Persistent Fraction Research Articles

The In vitro Antibiotic Tolerant Persister Population in Burkholderia pseudomallei is Altered by Environmental Factors

Bacterial persistence due to antibiotic tolerance is a critical aspect of antibiotic treatment failure, disease latency, and chronic or reemergent infections. The levels of persisters is especially notable for the opportunistic Gram-negative pathogens from the Burkholderia and Pseudomonas genera. We examined the rate of drug tolerant persisters in Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia cepacia complex organisms, and Pseudomonas aeruginosa at mid-log growth in LB broth culture. We found that a fraction of the antibiotic-sensitive cells from every species were tolerant to a 24 h high-dose antibiotic challenge. All tested Burkholderia strains demonstrated a drug tolerant persister population at a rate that was at least 100–500 times higher than P. aeruginosa. When challenged with at least a 10X minimum inhibitory concentration (MIC) 24 h exposure to three different antibiotics with different modes of action we found that in B. pseudomallei Bp82 each of the tree antibiotics revealed different persister fractions at each of two different growth states. This observation suggests that our assay is detecting heterogeneous persister subpopulations. Persistence in B. pseudomallei Bp82 was highly dependent on growth stage, with a surprisingly high persister fraction of >64% of the late stationary phase cells being antibiotic tolerant to 100XMIC cefotaxime. Adaptation of B. pseudomallei to distilled water storage resulted in a population of drug tolerant cells up to 100% of the non-drug-challenged viable cell count in the same cefotaxime assay. Cultivation of B. pseudomallei with a sub-inhibitory concentration of several antibiotics resulted in altered persister fractions within the population relative to cultures lacking the antibiotic. Our study provides insight into the sensitivity of the persister fraction within the population of B. pseudomallei due to environmental variables and suggests diversity within the persister population revealed by different challenge antibiotics.

Solar photocatalytic degradation of naphthenic acids in oil sands process-affected water.

Bitumen mining in the Canadian oil sands creates large volumes of oil sands process-affected water (OSPW), the toxicity of which is due in part to naphthenic acids (NAs) and other acid extractable organics (AEO). The objective of this work was to evaluate the potential of solar photocatalysis over TiO2 to remove AEO from OSPW. One day of photocatalytic treatment under natural sunlight (25 MJ/m(2) over ∼14 h daylight) eradicated AEO from raw OSPW, and acute toxicity of the OSPW toward Vibrio fischeri was eliminated. Nearly complete mineralization of organic carbon was achieved within 1-7 day equivalents of sunlight exposure, and degradation was shown to proceed through a superoxide-mediated oxidation pathway. High resolution mass spectrometry (HRMS) analysis of oxidized intermediate compounds indicated preferential degradation of the heavier and more cyclic NAs (higher number of double bond equivalents), which are the most environmentally persistent fractions. The photocatalyst was shown to be recyclable for multiple uses, and thus solar photocatalysis may be a promising "green" advanced oxidation process (AOP) for OSPW treatment.

Delicate Metabolic Control and Coordinated Stress Response Critically Determine Antifungal Tolerance of Candida albicans Biofilm Persisters.

Candida infection has emerged as a critical health care burden worldwide, owing to the formation of robust biofilms against common antifungals. Recent evidence shows that multidrug-tolerant persisters critically account for biofilm recalcitrance, but their underlying biological mechanisms are poorly understood. Here, we first investigated the phenotypic characteristics of Candida biofilm persisters under consecutive harsh treatments of amphotericin B. The prolonged treatments effectively killed the majority of the cells of biofilms derived from representative strains of Candida albicans, Candida glabrata, and Candida tropicalis but failed to eradicate a small fraction of persisters. Next, we explored the tolerance mechanisms of the persisters through an investigation of the proteomic profiles of C. albicans biofilm persister fractions by liquid chromatography-tandem mass spectrometry. The C. albicans biofilm persisters displayed a specific proteomic signature, with an array of 205 differentially expressed proteins. The crucial enzymes involved in glycolysis, the tricarboxylic acid cycle, and protein synthesis were markedly downregulated, indicating that major metabolic activities are subdued in the persisters. It is noteworthy that certain metabolic pathways, such as the glyoxylate cycle, were able to be activated with significantly increased levels of isocitrate lyase and malate synthase. Moreover, a number of important proteins responsible for Candida growth, virulence, and the stress response were greatly upregulated. Interestingly, the persisters were tolerant to oxidative stress, despite highly induced intracellular superoxide. The current findings suggest that delicate metabolic control and a coordinated stress response may play a crucial role in mediating the survival and antifungal tolerance of Candida biofilm persisters.

