共查询到20条相似文献,搜索用时 15 毫秒
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Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens. 相似文献
3.
Plaque analysis allows the determination of phage titer and multiplicity of infection. Yet, this overnight assay provides only endpoint results, ignoring kinetic aspects. We introduce an alternative high-throughput and rapid method for kinetic analysis of lytic coliphage activity. Escherichia coli was infected with serial dilutions of MS2 coliphage, and bacterial growth was monitored using a multi-well plate reader providing within hours the equivalent data as obtained overnight. Additional information is yielded, including phage replication rate, progeny size per cycle, and viral propagation during bacterial growth. This method offers further insights into physicochemical mechanisms of lytic coliphage infection and temporal control. It also provides a virus–host interaction acumen. 相似文献
4.
《Biochemical and biophysical research communications》2020,521(2):527-532
To enable large-scale screening of signaling molecules and drugs that regulate cellular contractility-associated mechanotransduction, we previously modified, particularly in terms of the capability of efficiently collecting big data, conventional methodologies using wrinkled substrates to determine the cellular contractility. Here, we present a new system to perform the wrinkle-based cell force assay in a multi-well plate format conformed to standardized geometric configurations and compatible with available technologies such as automated plate readers. With this highly improved throughput in terms of hardware as well as software using a deep learning-based technology, we evaluated the effect of treating cells with various types of pharmacological inhibitors on the cellular contractility. We found opposite responses of cells to the inhibitors between the contractility and collective migration activities. While similar inverse relationships between the contractility and migration have been reported in separate studies, our results here with the high-throughput screening system more broadly generalized these observations. 相似文献
5.
Roberta L. Grant Daniel Acosta 《In vitro cellular & developmental biology. Animal》1997,33(4):256-260
Summary A number of methods have been developed to measure intracellular pH (pHi) because of its importance in intracellular events.
A major advance in accurate pHi measurement was the development of the ratiometric fluorescent indicator dye, 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein
(BCECF). We have used a fluorescence multi-well plate reader and a ratiometric method for determining pHi in primary cultures
of rabbit corneal epithelial (CE) cells with BCECF. Fluorescence was measured at excitation wavelengths of 485±11 nm and 395±12.5
nm, with emission detected at 530±15 nm. Cells grown in multi-well plates were loaded with 4 μM BCECF for 30 min at 37° C. Resting pHi was 7.34±0.03 (2 cultures, N=5 wells). Changes in pHi determined with the fluorescence multi-well plate reader after the addition and removal of NH4Cl or sodium lactate were comparable to changes in cells analyzed with a digitized fluorescence imaging system. A concentration-response
relationship involving changes in pHi was easily demonstrated in CE cells after treatment with ionomycin, a calcium ionophore.
Low doses of ionomycin (2.5–5 μM), produced a prolonged acidification; 7.5 μM ionomycin produced a transient acidification; and 10 μM ionomycin resulted in a slight alkalinization. We conclude that accurate pHi measurements can be obtained with a ratiometric
method with BCECF in a multi-well plate reader. This technology may simplify screening studies evaluating effects of hormones,
growth factors, or toxicants on pHi homeostasis. 相似文献
6.
Evolutionary theory predicts that adaptations, including antibiotic resistance, should come with associated fitness costs; yet, many resistance mutations seemingly contradict this prediction by inducing no growth rate deficit. However, most growth assays comparing sensitive and resistant strains have been performed under a narrow range of environmental conditions, which do not reflect the variety of contexts that a pathogenic bacterium might encounter when causing infection. We hypothesized that reduced niche breadth, defined as diminished growth across a diversity of environments, can be a cost of antibiotic resistance. Specifically, we test whether chloramphenicol-resistant Escherichia coli incur disproportionate growth deficits in novel thermal conditions. Here we show that chloramphenicol-resistant bacteria have greater fitness costs at novel temperatures than their antibiotic-sensitive ancestors. In several cases, we observed no resistance cost in growth rate at the historic temperature but saw diminished growth at warmer and colder temperatures. These results were consistent across various genetic mechanisms of resistance. Thus, we propose that decreased thermal niche breadth is an under-documented fitness cost of antibiotic resistance. Furthermore, these results demonstrate that the cost of antibiotic resistance shifts rapidly as the environment changes; these context-dependent resistance costs should select for the rapid gain and loss of resistance as an evolutionary strategy.Subject terms: Bacterial evolution, Microbial ecology, Antibiotics 相似文献
7.
