Persistent Fraction Research Articles

Controlled cellular redox, repressive hemin utilization and adaptive stress responses are crucial to metronidazole tolerance of Porphyromonas gingivalis persisters

Antimicrobial-tolerant microbial persisters critically account for various infections and inflammation. This study identified the characteristics of Porphyromonas gingivalis persisters, and explored their underlying survival mechanisms through proteomic profiling. Porphyromonas gingivalis cultured with different concentrations of hemin was treated with 100μg/ml of metronidazole (MTZ). The viability of P.gingivalis persisters was determined by colony-forming unit assay and LIVE/DEAD staining. The proteomic signature of P.gingivalis persister fractions was examined using LC-MS/MS and bioinformatic analysis. A small fraction of P.gingivalis persisters survived from lethal MTZ treatment without heritability. At late exponential phase, the frequency of these persisters significantly increased when incubated with 1μg/ml of hemin compared to 10μg/ml. Higher levels of P.gingivalis persisters formed at stationary phase than the late exponential phase. High-throughput proteomic analysis showed that the persisters markedly downregulated multiple proteins involved in electron transfer and heme/iron utilization essential for redox regulation and MTZ activation. Moreover, the persisters enabled to shut down major cellular activities (e.g. translation) and overexpress stress proteins. The presence and survival of metronidazole-tolerant P.gingivalis persisters may be dominated by regulation of cellular redox state and enhanced via repression of heme/iron utilization, dormancy and stress responses.

A comparison of coarse woody debris volume and variety between old-growth and secondary longleaf pine forests in the southeastern United States

Few efforts have been made to quantify the amount and variety of deadwood in frequently burned ecosystems, particularly the longleaf pine (Pinus palustris Mill.) ecosystem of the southeastern United States. Moreover, comparisons of coarse woody debris between old-growth and secondary longleaf pine forests are lacking despite the widely recognized value of deadwood to biodiversity in many forest types. We measured standing and fallen deadwood in three old-growth and four mature (100–125 years-old) secondary forests in two landscapes characterized by either sandy or clayey soils within the historic range of P. palustris. Downed coarse woody debris volume was variable at the old-growth locations, ranging from 2.51 ± 0.79 to 29.10 ± 14.55 m3 per ha, which includes perhaps the lowest values ever reported from any old-growth forest. Factors likely contributing to these low volumes include frequent fire, the low basal area characteristic of this forest type, subtropical climatic conditions of the southeastern Coastal Plain, and large termite populations. The high variability observed among the three old-growth locations probably reflect interactions between fire and other disturbances (e.g., wind damage). The old-growth location on sandy soils had significantly higher coarse woody debris volume and deadwood variety (e.g., diameter increments, posture, tree genera and decay classes) than secondary forests sampled nearby. Highly resinous heartwood is a significant indicator of old-growth conditions relative to secondary locations, appearing to accumulate as a persistent fraction of the deadwood pool over time.

The Putative De-N-acetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones.

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1–3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5–7 fold) and mexX (3.6–5.4 fold) in the parental strain but to a lower extent (3–4-fold for gyrA and 1.8–2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target.

Endo or Exo? Structures of Gas-Phase Alkali Metal Cation/Aromatic Half-Belt Complexes.

1,1,9,9-Tetramethyl[9](2,11)teropyrenophane (TM9TP), a belt-shaped molecule, has a sizable cavity that molecules or ions could occupy. In this study, the question of whether TM9TP forms gas-phase ion-molecule complexes with metal cations (K+ , Rb+ , Cs+ ) situated inside or outside the TM9TP cavity was addressed using both experimental and computational methods. Complexes were trapped in a Fourier transform ion cyclotron resonance mass spectrometer and their structures were explored by some novel physical chemistry/mass spectrometry methods. Blackbody infrared radiative dissociation kinetics reveal two populations of ions, a fast dissociating fraction and a persistent fraction. Infrared multiphoton dissociation spectra (vibrational spectra) provide very strong evidence that the most abundant population is a complex where the metal cation is inside the TM9TP cavity, endo-TM9TP. Red-shifted C-H stretching bands present in the gas-phase vibrational spectra of these ionic complexes show that there is an interaction between the metal cation and bridge C-H bonds due to the cation sitting inside the cavity of TM9TP. B3LYP/6-31+G(d,p) calculations showed the endo complexes to be the lowest in energy; about 60 kJ mol-1 more thermodynamically stable and more than 120 kJ mol-1 kinetically more stable than the exo complex.