Isolation of Asphaltene-Degrading Bacteria from Sludge Oil

Sludge oil contains 30%–50% hydrocarbon fractions that comprise saturated fractions, aromatics, resins, and asphaltene. Asphaltene fraction is the most persistent fraction. In this research, the indigenous bacteria that can degrade asphaltene fractions from a sludge oil sample from Balikpapan that was isolated using BHMS medium ( Bushnell-Hass Mineral Salt ) with 0.01% ( w /v) yeast extract, 2% ( w /v) asphaltene extract, and 2% ( w /v) sludge oil. The ability of the four isolates to degrade asphaltene fractions was conducted by the biodegradation asphaltene fractions test using liquid cultures in a BHMS medium with 0.01% (w/v) yeast extract and 2% (w/v) asphaltene extract as a carbon source. The parameters measured during the process of biodegradation of asphaltene fractions include the quantification of Total Petroleum Hydrocarbon (g), log total number of bacteria (CFU/ml), and pH. There are four bacteria (isolates 1, 2, 3, and 4) that ha ve been cha r acterized to degrade asphaltic fraction and have been identified as Bacillus sp. Lysinibacillus fusiformes, Acinetobacter sp., and Mycobacterium sp. , respectively. The results showed that the highest ability to degrade asphaltene fractions is that of Bacillus sp. (isolate 1) and Lysinibacillus fusiformes (Isolat e 2), with biodegradation percentages of asphaltene fractions being 50% and 55%, respectively, and growth rate at the exponential phase is 7.17x10 7 CFU/mL.days and 4.21x10 7 CFU/mL.days, respectively.

Evolution of brown carbon in wildfire plumes

AbstractParticulate brown carbon (BrC) in the atmosphere absorbs light at subvisible wavelengths and has poorly constrained but potentially large climate forcing impacts. BrC from biomass burning has virtually unknown lifecycle and atmospheric stability. Here, BrC emitted from intense wildfires was measured in plumes transported over 2 days from two main fires, during the 2013 NASA SEAC4RS mission. Concurrent measurements of organic aerosol (OA) and black carbon (BC) mass concentration, BC coating thickness, absorption Ångström exponent, and OA oxidation state reveal that the initial BrC emitted from the fires was largely unstable. Using back trajectories to estimate the transport time indicates that BrC aerosol light absorption decayed in the plumes with a half‐life of 9 to 15 h, measured over day and night. Although most BrC was lost within a day, possibly through chemical loss and/or evaporation, the remaining persistent fraction likely determines the background BrC levels most relevant for climate forcing.