Joshua G. Schraiber Angela N. Kaczmarczyk Ricky Kwok Miran Park Rachel Silverstein Florentine U. Rutaganira Taruna Aggarwal Michael A. Schwemmer Carole L. Hom Richard K. Grosberg Sebastian J. Schreiber 《Journal of theoretical biology》2012
Dengue fever, a viral disease spread by the mosquito Aedes aegypti, affects 50–100 million people a year in many tropical countries. Because the virus must incubate within mosquito hosts for two weeks before being able to transmit the infection, shortening the lifespan of mosquitoes may curtail dengue transmission. We developed a continuous time reaction-diffusion model of the spatial spread of Wolbachia through a population of A. aegypti. This model incorporates the lifespan-shortening effects of Wolbachia on infected A. aegypti and the fitness advantage to infected females due to cytoplasmic incompatibility (CI). We found that local establishment of the Wolbachia infection can occur if the fitness advantage due to CI exceeds the fitness reduction due to lifespan-shortening effects, in accordance with earlier results concerning fecundity reduction. However, spatial spread is possible only if the fitness advantage due to CI is twice as great as the fitness reduction due to lifespan shortening effects. Moreover, lifespan-shortening and fecundity-reduction can have different effects on the speed of wave-retreat. Using data from the literature, we estimated all demographic parameters for infected and uninfected mosquitoes and computed the velocities of spread of infection. Our most optimistic estimates suggest that the spatial spread of lifespan-shortening Wolbachia may be so slow that efficient spatial spread would require a prohibitively large number of point releases. However, as these estimates of demographic parameters may not accurately reflect natural conditions, further research is necessary to corroborate these predictions. 相似文献
8.
Kumiko Kurabayashi Yuko Hirakawa Koichi Tanimoto Haruyoshi Tomita Hidetada Hirakawa 《Journal of bacteriology》2014,196(2):248-256
Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. 相似文献
9.
Garbeva P Tyc O Remus-Emsermann MN van der Wal A Vos M Silby M de Boer W 《PloS one》2011,6(11):e27266
Background
Many soil-inhabiting bacteria are known to produce secondary metabolites that can suppress microorganisms competing for the same resources. The production of antimicrobial compounds is expected to incur fitness costs for the producing bacteria. Such costs form the basis for models on the co-existence of antibiotic-producing and non-antibiotic producing strains. However, so far studies quantifying the costs of antibiotic production by bacteria are scarce. The current study reports on possible costs, for antibiotic production by Pseudomonas fluorescens Pf0-1, a soil bacterium that is induced to produce a broad-spectrum antibiotic when it is confronted with non-related bacterial competitors or supernatants of their cultures.Methodology and Principal Findings
We measured the possible cost of antibiotic production for Pseudomonas fluorescens Pf0-1 by monitoring changes in growth rate with and without induction of antibiotic production by supernatant of a bacterial competitor, namely Pedobacter sp.. Experiments were performed in liquid as well as on semi-solid media under nutrient-limited conditions that are expected to most clearly reveal fitness costs. Our results did not reveal any significant costs for production of antibiotics by Pseudomonas fluorescens Pf0-1. Comparison of growth rates of the antibiotic-producing wild-type cells with those of non-antibiotic producing mutants did not reveal costs of antibiotic production either.Significance
Based on our findings we propose that the facultative production of antibiotics might not be selected to mitigate metabolic costs, but instead might be advantageous because it limits the risk of competitors evolving resistance, or even the risk of competitors feeding on the compounds produced. 相似文献10.
The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods. 相似文献
11.
Measurement of Fitness and Its Components in Six Laboratory Strains of DROSOPHILA MELANOGASTER 总被引:2,自引:2,他引:0
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Yamazaki T 《Genetics》1984,108(1):201-211
Six laboratory strains of Drosophila melanogaster were used to measure "net fitness" and its components by interspecific competition with D. hydei using 100 experimental populations. The "total competitive ability," an estimate of net fitness measured in these competition experiments, was tightly correlated with another measure of net fitness, the population size, in single-species experiments (phenotypic correlation rp = 0.675 and genotypic correlation rg = 0.997). Other components of fitness were also measured simultaneously, and the correlation with the net fitness was calculated. The very high correlation between two measurements of net fitness and lower correlations between net fitness and components of fitness suggests that these net fitness measures are more reliable estimates of the "real net fitness" than the components of fitness. 相似文献
12.