Pyrogenic Carbon Lacks Long-Term Persistence in Temperate Arable Soils

Pyrogenic organic carbon (PyOC) derived from incomplete burning of biomass is considered the most persistent fraction of soil organic carbon (SOC), being expected to remain in soil for centuries. However, PyOC persistence has seldom been evaluated under field conditions. Based on a unique set of soils from five European long-term bare fallows (LTBF), i.e., vegetation-free field experiments, we provide the first direct comparison between PyOC and SOC persistence in temperate arable soils. We found that soil PyOC contents decreased more rapidly than expected from current concepts, the mean residence time (MRT) of native PyOC being just 1.6 times longer than that of SOC. At the oldest experimental site, 55% of the initial PyOC remained after 80 years of bare fallow. Our results suggest that while the potential for long-term C storage exists, the persistence of PyOC in soil may currently be overestimated.

Colloidal Stability and Mobility of Extracellular Polymeric Substance Amended Hematite Nanoparticles

Core Ideas Extracellular polymeric substances (EPS) sorb to hematite nanoparticle surfaces. High EPS loadings on hematite cause an increase in colloidal stability. Hematite is immobilized in glass bead porous media during transport. Colloidally stable EPS–hematite associates are mobile in glass bead porous media. Colloidal stability and the mobility of mineral nanoparticles largely depend on the properties of the nanoparticle surface. Thus, any modification of the surface due to adsorption will alter the fate of nanoparticles in the environment. In this study, we explored the effect of extracellular polymeric substances (EPS) on the colloidal stability and the mobility of hematite nanoparticles (HNP). Hematite nanoparticles were amended with increasing concentrations of EPS (20, 200, and 500 mg L−1 C) that resulted in EPS‐C to HNP‐Fe mass ratios of 1:5, 2:1, and 5:1 (HNP 1:5, HNP 2:1, and HNP 5:1). With increasing EPS loadings, the isoelectric point of the EPS–HNP associates shifted from 9.8 (pure HNP without EPS) to ?2 (HNP 5:1), resulting in an aggregation of HNP 1:5 with an isoelectric point near the solution pH of 7. In NaCl solution, both the aggregation kinetics and the critical coagulation concentration (CCC) increased with increasing loadings of EPS. Conversely, in CaCl2 solution, the CCC decreased with increasing EPS loadings due to the formation of Ca bridges. The mobility of pure HNP and EPS–HNP associates was explored with transport experiments in closed‐flow mode at neutral pH in a porous medium made of glass beads. Pure HNP were colloidally stable but completely retained in the porous medium due to strong electrostatic interactions between the HNP and the glass surfaces. In contrast, for colloidally stable EPS–HNP associates (HNP 2:1 and HNP 5:1), a persistent mobile fraction and surface attachment was observed. This highlights that EPS association promotes mobility of HNP that were otherwise rendered immobile.

3. Clinical features and outcome of patients with recurrent myocarditis

Clinical research. Oral presentation. Myocarditis can be associated with short and long term major cardiac event. However, most episodes are self-limited and rarely recurrent. The aim of this analysis was to describe a series of six patients who had multiple episodes of recurrent myocarditis and characterize their clinical, laboratory, electrocardiographic (ECG) and imaging features. The patients were identified from cardiac MRI (CMR) database during the period of 2007–2016. Patients records were reviewed for demographic data, laboratory results including serum troponin I, ECG findings, coronary angiography, imaging findings from echocardiography and CMR. The diagnosis of acute myocarditis was ascertained by a combined clinical and imaging findings. Patients with myocarditis related to connective tissue disease were excluded. During the study period, a total of 37 patients presented with acute myocarditis of which 6 (16.2%) patients (mean age 31.7 ± 10.4 years, 100% males) had multiple recurrent episodes; twice in 4 patients and 4 episodes in 2 patients (Table 1). The mean interval period between the first and last attack was 3.3 years (range was 0.8–5.0). After a median follow-up of 4.4 years, there was evidence of new epicardial or mid-wall myocardial delayed enhancement (MDE) with every new episode and all patients had persistent MDE on the last CMR. However, no patient developed persistent heart failure symptoms or left ventricular ejection fraction <40%. Only one patient had transient heart failure symptoms which improved on angiotensin converting enzyme inhibitors and beta blockers. Table 1 demonstrates imaging findings from the last echocardiography and CMR examinations. Recurrent myocarditis in our population is probably under-reported. One in six patients with myocarditis had clinical and imaging proven recurrence. The use of CMR may help to ascertain the diagnosis in otherwise unexplained elevated cardiac markers. The long term management and outcome of recurrent myocarditis requires further study.

