Non-growing State Research Articles

A Novel Method for Long-Term Analysis of Lactic Acid and Ammonium Production in Non-growing Lactococcus lactis Reveals Pre-culture and Strain Dependence.

In various (industrial) conditions, cells are in a non-growing but metabolically active state in which de novo protein synthesis capacity is limited. The production of a metabolite by such non-growing cells is dependent on the cellular condition and enzyme activities, such as the amount, stability, and degradation of the enzyme(s). For industrial fermentations in which the metabolites of interest are mainly formed after cells enter the stationary phase, the investigation of prolonged metabolite production is of great importance. However, current batch model systems do not allow prolonged measurements due to metabolite accumulation driving product-inhibition. Here we developed a protocol that allows high-throughput metabolic measurements to be followed in real-time over extended periods (weeks). As a validation model, sugar utilization and arginine consumption by a low density of translationally blocked Lactococcus lactis was designed in a defined medium. In this system L. lactis MG1363 was compared with its derivative HB60, a strain described to achieve higher metabolic yield through a shift toward heterofermentative metabolism. The results showed that in a non-growing state HB60 is able to utilize more arginine than MG1363, and for both strains the decay of the measured activities were dependent on pre-culture conditions. During the first 5 days of monitoring a ∼25-fold decrease in acidification rate was found for strain HB60 as compared to a ∼20-fold decrease for strain MG1363. Such measurements are relevant for the understanding of microbial metabolism and for optimizing applications in which cells are frequently exposed to long-term suboptimal conditions, such as microbial cell factories, fermentation ripening, and storage survival.

Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms

The water-borne bacterium Legionella pneumophila is the causative agent of Legionnaires' disease. In the environment, the opportunistic pathogen colonizes different niches, including free-living protozoa and biofilms. The physiological state(s) of sessile Legionella in biofilms and their functional consequences are not well understood. Using single-cell techniques and fluorescent growth rate probes as well as promoter reporters, we show here that sessile L. pneumophila exhibits phenotypic heterogeneity and adopts growing and nongrowing ("dormant") states in biofilms and microcolonies. Phenotypic heterogeneity is controlled by the Legionella quorum sensing (Lqs) system, the transcription factor LvbR, and the temperature. The Lqs system and LvbR determine the ratio between growing and nongrowing sessile subpopulations, as well as the frequency of growth resumption ("resuscitation") and microcolony formation of individual bacteria. Nongrowing L. pneumophila cells are metabolically active, express virulence genes and show tolerance toward antibiotics. Therefore, these sessile nongrowers are persisters. Taken together, the Lqs system, LvbR and the temperature control the phenotypic heterogeneity of sessile L. pneumophila, and these factors regulate the formation of a distinct subpopulation of nongrowing, antibiotic tolerant, virulent persisters. Hence, the biofilm niche of L. pneumophila has a profound impact on the ecology and virulence of this opportunistic pathogen.

Intracellular Staphylococcus aureus persisters upon antibiotic exposure

Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here, we provide in vitro evidence of Staphylococcus aureus persisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. Intracellular S. aureus persisters remain metabolically active but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell wall stress, SOS and heat shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellular S. aureus persisters may constitute a reservoir for relapsing infection and could contribute to therapeutic failures.

Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis.

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+ Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells.IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

Stochastic Variation in Expression of the Tricarboxylic Acid Cycle Produces Persister Cells.

Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.IMPORTANCE Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.

MRNA Degradation Rates Are Coupled to Metabolic Status in Mycobacterium smegmatis.

The success of Mycobacterium tuberculosis as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states, M. tuberculosis can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including M. tuberculosis, have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remain unknown. To better understand the biology of nongrowing mycobacteria, we sought to identify the mechanistic basis of mRNA stabilization in the nonpathogenic model Mycobacterium smegmatis We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global stabilization was rapidly reversed when hypoxia-adapted cultures were reexposed to oxygen, even in the absence of new transcription. The stringent response and RNase levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activities of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status but rather is dependent on the status of energy metabolism.IMPORTANCE The logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months. Mycobacterium tuberculosis survives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on how M. tuberculosis survives during these low-metabolism states is incomplete, and we hypothesize that optimized energy resource management is important. Here, we report that slowed mRNA turnover is a common feature of mycobacteria under energy stress but is not dependent on the mechanisms that have generally been postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rate per se Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

Study on the cyanobacterial toxin metabolism of Microcystis aeruginosa in nitrogen-starved conditions by a stable isotope labelling method.