R. E. LENSKI V. SOUZA L. P. DUONG Q. G. PHAN T. N. M. NGUYEN K. P. BERTRAND 《Molecular ecology》1994,3(2):127-135
We have been studying the effects of expression of plasmid-borne, Tn10-encoded, tetracycline resistance on the fitness of Escherichia coli K12. We previously demonstrated large reductions in fitness resulting from induced or constitutive expression of the resistance protein; however, any residual expression by the repressed operon was so slight that possession of an inducible resistance function imposed essentially no burden in the absence of antibiotic. Here, we demonstrate two distinct disadvantages for inducible genotypes relative to isogenic constitutive constructs. During the transition from antibiotic-free to antibiotic-containing media, the inducible genotype experiences a longer lag phase prior to growth. In the sustained presence of antibiotic, full induction of the resistance function in the inducible genotype is prevented by the continued action of its repressor. However, these disadvantages may be reduced by increasing the strength of the promoter for the resistance gene in the inducible genotype. Simultaneous consideration of the mode of gene regulation (i.e. constitutive or inducible) and the strength of the resistance-gene promoter (i.e. maximum level of expression) indicates an adaptive landscape with very strong epistasis and, perhaps, multiple fitness peaks. 相似文献
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14.
Jay M. Fitzsimmons Sijmen E. Schoustra Jeremy T. Kerr Rees Kassen 《Evolutionary ecology》2010,24(1):227-236
What are the effects of a mutational event on population dynamics? This eco-evolutionary question has relevance not only to basic biological theories but also to conservation applications. We evaluated the relationship between maximum population growth rate (r max) and carrying capacity (K) among strains of the bacterium Pseudomonas fluorescens. Each of 65 strains differed from their common ancestor by one naturally acquired phenotypic change conferring antibiotic resistance, brought about by a single mutational event, and each was grown in isolation in four environments. We found no evidence of a trade-off between r max and K. Rather, strains with rapid growth rates also had high carrying capacity, with little interaction between strain and environment. We conclude that the extensive variation in overall fitness resulting from single mutational events likely masks whatever population trade-offs may exist. 相似文献
15.
Melissa Lasaro Zhi Liu Rima Bishar Kathryn Kelly Sujay Chattopadhyay Sandip Paul Evgeni Sokurenko Jun Zhu Mark Goulian 《Journal of bacteriology》2014,196(9):1723-1732
The biology of Escherichia coli in its primary niche, the animal intestinal tract, is remarkably unexplored. Studies with the streptomycin-treated mouse model have produced important insights into the metabolic requirements for Escherichia coli to colonize mice. However, we still know relatively little about the physiology of this bacterium growing in the complex environment of an intestine that is permissive for the growth of competing flora. We have developed a system for studying colonization using an E. coli strain, MP1, isolated from a mouse. MP1 is genetically tractable and does not require continuous antibiotic treatment for stable colonization. As an application of this system, we separately knocked out each two-component system response regulator in MP1 and performed competitions against the wild-type strain. We found that only three response regulators, ArcA, CpxR, and RcsB, produce strong colonization defects, suggesting that in addition to anaerobiosis, adaptation to cell envelope stress is a critical requirement for E. coli colonization of the mouse intestine. We also show that the response regulator OmpR, which had previously been hypothesized to be important for adaptation between in vivo and ex vivo environments, is not required for MP1 colonization due to the presence of a third major porin. 相似文献
16.
Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. We used computer simulations of a mathematical model for phage growth to predict which features of bacterial growth curves were best associated with differences in growth among phage strains. We then tested these predictions using the in vitro method to confirm which of the inferred viral growth traits best reflected known fitness differences among genotypes of the RNA phage phi-6, when infecting a Pseudomonas syringae host. Results showed that the inferred phage trait of time-to-extinction (time required to drive bacterial density below detectable optical density) reliably correlated with genotype rankings based on absolute fitness (phage titer per ml). These data suggested that the high-throughput analysis was valuable for identifying growth differences among virus strains, and that the method may be especially useful for high throughput analyses of fitness differences among phage strains cultured and/or evolved in liquid (unstructured) environments. 相似文献
17.