5. Incidence of herceptin induced cardiomyopathy in treated breast cancer patients: A prospective single center study in Saudi Arabia

Clinical Research. Digital Poster. Herceptin therapy is currently a standard therapy for receptor positive breast cancer. Prior studies suggested that Herceptin reduced risk of breast cancer recurrence. Nevertheless, cardiotoxicity is a well-known adverse effect of cancer related therapies. Early identification of patients at risk for cardiotoxicity could prevent late cardiovascular complications. In Saudi Arabia, there is limited published data about Herceptin cardiotoxicity. Thus, the aim of this study is to estimate the incidence of Herceptin induced cardiomyopathy in a prospective well controlled manner. This is a prospective study conducted at king abdulaziz medial city between January 2012 and December 2013. The study included all patients who had proven breast cancer and were eligible for Herceptin therapy. Patients with prior cardiovascular diseases or pregnant patients were excluded. Herceptin therapy was administered according to standard guideline for management. Patients were followed up for one year: clinically every three weeks in the first three month then every three months for one year. Furthermore, Echocardiography and troponin evaluation were obtained every three months, and cardiac magnetic resonance scanning every 6 months. A total of 50 patients (mean age 47 ± 10 years, 96% were Saudis) without previous cardiovascular diseases were included. Using echocardiography criteria, 4(8%) patients developed cardiomyopathy. During follow-up, 75% of them had a persistent low left ventricular ejection fraction (LVEF) less than 50%, while only one patient recovered her LVEF. Global longitudinal strain (GLS) was done in 45 patients, one third had normal GLS (<−18). On the other hand, using CMR, only one patient had a significant dropped in the LVEF (from 61% to 48%) while another patient developed new myocardial delayed enhancement. Conclusion Our study showed low incidence rate of cardiomyopathy toxicity due to Herceptin therapy in Saudi individuals. However, the rate of cardiomyopathy differs by the criteria used as well as the imaging modality used to assess LV function.

On the dynamics of instabilities in two-fluid models for bubbly flows

In this paper we look at instabilities in bubbly gas-liquid flows and investigate the emergence and characteristics of phase heterogeneities. We apply a shared-pressure two-fluid model to low bubble loadings and demonstrate the existence of persistent gas fraction instabilities of a characteristic size larger than the applied computational grid. In particular, we investigate the influence of a virtual mass effect on the stability of the two-fluid model and we demonstrate a change in the emergence and the dynamics of the phase heterogeneities. The change is accounted to a difference in the degree of hyperbolicity due to the inclusion of the virtual mass force. Furthermore, the results indicate that an initial instability, concluded as numerical in its character, evolves into a state with a physical character of the heterogeneities. We discuss implications of the existence and dynamics of the heterogeneities and the importance of the numerical behavior for interpretation of the results. In particular, we argue that underlying characteristics of the model cannot and should not be concealed with additional sub-models (such as momentum exchange terms) but must be acknowledged in the analysis of results from the two-fluid model for bubbly flows.

Myocardial Damage Detected by Late Gadolinium Enhancement Cardiac Magnetic Resonance Is Uncommon in Peripartum Cardiomyopathy.

