Reverting Antibiotic Tolerance of Pseudomonas aeruginosa PAO1 Persister Cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one

Background

Bacteria are well known to form dormant persister cells that are tolerant to most antibiotics. Such intrinsic tolerance also facilitates the development of multidrug resistance through acquired mechanisms. Thus persister cells are a promising target for developing more effective methods to control chronic infections and help prevent the development of multidrug-resistant bacteria. However, control of persister cells is still an unmet challenge.

Methodology/Principal Findings

We show in this report that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can restore the antibiotic susceptibility of Pseudomonas aeruginosa PAO1 persister cells at growth non-inhibitory concentrations. Persister control by BF8 was found to be effective against both planktonic and biofilm cells of P. aeruginosa PAO1. Interestingly, although BF8 is an inhibitor of quorum sensing (QS) in Gram-negative bacteria, the data in this study suggest that the activities of BF8 to revert antibiotic tolerance of P. aeruginosa PAO1 persister cells is not through QS inhibition and may involve other targets.

Conclusion

BF8 can sensitize P. aeruginosa persister cells to antibiotics.     

Structural effects on persister control by brominated furanones

Bacterial persister cells are a small population of dormant cells that are tolerant to essentially all antibiotics. Recently, we reported that a quorum sensing (QS) inhibitor, (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8), can revert antibiotic tolerance of Pseudomonas aeruginosa persister cells. To better understand this phenomenon, several synthetic brominated furanones with similar structures were compared for their activities in persister control and inhibition of acyl-homoserine lactone (AHL) mediated QS. The results show that some other furanones in addition to BF8 are also AHL QS inhibitors and can revert antibiotic tolerance of P. aeruginosa PAO1 persister cells. However, not all QS inhibiting BFs can revert persistence at growth non-inhibitory concentrations, suggesting that QS inhibition itself is not sufficient for persister control.     

Controlling persister cells of Pseudomonas aeruginosa PDO300 by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one

Pseudomonas aeruginosa is a major pathogen causing chronic pulmonary infections; for example, 80% of cystic fibrosis patients get infected by this bacterium as the disease progresses. Such chronic infections are challenging because P. aeruginosa exhibits high-level tolerance to antibiotics by forming biofilms (multicellular structures attached to surfaces), by entering dormancy and forming antibiotic tolerant persister cells, and by conversion to the mucoid phenotype. Recently, we reported that a synthetic quorum sensing inhibitor, (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8), can sensitize both planktonic and biofilm-associated persister cells of P. aeruginosa PAO1 to antibiotics at the concentrations non-inhibitory to its growth. In this study, we further characterized the effects of this compound on the mucoid strain P. aeruginosa PDO300. BF8 was found to reduce persistence during the growth of PDO300 and effectively kill the persister cells isolated from PDO300 cultures. In addition to planktonic cells, BF8 was also found to inhibit biofilm formation of PDO300 and reduce associated persistence. These findings broaden the activities of this class of compounds and indicate that BF8 also has other targets in P. aeruginosa in addition to quorum sensing.     

Stabilization of Homoserine-O-Succinyltransferase (MetA) Decreases the Frequency of Persisters in Escherichia coli under Stressful Conditions

Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in Escherichia coli cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in E. coli growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native metA gene on the E. coli K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in E. coli K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.     

Escherichia coli persister cells suppress translation by selectively disassembling and degrading their ribosomes

Bacterial persisters are rare, phenotypically distinct cells that survive exposure to multiple antibiotics. Previous studies indicated that formation and maintenance of the persister phenotype are regulated by suppressing translation. To examine the mechanism of this translational suppression, we developed novel methodology to rapidly purify ribosome complexes from persister cells. We purified His‐tagged ribosomes from Escherichia coli cells that over‐expressed HipA protein, which induces persister formation, and were treated with ampicillin to remove antibiotic‐sensitive cells. We profiled ribosome complexes and analyzed the ribosomal RNA and protein components from these persister cells. Our results show that (i) ribosomes in persisters exist largely as inactive ribosomal subunits, (ii) rRNAs and tRNAs are mostly degraded and (iii) a small fraction of the ribosomes remain mostly intact, except for reduced amounts of seven ribosomal proteins. Our findings explain the basis for translational suppression in persisters and suggest how persisters survive exposure to multiple antibiotics.     

A Quorum Sensing Small Volatile Molecule Promotes Antibiotic Tolerance in Bacteria

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant “persister” trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2’ Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.     

The Formation of Persister Cells in Stationary-Phase Cultures of Escherichia Coli Is Associated with the Aggregation of Endogenous Proteins

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.     

SOS Response Induces Persistence to Fluoroquinolones in Escherichia coli

Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo.     

Bacterial persistence is an active σS stress response to metabolic flux limitation

While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high‐throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient‐rich conditions. The persister proteome is characterized by σ^S‐mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system‐level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti‐toxin systems, and the σ^S‐mediated stress response. Our system‐level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters.     