Andrea von Groll Anandi Martin Matthias Stehr Mahavir Singh Fran?oise Portaels Pedro Eduardo Almeida da Silva Juan Carlos Palomino 《PloS one》2010,5(4)
Background
Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains.Methodology/Principal Findings
Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80.Conclusions/Significance
It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes. 相似文献18.
Genes That Enhance the Ecological Fitness of Shewanella oneidensis MR-1 in Sediments Reveal the Value of Antibiotic Resistance 总被引:1,自引:0,他引:1
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Jennifer L. Groh Qingwei Luo Jimmy D. Ballard Lee R. Krumholz 《Applied microbiology》2007,73(2):492-498
Environmental bacteria persist in various habitats, yet little is known about the genes that contribute to growth and survival in their respective ecological niches. Signature-tagged mutagenesis (STM) of Shewanella oneidensis MR-1 coupled with a screen involving incubations of mutant strains in anoxic aquifer sediments allowed us to identify 47 genes that enhance fitness in sediments. Gene functions inferred from annotations provide us with insight into physiological and ecological processes that environmental bacteria use while growing in sediment ecosystems. Identification of the mexF gene and other potential membrane efflux components by STM demonstrated that homologues of multidrug resistance genes present in pathogens are required for sediment fitness of nonpathogenic bacteria. Further studies with a mexF deletion mutant demonstrated that the multidrug resistance pump encoded by mexF is required for resistance to antibiotics, including chloramphenicol and tetracycline. Chloramphenicol-adapted cultures exhibited mutations in the gene encoding a TetR family regulatory protein, indicating a role for this protein in regulating expression of the mexEF operon. The relative importance of mexF for sediment fitness suggests that antibiotic efflux may be a required process for bacteria living in sediment systems. 相似文献
19.
Sexually antagonistic genetic variation can pose limits to the independent evolution and adaptation of the sexes. The extent of sexually antagonistic variation is reflected in the intersex genetic correlation for fitness (rwFM). Previous estimates of this correlation have been mostly limited to populations in environments to which they are already well adapted, making it difficult to gauge the importance of sexually antagonistic genetic variance during the early stages of adaptation, such as that occurring following abrupt environmental change or upon the colonization of new habitat. Here we assayed male and female lifetime fitness in a population of Drosophila serrata in four novel laboratory environments. We found that rwFM varied significantly across environments, with point estimates ranging from positive to negative values of considerable magnitude. We also found that the variability among estimates was because, at least in part, of significant differences among environments in the genetic variances of both male and female fitness, with no evidence of any significant changes in the intersex covariance itself, although standard errors of these estimates were large. Our results illustrate the unpredictable nature of rwFM in novel environments and suggest that, although sexually antagonistic genetic variance can be pronounced in some novel environments, it may have little effect in constraining the early stages of adaptation in others. 相似文献
20.
Novel resistance to pathogens and pesticides is commonly associated with a fitness cost. However, measurements of the fitness costs of insecticide resistance have used diverse methods to control for genetic background and rarely assess the effects of environmental variation. Here, we explored how genetic background interacts with resource quality to affect the expression of the fitness costs associated with resistance. We used a serially backcrossed line of the diamondback moth, Plutella xylostella, resistant to the biopesticide Bacillus thuringiensis, to estimate the costs of resistance for insects feeding on two Brassica species. We found that fitness costs increased on the better-defended Brassica oleracea cultivars. These data were included in two meta-analyses of fitness cost experiments that used standardized protocols (and a common resistant insect stock) but which varied in the methodology used to control for the effects of genetic background. The meta-analysis confirmed that fitness costs were higher on the low-quality host (B. oleracea); and experimental methodology did not influence estimates of fitness costs on that plant species. In contrast, fitness costs were heterogeneous in the Brassica pekinensis studies: fitness costs in genetically homogenized lines were significantly higher than in studies using revertant insects. We hypothesize that fitness modifiers can moderate fitness costs on high-quality plants but may not affect fitness when resource quality is low. 相似文献