30 Primary Prevention ICD post-STEMI: Impact of Adoption of Nice vs ESC Guidelines

<h3>Background</h3> It has been demonstrated that implantable cardiac defibrillators (ICD) can be effective in the primary prevention of sudden cardiac death in patients with severe left ventricular systolic dysfunction (LVSD). However, studies have been unable to elucidate any benefit from ICD implantation prior to 40 days post-infarct. Therefore, ESC guidelines recommend ICD implantation in patients with a persistent ejection fraction (EF) &lt;/=40% on repeat LV assessment taken at least 40 days post-STEMI. The NICE guidelines are more restrictive, recommending ICD implantation in patients with EF &lt;/=35% and QRS duration &gt;120 ms. The extent to which these guidelines are followed in clinical practice is unclear. <h3>Aims</h3> To evaluate in a primary percutaneous coronary intervention (PPCI) cohort: Whether patients with LVSD are receiving a re-assessment of their EF at least 40 days post-STEMI. The impact of adoption of ESC versus NICE guidelines on ICD implantation rates for primary prevention. <h3>Methods</h3> This was a retrospective observational study carried out at a tertiary referral centre in South West England. Data was collected from consecutive patients referred for PPCI with a diagnosis of STEMI (January to December 2013). LVSD was defined as an EF &lt;/=40% according to echocardiography or cardiac MRI. ECG changes were preferentially sourced from the date closest to the follow-up LV assessment. <h3>Results</h3> 573 patients were included, of which, 74% were male and the median age was 64 years. 214 patients had LVSD on initial assessment and were alive after 40 days. 29% (62/214) of patients were followed up elsewhere (echo data not available) and hence excluded from further analysis. 76% (115/152) had a repeat assessment of their EF; however, only 70% (80/115) of reassessments were carried out at least 40 days after the acute event. Of these, 41% (33/80) had persistent LVSD. Thus, 28% (33/115) patients qualified for an ICD under ESC guidelines. On the contrary, 3% qualified under the NICE guidelines, as 17/80 had an EF &lt;/=35% at repeat echo and only 3 of these had a QRS duration &gt; 120 ms. <h3>Conclusion</h3> In a UK PPCI population, significantly more patients would be eligible for an ICD based on ESC compared to NICE guidelines (28% vs. 3%). This would have significant cost implications. The requirement for repeat echocardiography and ECG least 40 days after the STEMI emphasises the need for appropriate linking of care between the PPCI centre and those hospitals undertaking subsequent follow-up. This remains a challenge to the NHS but we need to find ways to achieve it!

SIMULTANEOUSNuSTAR/CHANDRAOBSERVATIONS OF THE BURSTING PULSAR GRO J1744-28 DURING ITS THIRD REACTIVATION

We report on a 10 ks simultaneous Chandra/HETG-NuSTAR observation of the Bursting Pulsar, GRO J1744-28, during its third detected outburst since discovery and after nearly 18 years of quiescence. The source is detected up to 60 keV with an Eddington persistent flux level. Seven bursts, followed by dips, are seen with Chandra, three of which are also detected with NuSTAR. Timing analysis reveals a slight increase in the persistent emission pulsed fraction with energy (from 10% to 15%) up to 10 keV, above which it remains constant. The 0.5-70 keV spectra of the persistent and dip emission are the same within errors, and well described by a blackbody (BB), a power-law with an exponential rolloff, a 10 keV feature, and a 6.7 keV emission feature, all modified by neutral absorption. Assuming that the BB emission originates in an accretion disc, we estimate its inner (magnetospheric) radius to be about 4x10^7 cm, which translates to a surface dipole field B~9x10^10 G. The Chandra/HETG spectrum resolves the 6.7 keV feature into (quasi-)neutral and highly ionized Fe XXV and Fe XXVI emission lines. XSTAR modeling shows these lines to also emanate from a truncated accretion disk. The burst spectra, with a peak flux more than an order of magnitude higher than Eddington, are well fit with a power-law with an exponential rolloff and a 10~keV feature, with similar fit values compared to the persistent and dip spectra. The burst spectra lack a thermal component and any Fe features. Anisotropic (beamed) burst emission would explain both the lack of the BB and any Fe components.

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance inPseudomonas aeruginosa

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.

Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

A Randomized Controlled Trial of Catheter Ablation Versus Medical Treatment of Atrial Fibrillation in Heart Failure (The CAMTAF Trial)

Restoring sinus rhythm in patients with heart failure (HF) and atrial fibrillation (AF) may improve left ventricular (LV) function and HF symptoms. We sought to compare the effect of a catheter ablation strategy with that of a medical rate control strategy in patients with persistent AF and HF. Patients with persistent AF, symptomatic HF, and LV ejection fraction <50% were randomized to catheter ablation or medical rate control. The primary end-point was the difference between groups in LV ejection fraction at 6 months. Baseline LV ejection fraction was 32±8% in the ablation group and 34±12% in the medical group. Twenty-six patients underwent catheter ablation, and 24 patients were rate controlled. Freedom from AF was achieved in 21/26 (81%) at 6 months off antiarrhythmic drugs. LV ejection fraction at 6 months in the ablation group was 40±12% compared with 31±13% in the rate control group (P=0.015). Ablation was associated with better peak oxygen consumption (22±6 versus 18±6 mL/kg per minute; P=0.014) and Minnesota living with HF questionnaire score (24±22 versus 47±22; P=0.001) compared with rate control. Catheter ablation is effective in restoring sinus rhythm in selected patients with persistent AF and HF, and can improve LV function, functional capacity, and HF symptoms compared with rate control. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT01411371.

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.

Molecular transformation and degradation of refractory dissolved organic matter in the Atlantic and Southern Ocean

More than 90% of the global ocean dissolved organic carbon (DOC) is refractory, has an average age of 4000–6000years and a lifespan from months to millennia. The fraction of dissolved organic matter (DOM) that is resistant to degradation is a long-term buffer in the global carbon cycle but its chemical composition, structure, and biochemical formation and degradation mechanisms are still unresolved. We have compiled the most comprehensive molecular dataset of 197 Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses from solid-phase extracted marine DOM covering two major oceans, the Atlantic sector of the Southern Ocean and the East Atlantic Ocean (ranging from 50°N to 70°S). Molecular trends and radiocarbon dating of 34 DOM samples (comprising Δ14C values from −229‰ to −495‰) were combined to model an integrated degradation rate for bulk DOC resulting in a predicted age of >24ka for the most persistent DOM fraction. First order kinetic degradation rates for 1557 mass peaks indicate that numerous DOM molecules cycle on timescales much longer than the turnover of the bulk DOC pool (estimated residence times of up to ~100ka) and the range of validity of radiocarbon dating. Changes in elemental composition were determined by assigning molecular formulae to the detected mass peaks. The combination of residence times with molecular information enabled modelling of the average elemental composition of the slowest degrading fraction of the DOM pool. In our dataset, a group of 361 molecular formulae represented the most stable composition in the oceanic environment (“island of stability”). These most persistent compounds encompass only a narrow range of the molecular elemental ratios H/C (average of 1.17±0.13), and O/C (average of 0.52±0.10) and molecular masses (360±28 and 497±51Da). In the Weddell Sea DOC concentrations in the surface waters were low (46.3±3.3μM) while the organic radiocarbon was significantly more depleted than that of the East Atlantic, representing average surface water DOM ages of 4920±180a. These results are in accordance with a highly degraded DOM in the Weddell Sea surface water as also shown by the molecular degradation index IDEG obtained from FT-ICR MS data. Further, we identified 339 molecular formulae which probably contribute to an increased DOC concentration in the Southern Ocean and potentially reflect an accumulation or enhanced sequestration of refractory DOC in the Weddell Sea. These results will contribute to a better understanding of the persistent nature of marine DOM and its role as an oceanic carbon buffer in a changing climate.

