures; however, knowledge of persister metabolism remains limited due to difficulties associated with isolating these rare and transient phenotypic variants. By using a technique to measure persister catabolic activity, which is based on the ability of metabolites to enable aminoglycoside (AG) killing of persisters, we investigated the role of seven global transcriptional regulators (ArcA, Cra, cyclic AMP [cAMP] receptor protein [CRP], DksA, FNR, Lrp, and RpoS) on persister metabolism. We found that removal of CRP resulted in a loss of AG potentiation in persisters for all metabolites tested. These results highlight a central role for cAMP/CRP in persister metabolism, as its perturbation can significantly diminish the metabolic capabilities of persisters and effectively eliminate the ability of AGs to eradicate these troublesome bacteria. Bacterial persisters are phenotypic variants that are highly tolerant to antibiotics (1). It is believed that they are a main culprit of the proclivity of biofilm infections to relapse, which imposes a substantial burden on the health care system (2, 3). When a bacterial population is treated with bactericidal antibiotics, biphasic killing is observed, where the death of normal cells is characterized by an initial, rapid killing rate, and the presence of persisters is illuminated by a second regime with a much lower rate of cell death (4, 5). When these survivors are recultured, the resulting population exhibits antibiotic sensitivity identical to that of the original culture, demonstrating that persisters are not antibiotic-resistant mutants but phenotypic variants (1, 5, 6).While persisters largely arise from dormant subpopulations (2,4,7,8), recent studies have demonstrated that they remain metabolically active, with the capacity to catabolize specific carbon sources and generate proton motive force through respiration (9, 10). This metabolic activity, specifically, proton motive force generation, enables aminoglycoside (AG) transport into cells that results in killing of persisters, and several enzymes needed for this process have been identified (9, 10). Knowledge of the enzymes and metabolic pathways present in persisters, as well as how they can be altered, could prove useful for the development of antipersister therapies. A fundamental question in this regard is, what are the cellular components responsible for defining the metabolic network in persisters? Due to the strong dependence of metabolism on transcriptional regulation (11, 12), the goal of this study was to determine the importance of several global transcriptional regulators to persister metabolism. To do this, we measured catabolic activity in persisters from ⌬arcA, ⌬cra, ⌬crp, ⌬dksA, ⌬fnr, ⌬lrp, and ⌬rpoS mutants and...

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Section: Discussionmentioning

confidence: 99%

Impacts of Global Transcriptional Regulators on Persister Metabolism

Mok

Orman

Brynildsen

2015

Antimicrob Agents Chemother

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“…A previous report demonstrated that medium alkalinization upon addition of the amino acid L-arginine enhanced the effects of aminoglycosides on killing Gram-negative and Gram-positive bacteria in vitro and in vivo during an infection (52). Therefore, to determine whether L-arginine was capable of restoring aminoglycoside susceptibility in the presence of eDNA, we assessed the MIC of P. aeruginosa in the presence of gentamicin and tobramycin.…”

Section: P Aeruginosa Biofilms Contain Acidic Microdomainsmentioning

confidence: 99%

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

Wilton

Charron-Mazenod

Moore

et al. 2016

Antimicrob Agents Chemother

197

188

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bBiofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2-to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

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“…Recently, the hipAB system of Shewanella oneidensis was characterized, and a ternary HipAB-DNA complex different from the one from E. coli was observed (46). Since persister cells are highly problematic with respect to antibiotic treatments of infectious diseases, much emphasis has been placed on the development of drugs that suppress the development and survival of persister cells (47)(48)(49)(50)(51)(52)(53).…”

Section: Persister Cells As a Classical Model For Phenotypic Heterogementioning

confidence: 99%

Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior

Grote

Krysciak

Streit

2015

Appl Environ Microbiol

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Phenotypic heterogeneity describes the occurrence of "nonconformist" cells within an isogenic population. The nonconformists show an expression profile partially different from that of the remainder of the population. Phenotypic heterogeneity affects many aspects of the different bacterial lifestyles, and it is assumed that it increases bacterial fitness and the chances for survival of the whole population or smaller subpopulations in unfavorable environments. Well-known examples for phenotypic heterogeneity have been associated with antibiotic resistance and frequently occurring persister cells. Other examples include heterogeneous behavior within biofilms, DNA uptake and bacterial competence, motility (i.e., the synthesis of additional flagella), onset of spore formation, lysis of phages within a small subpopulation, and others. Interestingly, phenotypic heterogeneity was recently also observed with respect to quorum-sensing (QS)-dependent processes, and the expression of autoinducer (AI) synthase genes and other QS-dependent genes was found to be highly heterogeneous at a single-cell level. This phenomenon was observed in several Gram-negative bacteria affiliated with the genera Vibrio, Dinoroseobacter, Pseudomonas, Sinorhizobium, and Mesorhizobium. A similar observation was made for the Gram-positive bacterium Listeria monocytogenes. Since AI molecules have historically been thought to be the keys to homogeneous behavior within isogenic populations, the observation of heterogeneous expression is quite intriguing and adds a new level of complexity to the QS-dependent regulatory networks. All together, the many examples of phenotypic heterogeneity imply that we may have to partially revise the concept of homogeneous and coordinated gene expression in isogenic bacterial populations. Bacteria have evolved multiple strategies to cope with rapid and frequent changes in their environment. These survival strategies may include spore formation, increased production of polysaccharides, biofilm formation or escape from biofilms (i.e., switching from a motile into a sessile form and vice versa), altered motility, change of metabolic capabilities, response to antibiotics, and many more. In general, these switches are initiated by environmental or bacterially produced signals that are perceived by a diverse array of regulators and delegated into the corresponding regulatory networks. This presumably results in altered transcription levels of different genes or operons, which eventually causes a change in the phenotype and a response to the environmental stimulus. Within this context, it is generally assumed that the majority of a population will undergo this switch within a short time period and that the population will show a mostly homogeneous expression profile. During the last decade, it became, however, evident that a certain fraction of cells in an isogenic population behaves differently from the others, even though the environment may not have changed significantly. The term "phenotypic heterogeneity" thereby descr...

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pH-Mediated Potentiation of Aminoglycosides Kills Bacterial Persisters and Eradicates In Vivo Biofilms

Abstract: Given that intravenous administration of L-arginine to human patients is well tolerated, combined use of aminoglycoside and the non-toxic adjuvant L-arginine as catheter lock solution could constitute a new option for the eradication of pathogenic biofilms.

Cited by 112 publications

References 41 publications

Impacts of Global Transcriptional Regulators on Persister Metabolism

Impacts of Global Transcriptional Regulators on Persister Metabolism

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior

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