BackgroundIn peripartum cardiomyopathy, the prevalence of focal myocardial damage detected by late gadolinium enhancement (LGE) cardiovascular magnetic resonance is important to elucidate mechanisms of myocardial injury and cardiac dysfunction. LGE equates irreversible myocardial injury, but LGE prevalence in peripartum cardiomyopathy is uncertain.Methods and ResultsAmong 100 women enrolled within the Investigations of Pregnancy Associated Cardiomyopathy cohort, we recruited 40 women at 13 centers to undergo LGE cardiovascular magnetic resonance, enrolled within the first 13 weeks postpartum. Follow‐up scans occurred at 6 months postpartum, and death/transplant rates at 12 months. Baseline characteristics did not differ significantly in the parent cohort according to cardiovascular magnetic resonance enrollment except for mechanical circulatory support. LGE was noted only in 2 women (5%) at baseline. While left ventricular dysfunction with enlargement was prevalent at baseline cardiovascular magnetic resonance scans (eg, ejection fraction 38% [Q1–Q3 31–50%], end diastolic volume index=108 mL/m2 [Q1–Q3 83–134 mL/m2]), most women demonstrated significant improvements at 6 months, consistent with a low prevalence of LGE. LGE was not related to baseline clinical variables, ejection fraction, New York Heart Association heart failure class, or mortality. Neither of the 2 women who died exhibited LGE. LGE was inversely associated with persistent left ventricular ejection fraction at 6 months (P=0.006).ConclusionsFactors other than focal myocardial damage detectable by LGE explain the initial transient depressions in baseline left ventricular ejection fraction, yet focal myocardial damage may contribute to persistent myocardial dysfunction and hinder recovery in a small minority. Most women exhibit favorable changes in ventricular function over 6 months.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01085955.

Effects of meropenem exposure in persister cells of Acinetobacter calcoaceticus-baumannii.

To evaluate the influence of meropenem in the Acinetobacter calcoaceticus-baumannii (ACB) persister levels. Persister levels in planktonic and biofilm cultures from ACB isolates were evaluated after exposure to different meropenem concentrations. A high variability of persister fractions was observed among the isolates cultured under planktonic and biofilm conditions. Meropenem concentration did not influence persister fractions, even when far above the MIC. No correlation was found between persister levels and biofilm biomass. The magnitude of persister levels from ACB planktonic and, particularly, biofilm cultures exposed to meropenem was independent of the antibiotic concentration, dosing regimen and biofilm biomass. These findings, in a context of meropenem failure to treat chronic infections, strengthen the importance of understanding persister behavior.

Carbon and nitrogen molecular composition of soil organic matter fractions resistant to oxidation

The methods used to isolate and characterise pyrogenic organic carbon (PyC) from soils vary widely, and there is little agreement in the literature as to which method truly isolates the most chemically recalcitrant (inferred from oxidative resistance) and persistent (inferred from radiocarbon abundance) fraction of soil organic matter. In addition, the roles of fire, fuel type and soil morphology in the preservation of PyC are not yet defined. In an attempt to elucidate the importance of oxidative recalcitrance, fuel type and soil morphology to the persistence of soil organic matter, we examined two strongly contrasting soils using a variety of PyC isolation techniques coupled with quantifications of the molecular structure and mean residence time of the isolated organic materials. Surface and subsurface soil samples were examined from a Red Chromosol soil and a Black Vertosol soil. The δ13C values suggest that PyC in the Red Chromosol was sourced from eucalyptus, whereas PyC in the Black Vertosol was formed from grass. Soils were sieved at 53µm, treated with hydrofluoric acid to remove organics associated with mineral surfaces, then subjected to three common ‘PyC isolation’ treatments: chromic acid, photo-oxidation and chromic acid followed by photo-oxidation. Molecular structure of the organic residues remaining after each treatment was quantified by solid-state 13C cross polarisation magic angle spinning nuclear magnetic resonance and near edge X-ray absorption fine structure spectroscopy, and the mean residence time of the organic residues was estimated based on radiocarbon abundance. In all cases, treatment with chromic acid followed by photo-oxidation isolated the smallest proportion of organic matter (5–10% of &lt;53µm C) which also had the longest mean residence time (estimated 600–3460 years). Additionally, molecular structure measurements indicated that this fraction was not composed solely of aromatic compounds, suggesting a non-homogenous source for the most oxidative-resistant fraction of soil organic matter.

Contract and Rent Dispersion in a Frictional Labor Market

This paper incorporates a risk-neutral principal-agent model with contractual constraints into a random search model to study the interaction between contracting and search in partial and general equilibrium. I introduce heterogeneity in principals' and agents' production technologies in terms of the distribution of output across states of nature and characterize equilibrium in this context. Principals tailor contracts to agents' production technologies to incentivize agents' effort, generating dispersion in optimal contracts. The resulting endogenous dispersion in agency rents introduces an option value of search, which can generate inefficient equilibria with lengthy search and a persistent fraction of unmatched principals and agents. I show that a reduction in search costs and contractual innovations can reduce welfare. The results have implications for labor and financial economics and imply a novel theory of specialization in decentralized markets.