Zweiphasiges Biogasverfahren: Hydrolyse unterschiedlicher Substrattypen

Bei einem zweiphasigen Biogasprozess laufen die einzelnen Phasen der anaeroben Vergarung raumlich getrennt ab, sodass die Prozessbedingungen an die spezifischen Anforderungen der beteiligten Mikrobiologie angepasst werden konnen. Zielsetzung ist es dabei, die Saurekonzentration durch eine gezielte Einstellung des pH-Wertes in der Hydrolysestufe zu kontrollieren. Die produzierten Sauren konnen zwischengespeichert werden, um nachfrageabhangig Biogas bzw. Strom zu produzieren. In der vorliegenden Studie wurden zwei Substratvarianten untersucht: ein schwer sowie ein leicht zu vergarendes Substrat. Steigende pH-Werte in der Hydrolysestufe beider Varianten waren mit einer gesteigerten Saurekonzentration bzw. einem hoheren Abbaugrad verbunden. Dabei konnte bei steigenden pH-Werten eine Tendenz zu einem verbesserten Aufschluss der Hemicellulose sowie der Cellulose aufgezeigt werden. In der Methanstufe entstand ein Biogas mit einem Methangehalt von bis zu 80 %.

Impact of Stepwise Ablation on the Biatrial Substrate in Patients With Persistent Atrial Fibrillation and Heart Failure

Ablation of persistent atrial fibrillation can be challenging, often involving not only pulmonary vein isolation (PVI) but also additional linear lesions and ablation of complex fractionated electrograms (CFE). We examined the impact of stepwise ablation on a human model of advanced atrial substrate of persistent atrial fibrillation in heart failure. In 30 patients with persistent atrial fibrillation and left ventricular ejection fraction ≤35%, high-density CFE maps were recorded biatrially at baseline, in the left atrium (LA) after PVI and linear lesions (roof and mitral isthmus), and biatrially after LA CFE ablation. Surface area of CFE (mean cycle length ≤120 ms) remote to PVI and linear lesions, defined as CFE area, was reduced after PVI (18.3±12.03 to 10.2±7.1 cm(2); P<0.001) and again after linear lesions (7.7±6.5 cm(2); P=0.006). Complete mitral isthmus block predicted greater CFE reduction (P=0.02). Right atrial CFE area was reduced by LA ablation, from 25.9±14.1 to 12.9±11.8 cm(2) (P<0.001). Estimated 1-year arrhythmia-free survival was 72% after a single procedure. Incomplete linear lesion block was an independent predictor of arrhythmia recurrence (hazard ratio, 4.69; 95% confidence interval, 1.05-21.06; P=0.04). Remote LA CFE area was progressively reduced following PVI and linear lesions, and LA ablation reduced right atrial CFE area. Reduction of CFE area at sites remote from ablation would suggest either regression of the advanced atrial substrate or that these CFE were functional phenomena. Nevertheless, in an advanced atrial fibrillation substrate, linear lesions after PVI diminished the target area for CFE ablation, and complete lesions resulted in a favorable clinical outcome.

Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

Quantitative analysis of persister fractions suggests different mechanisms of formation among environmental isolates of E. coli

BackgroundBacterial persistence describes a phenomenon wherein a small subpopulation of cells is able to survive a challenge with high doses of an antibiotic (or other stressor) better than the majority of the population. Previous work has shown that cells that are in a dormant or slow-growing state are persistent to antibiotic treatment and that populations with higher fractions of dormant cells exhibit higher levels of persistence. These data suggest that a major determinant of the fraction of persisters within a population is the rate at which cells enter and exit from dormancy. However, it is not known whether there are physiological changes in addition to dormancy that influence persistence. Here, we use quantitative measurements of persister fractions in a set of environmental isolates of E. coli together with a mathematical model of persister formation to test whether a single general physiological change, such as cell dormancy, can explain the differences in persister phenotypes observed in different strains.ResultsIf a single physiological change (e.g. cell dormancy) underlies most persister phenotypes, then strains should exhibit characteristic fractions of persister cells: some strains will consistently have high fractions of persisters (dormant cells), whereas others will have low fractions. Although we found substantial variation in the fraction of persisters between different environmental isolates of E. coli, these fractions were not correlated across antibiotics. Some strains exhibited high persister fractions in one antibiotic, but low persister fractions in a second antibiotic. Surprisingly, no correlation in persister fractions was observed between any two drugs, even for antibiotics with nearly identical modes of action (ciprofloxacin and nalidixic acid).ConclusionsThese data support the hypothesis that there is no single physiological change that determines the persistence level in a population of cells. Instead, the fraction of cells that survive antibiotic treatment (persist) depends critically on the specific antibiotic that is used, suggesting that physiological changes in addition to dormancy can underlie persister phenotypes.