In this study, the biosynthesis of microcystins (MCs) was investigated after long-term nitrogen-starved conditions in cyanobacterium Microcystis aeruginosa. The results demonstrated that the algal cells were able to survive in a non-growing state with nitrogen starvation for more than one month. The physiological properties of the algal cells were studied to elucidate the mechanisms of viability under nitrogen-deprivation conditions. After the state of nitrogen chlorosis, new toxins could be resynthesized and tracked using 15N-stable isotope-labelled nitrogen. Nitrogen starvation of nutritionally replete cells resulted in a significant increase of microcystin-LY (MC-LY), thereby suggesting that MC-LY may undergo catabolism to provide nitrogen or that MC-LY may be produced to play an important role in the cell in response to nitrogen deprivation. The rank order of different types of nitrogen in algal cells assimilation was N-ammonium > N-urea > N-nitrate > N-alanine. The relationship between the production of toxin variants and various environmental conditions is an interesting issue for future research and may help improve the understanding of the ecological role of cyanobacterial toxins.

Magnesium Flux Modulates Ribosomes to Increase Bacterial Survival

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

Regrowth-delay body as a bacterial subcellular structure marking multidrug-tolerant persisters

Bacteria have long been recognized to be capable of entering a phenotypically non-growing persister state, in which the cells exhibit an extended regrowth lag and a multidrug tolerance, thus posing a great challenge in treating infectious diseases. Owing to their non-inheritability, low abundance of existence, lack of metabolic activities, and high heterogeneity, properties of persisters remain poorly understood. Here, we report our accidental discovery of a subcellular structure that we term the regrowth-delay body, which is formed only in non-growing bacterial cells and sequesters multiple key proteins. This structure, that dissolves when the cell resumes growth, is able to be viewed as a marker of persisters. Our studies also indicate that persisters exhibit different depth of persistence, as determined by the status of their regrowth-delay bodies. Our findings imply that suppressing the formation and/or promoting the dissolution of regrowth-delay bodies could be viable strategies for eradicating persisters.

Heterotrophic substrate specificity in the aquatic environment: The role of microscale patchiness investigated using modelling.

Recent observations of natural bacterial communities show high genetic diversity in organic carbon uptake systems (microdiversity) and specificity in substrate species taken up. This seemingly contradicts a large body of literature from laboratory experiments under nutrient-limiting conditions, where mixed substrate use is the rule. This apparent discrepancy can be resolved by realizing that bacteria in the natural aquatic environment encounter nutrients as high-concentration patches. They may live in an ecologically nutrient-limiting environment, but they are rarely in a biologically nutrient-limited state. Rather they switch between non-growing and nutrient-replete states. During nutrient-replete growth the metabolism is saturated and assimilating additional substrates does not increase the growth rate, but carrying the assimilation system constitutes a cost. Consequently, the specialist strategy is beneficial, which is consistent with observations from laboratory experiments. When the bacteria are not growing, the added cost also reduces the fitness of the generalist species. A simple mathematical model encompassing the relevant mechanisms is developed and parameterized realistically based on the literature. The model predicts that, under pulsed conditions, specialization is beneficial when the metabolic cost of an additional uptake system is more than ~0.5% of the total metabolic cost, which is a reasonable estimate and illustrates that this is a plausible hypothesis.

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production.

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions.

Quiescent Body as a Reversible Subcellular Structure Formed in Nongrowing Bacterial Cells

Summary Bacterial cells take a variable lag time and maintain a multi-drug tolerant non-growing state before resuming growth when encountering growth-supportive conditions. Some of them exhibit an extraordinarily long lag, as pathogenic persisters do. It remains unknown on what determines lag time duration. Here, we unveiled a subcellular structure, termed quiescent body, that is formed in bacterial cells entering non-growing state and sequesters selected proteins essential for cell growth. Their formation occurs progressively in each cell and heterogeneously among individual cells. They only dissolve in re-growing cells to release proteins for immediate re-functioning when conditions become fit. Quiescent body, whose degree of formation is highly correlated with duration of lag time or level of antibiotic tolerance, apparently functions as a biological timer for bacterial growth resumption. Further, suppressing their formation, which directly relies on cellular respiration complexes, or promoting their dissolution might be a viable strategy to eradicate persisters.

Molecular Basis of Bacterial Longevity.

ABSTRACTIt is well known that many bacteria can survive in a growth-arrested state for long periods of time, on the order of months or even years, without forming dormant structures like spores or cysts. How is such longevity possible? What is the molecular basis of such longevity? Here we used the Gram-negative phototrophic alphaproteobacterium Rhodopseudomonas palustris to identify molecular determinants of bacterial longevity. R. palustris maintained viability for over a month after growth arrest due to nutrient depletion when it was provided with light as a source of energy. In transposon sequencing (Tn-seq) experiments, we identified 117 genes that were required for long-term viability of nongrowing R. palustris cells. Genes in this longevity gene set are annotated to play roles in a number of cellular processes, including DNA repair, tRNA modification, and the fidelity of protein synthesis. These genes are critically important only when cells are not growing. Three genes annotated to affect translation or posttranslational modifications were validated as bona fide longevity genes by mutagenesis and complementation experiments. These genes and others in the longevity gene set are broadly conserved in bacteria. This raises the possibility that it will be possible to define a core set of longevity genes common to many bacterial species.