Drug Combinations against Borrelia burgdorferi Persisters In Vitro: Eradication Achieved by Using Daptomycin,Cefoperazone and Doxycycline

Although most Lyme disease patients can be cured with antibiotics doxycycline or amoxicillin using 2-4 week treatment durations, some patients suffer from persistent arthritis or post-treatment Lyme disease syndrome. Why these phenomena occur is unclear, but possibilities include host responses, antigenic debris, or B. burgdorferi organisms remaining despite antibiotic therapy. In vitro, B. burgdorferi developed increasing antibiotic tolerance as morphology changed from typical spirochetal form in log phase growth to variant round body and microcolony forms in stationary phase. B. burgdorferi appeared to have higher persister frequencies than E. coli as a control as measured by SYBR Green I/propidium iodide (PI) viability stain and microscope counting. To more effectively eradicate the different persister forms tolerant to doxycycline or amoxicillin, drug combinations were studied using previously identified drugs from an FDA-approved drug library with high activity against such persisters. Using a SYBR Green/PI viability assay, daptomycin-containing drug combinations were the most effective. Of studied drugs, daptomycin was the common element in the most active regimens when combined with doxycycline plus either beta-lactams (cefoperazone or carbenicillin) or an energy inhibitor (clofazimine). Daptomycin plus doxycycline and cefoperazone eradicated the most resistant microcolony form of B. burgdorferi persisters and did not yield viable spirochetes upon subculturing, suggesting durable killing that was not achieved by any other two or three drug combinations. These findings may have implications for improved treatment of Lyme disease, if persistent organisms or detritus are responsible for symptoms that do not resolve with conventional therapy. Further studies are needed to validate whether such combination antimicrobial approaches are useful in animal models and human infection.     

A molecular model for persister in E. coli

Like many other bacteria, Escherichia coli remain as tiny viable individuals named persisters after being exposed to an antibiotic. These persisters are believed to be phenotypic heterogeneous one rather than mutants, because their progenies are as susceptible to antibiotics as their ancestors. Recently, two persister-related genes (hipB/hipA) were confirmed to belong to a toxin-antitoxin (TA) module. Their control circuit was believed to be responsible for generation of the persister subpopulation. For the well-studied TA module, we build a simple genetic regulation model to explain the phenotypic heterogeneity. We find that a sole double-negative feedback loop is not enough to explain the phenotypic heterogeneity; the cooperation mechanisms in HipB and HipA are indispensable. Moreover, our model illustrates an important persister-related experimental phenomenon: the emergence of the persister depends on the growth rate in continuous culture.     

Survival of Bactericidal Antibiotic Treatment by a Persister Subpopulation of Listeria monocytogenes

Listeria monocytogenes can cause the serious infection listeriosis, which despite antibiotic treatment has a high mortality. Understanding the response of L. monocytogenes to antibiotic exposure is therefore important to ensure treatment success. Some bacteria survive antibiotic treatment by formation of persisters, which are a dormant antibiotic-tolerant subpopulation. The purpose of this study was to determine whether L. monocytogenes can form persisters and how bacterial physiology affects the number of persisters in the population. A stationary-phase culture of L. monocytogenes was adjusted to 10⁸ CFU ml⁻¹, and 10³ to 10⁴ CFU ml⁻¹ survived 72-h treatment with 100 μg of norfloxacin ml⁻¹, indicating a persister subpopulation. This survival was not caused by antibiotic resistance as regrown persisters were as sensitive to norfloxacin as the parental strain. Higher numbers of persisters (10⁵ to 10⁶) were surviving when older stationary phase or surface-associated cells were treated with 100 μg of norfloxacin ml⁻¹. The number of persisters was similar when a ΔsigB mutant and the wild type were treated with norfloxacin, but the killing rate was higher in the ΔsigB mutant. Dormant norfloxacin persisters could be activated by the addition of fermentable carbohydrates and subsequently killed by gentamicin; however, a stable surviving subpopulation of 10³ CFU ml⁻¹ remained. Nitrofurantoin that has a growth-independent mode of action was effective against both growing and dormant cells, suggesting that eradication of persisters is possible. Our study adds L. monocytogenes to the list of bacterial species capable of surviving bactericidal antibiotics in a dormant stage, and this persister phenomenon should be borne in mind when developing treatment regimens.     

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.     

Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins

Bacterial populations contain persisters, cells which survive exposure to bactericidal antibiotics and other lethal factors. Persisters do not have a genetic resistance mechanism, and their means to tolerate killing remain unknown. In exponentially growing populations of Escherichia coli the frequency of persister formation usually is 10⁻⁷ to 10⁻⁵. It has been shown that cells overexpressing either of the toxic proteins HipA and RelE, both members of the bacterial toxin-antitoxin (TA) modules, have the ability to form more persisters, suggesting a specific role for these toxins in the mechanism of persistence. However, here we show that cells expressing proteins that are unrelated to TA modules but which become toxic when ectopically expressed, chaperone DnaJ and protein PmrC of Salmonella enterica, also form 100- to 1,000-fold more persisters. Thus, persistence is linked not only to toxicity caused by expression of HipA or dedicated toxins but also to expression of other unrelated proteins.     

Heterogeneous Persister Cells Formation in Acinetobacter baumannii

Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%–11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B – an antibiotic that is believed to act on non-dividing cells as well – failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.