Soil Microbe Active Community Composition and Capability of Responding to Litter Addition after 12 Years of No Inputs

One explanation given for the high microbial diversity found in soils is that they contain a large inactive biomass that is able to persist in soils for long periods of time. This persistent microbial fraction may help to buffer the functionality of the soil community during times of low nutrients by providing a reservoir of specialized functions that can be reactivated when conditions improve. A study was designed to test the hypothesis: in soils lacking fresh root or detrital inputs, microbial community composition may persist relatively unchanged. Upon addition of new inputs, this community will be stimulated to grow and break down litter similarly to control soils. Soils from two of the Detrital Input and Removal Treatments (DIRT) at the H. J. Andrews Experimental Forest, the no-input and control treatment plots, were used in a microcosm experiment where Douglas-fir needles were added to soils. After 3 and 151 days of incubation, soil microbial DNA and RNA was extracted and characterized using quantitative PCR (qPCR) and 454 pyrosequencing. The abundance of 16S and 28S gene copies and RNA copies did not vary with soil type or amendment; however, treatment differences were observed in the abundance of archaeal ammonia-oxidizing amoA gene abundance. Analysis of ∼110,000 bacterial sequences showed a significant change in the active (RNA-based) community between day 3 and day 151, but microbial composition was similar between soil types. These results show that even after 12 years of plant litter exclusion, the legacy of community composition was well buffered against a dramatic disturbance.

Up-Regulation of Heme Oxygenase-1 Is Induced by the Leukemia Stroma-Cell Interaction and Rescues Acute Myeloid Leukaemia (AML) Cells From Cytarabin Induced Cell Death

AbstractAbstract 533Although the majority of patients achieve a complete remission after induction chemotherapy with cytarabine and antracyclines relapse is still the major cause of death in acute myeloid leukemia (AML). In order to address the question of resistance mechanisms in residual leukemic cells we employed a murine leukemia model system. We transplanted Rag2−/−gc−/− mice with human KG1a leukemia cells and treated them with four doses of cytarabine at the onset of AML. Residual human leukemic cells were collected from murine marrow and spleen and RNA was further subjected to whole genome microarray analysis. Heme Oxygenase-1 (HO-1) was found to be highly up-regulated in the persistent cell fraction that had survived cytarabine chemotherapy in vivo. The clinical relevance of HO-1 up-regulation was confirmed with two AML data sets. In a small set of 31 unselected AML patients treated in one institution, overall survival (OS) was significantly worse in patients with high HO-1 expression as determined by qPCR analysis (log rank: p < 0.0001). A second patient cohort of 286 patients was therefore retrospectively analyzed for HO-1 expression based on whole genome expression profiling. Again, OS was inferior in patients expressing HO-1 at high levels (p<0.005).HO-1 is a well-known stress-response protein also termed heat-shock protein 32 (hsp32) that catalyzes the enzymatic cleavage of heme into biliverdin, carbon monoxide and iron, but also decreases cellular levels of reactive oxygen species (ROS). Employing an in vitro co-culture system we show that mesenchymal stem cells (MSC) increase HO-1 expression in leukemic cells causing a decrease in ROS levels and protection from apoptosis particularly in the adherent fraction of leukemic cells. The cytoprotective effect induced by leukemia stromal cell interaction can be overcome by concomitant treatment of cytarabin together with zinc protophoryrin (ZnPP), a specific competitive inhibitor of HO-1. Taken together we demonstrate that expression of HO-1 may play a critical role for chemotherapy resistance in AML in particular with regard to the leukemia stromal cell interaction. ZnPP is a specific HO-1 inhibitor that has already proven anti-tumor activity in vivo and might therefore be a promising candidate for further targeted drug therapy strategies that intend to eradicate persistent leukemic cells. Disclosures:No relevant conflicts of interest to declare.