Starvation- and antibiotics-induced formation of persister cells in Pseudomonas aeruginosa.

Planktonic stationary and exponential cultures of Pseudomonas aeruginosa are highly resistant to killing by bactericidal antimicrobials because of the presence of persisters, cells that are multidrug tolerant and play a key role in the recalcitrance of biofilm infections. The aim of this study was to investigate the formation of persister cells in P. aeruginosa stationary vs. exponential cultures using different class antimicrobials. The susceptibilities of P. aeruginosa PAO1 wild-type and mutant strains to antimicrobials were determined by standard microtiter broth dilution method. In order to determine persister formation, dose- and time-dependent killing experiments were performed with antibiotics. Ceftazidime (Cephalosporin) showed little efficacy against either culture. Stationary-phase cells were more tolerant to imipenem (Carbapenem) than exponential cells, leaving a small fraction of persisters at high imipenem concentration in both populations. Polymyxin B (Polymyxin) appeared to be ineffective at low concentrations against both cell populations. Very high polymyxin B concentration completely eradicated exponential cells and regrowth was seen in a stationary population. Stationary cells were more tolerant to tobramycin (Aminoglycoside) than exponential cells but a higher concentration of tobramycin completely eliminated survivors. Ciprofloxacin (Fluoroquinolone) at a low concentration resulted in killing of both cultures of P. aeruginosa, producing persisters that were invulnerable to killing. Stationary cells appear to be somewhat more tolerant than exponential cells in all of these assays. We also showed that nutrient deprivation (serine starvation) regulated by stringent and general stress response, contribute to the increased tolerance of P. aeruginosa exponential and stationary planktonic cells via production of persisters.

Enhancing sewage sludge anaerobic “re-digestion” with combinations of ultrasonic, ozone and alkaline treatments

This study investigated the feasibility of using ultrasonic (ULS), ozone assisted ultrasonic (ULS-Ozone) and alkaline assisted ultrasonic (ULS+ALK) post-treatment to target the persistent organic fraction in anaerobically digested sludge in order to increase methane recovery. Synergistic COD solubilization in digested sludge was observed when ozone (0.012g O3g−1 TS) and alkaline (0.02M for 10min) treatment was combined with ULS treatment. The digested sludge Soluble Chemical Oxygen Demand (SCOD) increased from 200mg/L to 1500, 2600 and 2650mg/L after the ULS, ULS-Ozone and ULS+ALK treatments, respectively. Different compounds were, however, solubilized after the ULS-Ozone and ULS+ALK treatments as indicated by size exclusion chromatography (SEC). ULS+ALK treatment primarily solubilized macromolecules with molecular weight (MW) over 500kDa; while, the ULS-Ozone treatment solubilized macromolecules with MW higher than 500kDa and also organics with MW around 103kDa. The methane production from “re-digestion” of the treated digested sludge increased by 28.3%, 48.3% and 39.5% after the ULS, ULS-Ozone and ULS+ALK treatments, respectively.

OBTENCIÓN DE UN MEDIO ENRIQUECIDO PARA HACER MÁS EFICIENTE LA BIODISPONIBILIDAD DE LOS HIDROCARBUROS INTEMPERIZADOS EN UN SUELO COSTERO

The weathered hydrocarbons are mainly constituted of highly persistent heavy petroleum fractions. Thus, the soil chemical complexity delays the hydrocarbon biodegradation. The objective of this study was to increase the bioavailability of highly persistent compounds by obtaining the greatest nutritional conditions for indigenous microorganisms. A consortium was previously adapted in order to achieve the full potential of degradation through the biostimulation process which was carried out by the Plackett-Burman analytic method experiments. These experiments conducted to a specific combination of nutrients, which provided the minimum requirements for the hydrocarbonoclastic bacteria to efficiently improve their metabolic processes. The biodegradation of hydrocarbons were estimated based on the heavy oil/kg dry soil, CO 2 production/kg dry soil, as well as the alteration of certain physicochemical characteristics, such as pH, EC (μS/cm), moisture and organic matter percentage. In conclusion, these analyses provide a contribution to the study of bioavailability of weathered hydrocarbons by controlling conditions in a nutrient biostimulation process and through the design of experiments. Likewise, a microbial consortium adapted to a 1 % concentration of Mayan crude, resulted in an up to 97 % increase in hydrocarbon degradation in the coastal weathered soil, which were expressed as a heavy fraction of hydrocarbons in a period of 28 days of degradation.