Efficient Killing of Planktonic and Biofilm-Embedded Coagulase-Negative Staphylococci by Bactericidal Protein P128.

Coagulase-negative staphylococci (CoNS) are the major causative agents of foreign-body-related infections, including catheter-related bloodstream infections. Because of the involvement of biofilms, foreign-body-related infections are difficult to treat. P128, a chimeric recombinant phage-derived ectolysin, has been shown to possess bactericidal activity on strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). We tested the killing potential of P128 on three clinically significant species of CoNS, S. epidermidis, S. haemolyticus, and S. lugdunensis, under a variety of physiological conditions representing growing and nongrowing states. The MIC90 and minimum bactericidal concentration at which 90% of strains tested are killed (MBC90) of P128 on 62 clinical strains of CoNS were found to be 16 and 32 μg/ml (0.58 and 1.16 μM), respectively, demonstrating the bactericidal nature of P128 on CoNS strains. Serum showed a potentiating effect on P128 inhibition, as indicated by 4- to 32-fold lower MIC values observed in serum. P128 caused a rapid loss of viability in all CoNS strains tested. Persisters of CoNS that were enriched in the presence of vancomycin or daptomycin were killed by P128 at 1× the MIC in a rapid manner. Low concentrations of P128 caused a 2- to 5-log reduction in CFU in stationary-phase or poorly metabolizing CoNS cultures. P128 at low concentrations eliminated CoNS biofilms in microtiter plates and on the surface of catheters. Combinations of P128 and standard-of-care (SoC) antibiotics were highly synergistic in inhibiting growth in preformed biofilms. Potent activity on planktonic cells, persisters, and biofilms of CoNS suggests that P128 is a promising candidate for the clinical development of treatments for foreign-body-related and other CoNS infections.

Effects of biocide exposure on P. Aeruginosa, E. coli and A. Baumannii complex isolates from hospital and household environments

Background: Bacterial responses to biocide exposure and its effects on survival and persistence remain to be studied in greater detail.Aim: To analyse the viability and survival of environmental isolates from household and hospital settings after biocide exposure.Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chlorhexidine (CHxG), benzalkonium chloride (BAC) and triclosan (TC) were determined in isolates of Pseudomonas aeruginosa, Acinetobacter baumannii complex and Escherichia coli collected from hospital and households environments. Viability was monitored after exposure and removal of biocides using agar cultures and flow cytometry.Findings: P. aeruginosa isolates showed greater tolerance for all biocides tested whereas A. baumannii complex and E. coli were less tolerant.When compared with reference strains, biocide tolerance was up to 8 to 13-fold higher for TC and BAC respectively. Flow cytometry showed that biocide exposure may induce viable but non-growing states in P. aeruginosa and E. coli isolates before becoming fully replicative. Changes in the susceptibility profile in one isolate of A. baumannii complex were observed after biocide exposure.Discussion: Bacteria isolates from hospital and households were able to recover after biocide exposure at bactericidal concentrations favouring persistence and spread of biocide-tolerant strains. This study reinforces that cleaning compliance should be monitored by non-culture based tests. Novel formulations in cleaning and disinfection protocols should be revisited in hospitals harbouring P. aeruginosa and A. baumannii multidrug resistant isolates.

Initiator tRNA genes template the 3' CCA end at high frequencies in bacteria.

BackgroundWhile the CCA sequence at the mature 3′ end of tRNAs is conserved and critical for translational function, a genetic template for this sequence is not always contained in tRNA genes. In eukaryotes and Archaea, the CCA ends of tRNAs are synthesized post-transcriptionally by CCA-adding enzymes. In Bacteria, tRNA genes template CCA sporadically.ResultsIn order to understand the variation in how prokaryotic tRNA genes template CCA, we re-annotated tRNA genes in tRNAdb-CE database version 0.8. Among 132,129 prokaryotic tRNA genes, initiator tRNA genes template CCA at the highest average frequency (74.1%) over all functional classes except selenocysteine and pyrrolysine tRNA genes (88.1% and 100% respectively). Across bacterial phyla and a wide range of genome sizes, many lineages exist in which predominantly initiator tRNA genes template CCA. Convergent and parallel retention of CCA templating in initiator tRNA genes evolved in independent histories of reductive genome evolution in Bacteria. Also, in a majority of cyanobacterial and actinobacterial genera, predominantly initiator tRNA genes template CCA. We also found that a surprising fraction of archaeal tRNA genes template CCA.ConclusionsWe suggest that cotranscriptional synthesis of initiator tRNA CCA 3′ ends can complement inefficient processing of initiator tRNA precursors, “bootstrap” rapid initiation of protein synthesis from a non-growing state, or contribute to an increase in cellular growth rates by reducing overheads of mass and energy to maintain nonfunctional tRNA precursor pools. More generally, CCA templating in structurally non-conforming tRNA genes can afford cells robustness and greater plasticity to respond rapidly to environmental changes and stimuli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3314-x) contains supplementary material, which is available to authorized users.