Does Initial or Persistent DNA damage Correlate With the Enhanced RBE of Carbon Versus Photon Radiation Therapy?

Purpose/Objective(s): Induction of DNA-double-strand-breaks (DSB) is considered a determinant of radiation-mediated cell killing. The relative biological efficacy (RBE) of high linear energy transfer (LET) irradiation versus low-LET irradiation is estimated to be w3. However, quantitative analysis of DSB directly after irradiation could not explain the biological differences in clonogenic survival. The higher complexity of DSB, reported as prolonged or persistent DNA damage after high-LET irradiation, is a candidate determinant of its enhanced RBE. We therefore sought to validate the role of complex DNA damage and differences in repair kinetics in radiation cell killing by correlating clonogenic survival with both initial and persistent radiation-induced DSB. Materials/Methods: To this end, human non small cell lung cancer cells (A549), epidermoid cancer cells (A431) and human umbilical vein endothelial cells (HUVEC) were irradiated with either high-LET Carbon ions (260 MeV/u, 100 keV/mm) or low-LET photon (250 keV X-rays, 2 keV/mm) irradiation. Sma-560 mouse glioma cells were irradiated with carbon irradiation of different LET (30 170 keV/mm). DSB were detected by gamma-H2AX staining followed by microscopy and flow cytometry at different time points post-irradiation. The persistent damage fraction (PDF) was determined as the ratio of DSB detected at 24 hours versus 30 minutes post-irradiation. To detect biological differences in radiation response, clonogenic survival assays were performed in parallel. Results: DNA repair kinetics analysis confirmed the high amount of persistent damage for high-LET carbon irradiation compared to low-LET irradiation. The RBE for clonogenic survival was between 2.0-2.5 for highLET carbon irradiation among different cell types. The “Damage RBE” ranged from 1.8 2.2 for initial damage and from 2.5 5.1 for persistent damage. In contrast, in Sma-560 and for carbon irradiation with a range of different LET, the RBE for clonogenic survival correlated with persistent DNA damage. The PDF showed a direct dose-dependency for low-LET irradiation (range from 5 20%), while the PDF for high-LET carbon irradiation was independent of dose at w20%. Conclusions: Quantitative DSB-repair studies with different LET carbon irradiation, so far performed only in Sma-560, indicated that the amount of persistent DNA damage, but not initial DNA damage may correlate with radiation cell killing of high-LET carbon irradiation. The PDF was dosedependent and low for low-LET irradiation while dose-independent and high for high-LET irradiation. These data suggest that neither initialnor persistent DNA damage may provide a plausible explanation for the enhanced RBE of high-LET carbon versus low-LET photon irradiation. Author Disclosure: P. Seidel: None. J. Debus: None. A. Abdollahi: None.

Regulation of Virus Neutralization and the Persistent Fraction by TRIM21

Despite a central role in immunity, antibody neutralization of virus infection is poorly understood. Here we show how the neutralization and persistence of adenovirus type 5, a prevalent nonenveloped human virus, are dependent upon the intracellular antibody receptor TRIM21. Cells with insufficient amounts of TRIM21 are readily infected, even at saturating concentrations of neutralizing antibody. Conversely, high TRIM21 expression levels decrease the persistent fraction of the infecting virus and allows neutralization by as few as 1.6 antibody molecules per virus. The direct interaction between TRIM21 and neutralizing antibody is essential, as single-point mutations within the TRIM21-binding site in the Fc region of a potently neutralizing antibody impair neutralization. However, infection at high multiplicity can saturate TRIM21 and overcome neutralization. These results provide insight into the mechanism and importance of a newly discovered, effector-driven process of antibody neutralization of nonenveloped viruses.