Atrial fibrillation per se was a major determinant of global left ventricular longitudinal systolic strain.

Atrial fibrillation (AF) may cause systolic abnormality via inadequate diastolic filling and tachycardia-induced cardiomyopathy. Global longitudinal strain (GLS) is a very sensitive method for detecting subtle left ventricular systolic dysfunction. Hence, this study aimed to evaluate whether AF patients had a more impaired GLS, AF was a major determinant of GLS, and determine the major correlates of GLS in AF patients.The study included 137 patients with persistent AF and left ventricular ejection fraction (LVEF) above 50% and 137 non-AF patients matched according to age, gender, and LVEF. Comprehensive echocardiography with GLS assessment was performed for all cases.Compared with non-AF patients, AF patients had a more impaired GLS, a larger left atrial volume index, higher transmitral E wave velocity (E), and early diastolic mitral velocity (Ea) (all P < 0.001) but comparable E/Ea. After adjustment for baseline and echocardiographic characteristics, the presence of AF remained significantly associated with impaired GLS (β = 0.533, P < 0.001). In addition, multivariate analysis of AF patients indicated that faster heart rates and decreased E, Ea, and LVEF were associated with more impaired GLS.This study demonstrated that AF patients had a more impaired GLS than non-AF patients, although LVEF was comparable between the 2 groups. AF was a major determinant of GLS even after adjustment for relevant clinical and echocardiographic parameters.

Novel Inhibitors of Toxin HipA Reduce Multidrug Tolerant Persisters

Persisters are a small fraction of drug-tolerant bacteria without any genotype variations. Their existence in many life-threatening infectious diseases presents a major challenge to antibiotic therapy. Persistence is highly related to toxin-antitoxin modules. HipA (high persistence A) was the first toxin found to contribute to Escherichia coli persistence. In this study, we used structure-based virtual screening for HipA inhibitors discovery and identified several novel inhibitors of HipA that remarkably reduced E. coli persistence. The most potent one decreased the persister fraction by more than five-fold with an in vitro K D of 270 ± 90 nM and an ex vivo EC50 of 46 ± 2 and 28 ± 1 μM for ampicillin and kanamycin screening, respectively. These findings demonstrated that inhibition of toxin can reduce bacterial persistence independent of the antibiotics used and provided a framework for persistence treatment by interfering with the toxin-antitoxin modules.

Protocol for Determination of the Persister Subpopulation in Candida Albicans Biofilms.

In contrast to planktonic cultures of the human fungal pathogen Candida albicans, C. albicans biofilms can contain a persister subpopulation that is tolerant to high concentrations of currently used antifungals. In this chapter, the method to determine the persister fraction in a C. albicans biofilm treated with an antifungal compound is described. To this end, a mature biofilm is developed and subsequently treated with a concentration series of the antifungal compound of interest. Upon incubation, the fraction of surviving biofilm cells is determined by plating and plotted versus the used concentrations of the antifungal compound. If a persister subpopulation in the biofilm is present, the dose-dependent killing of the biofilm cells results in a biphasic killing pattern.

A Whole-Cell-Based High-Throughput Screening Method to Identify Molecules Targeting Pseudomonas aeruginosa Persister Cells.

Despite its clinical relevance and the fact that the phenomenon of persistence was discovered in the 1940s, little is known about the mechanisms behind persister cell formation. Research in this field has mainly focused on the model organism Escherichia coli and few genetic determinants of persistence have been described in other bacterial species, impairing the development of target-based strategies to combat these antibiotic-tolerant cells. In this chapter we describe a top-down large-scale screening method capable of specifically identifying small molecule compounds that, in combination with conventional antibiotics, significantly reduce the persister fraction in Pseudomonas aeruginosa. The method is readily adaptable for other species. Further characterization and analysis of the mode of action of the identified compounds can provide additional insight into the mechanisms behind persister formation and can guide the development of future anti-persister therapies.