The physiology of growth arrest: uniting molecular and environmental microbiology.

Most bacteria spend the majority of their time in prolonged states of very low metabolic activity and little or no growth, in which electron donors, electron acceptors and/or nutrients are limited, but cells are poised to undergo rapid division cycles when resources become available. These non-growing states are far less studied than other growth states, which leaves many questions regarding basic bacterial physiology unanswered. In this Review, we discuss findings from a small but diverse set of systems that have been used to investigate how growth-arrested bacteria adjust metabolism, regulate transcription and translation, and maintain their chromosomes. We highlight major questions that remain to be addressed, and suggest that progress in answering them will be aided by recent methodological advances and by dialectic between environmental and molecular microbiology perspectives.

Uropathogenic Escherichia coli Metabolite-Dependent Quiescence and Persistence May Explain Antibiotic Tolerance during Urinary Tract Infection.

In the present study, it is shown that although Escherichiacoli CFT073, a human uropathogenic (UPEC) strain, grows in liquid glucose M9 minimal medium, it fails to grow on glucose M9 minimal medium agar plates seeded with ≤10(6)CFU. The cells on glucose plates appear to be in a "quiescent" state that can be prevented by various combinations of lysine, methionine, and tyrosine. Moreover, the quiescent state is characteristic of ~80% of E.coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community-acquired urinary tract infections in Denmark. In addition, E.coli CFT073 quiescence is not limited to glucose but occurs on agar plates containing a number of other sugars and acetate as sole carbon sources. It is also shown that a number of E.coli CFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) are nonquiescent on glucose M9 minimal agar plates and that quiescence requires a complete oxidative tricarboxylic acid (TCA) cycle. In addition, evidence is presented that, although E.coli CFT073 quiescence and persistence in the presence of ampicillin are alike in that both require a complete oxidative TCA cycle and each can be prevented by amino acids, E.coli CFT073 quiescence occurs in the presence or absence of a functional rpoS gene, whereas maximal persistence requires a nonfunctional rpoS. Our results suggest that interventions targeting specific central metabolic pathways may mitigate UPEC infections by interfering with quiescence and persistence. IMPORTANCE Recurrent urinary tract infections (UTIs) affect 10 to 40% of women. In up to 77% of those cases, the recurrent infections are caused by the same uropathogenic E.coli (UPEC) strain that caused the initial infection. Upon infection of urothelial transitional cells in the bladder, UPEC appear to enter a nongrowing quiescent intracellular state that is thought to serve as a reservoir responsible for recurrent UTIs. Here, we report that many UPEC strains enter a quiescent state when ≤10(6)CFU are seeded on glucose M9 minimal medium agar plates and show that mutations in several genes involved in central carbon metabolism prevent quiescence, as well as persistence, possibly identifying metabolic pathways involved in UPEC quiescence and persistence in vivo.

Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

Does credit create a ‘growth imperative’? A quasi-stationary economy with interest-bearing debt

This paper addresses the question of whether a capitalist economy can ever sustain a ‘stationary’ (or non-growing) state, or whether, as often claimed, capitalism has an inherent ‘growth imperative’ arising from the charging of interest on debt. We outline the development of a dedicated system dynamics macro-economic model for describing Financial Assets and Liabilities in a Stock-Flow consistent Framework (FALSTAFF) and use this model to explore the potential for stationary state outcomes in an economy with balanced trade, credit creation by banks, and private equity. Contrary to claims in the literature, we find that neither credit creation nor the charging of interest on debt creates a ‘growth imperative’ in and of themselves. This finding remains true even when capital adequacy and liquidity requirements are imposed on banks. We test the robustness of our results in the face of random variations and one-off shocks. We show further that it is possible to move from a growth path towards a stationary state without either crashing the economy or dismantling the system. Nonetheless, there remain several good reasons to support the reform of the monetary system. Our model also supports critiques of austerity and underlines the value of countercyclical spending by government.