scholarly journals Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Esha Atolia ◽  
Spencer Cesar ◽  
Heidi A. Arjes ◽  
Manohary Rajendram ◽  
Handuo Shi ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Growth Rate ◽  
High Throughput ◽  
Bacterial Growth ◽  
Physiological State ◽  
Maximal Growth ◽  
Content Type ◽  
Initial Cell ◽  
Maximal Growth Rate

ABSTRACT Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

scholarly journals Environmental and physiological factors affecting high-throughput measurements of bacterial growth

2020 ◽  
Author(s):  
Esha Atolia ◽  
Spencer Cesar ◽  
Heidi A. Arjes ◽  
Manohary Rajendram ◽  
Handuo Shi ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Escherichia Coli ◽  
Growth Rate ◽  
High Throughput ◽  
Bacterial Growth ◽  
Physiological State ◽  
Maximal Growth ◽  
Initial Cell ◽  
Maximal Growth Rate

AbstractBacterial growth in nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multi-well plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.Abstract ImportanceHow starved bacteria adapt to and multiply in replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allows for accurate growth-rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic-acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

scholarly journals Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

1999 ◽  
Vol 65 (6) ◽  
pp. 2631-2635 ◽  
Author(s):  
Sonja Isken ◽  
Antoine Derks ◽  
Petra F. G. Wolffs ◽  
Jan A. M. de Bont
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Organic Solvents ◽  
Critical Concentration ◽  
Bacterial Membrane ◽  
Maximal Growth ◽  
Wild Type ◽  
Active Efflux ◽  
Maximal Growth Rate ◽  
Solvent Tolerant

ABSTRACT Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

Applying the Fama and French three-factor model to analyze risk/reward in the Spanish REITs: an ARDL approach

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Zhenyu Su ◽  
Paloma Taltavull
Keyword(s):  
Growth Rate ◽  
Factor Model ◽  
Explanatory Power ◽  
Gdp Growth ◽  
Size Factor ◽  
Content Type ◽  
Gdp Growth Rate ◽  
Fama And French ◽  
Three Factor

Purpose This paper aims to analyse the risk and excess returns of the Spanish real estate investment trusts (S-REITs) using various methods, though focusing primarily on the Fama-French three-factor (FF3) model, over the period from 2007Q3 to 2017Q2. Design/methodology/approach The autoregressive distributed lag model is used for the empirical analysis to test long-term stable relationships between variables. Findings The findings indicate that the FF3 model is suitable for the S-REITs market, better explaining the S-REITs’ returns variation than the traditional single-index capital asset pricing model (CAPM) and the Carhart four-factor model. The empirical evidence is reasonably consistent with the FF3 model; the values for the market, size and value are highly statistically significant over the analysis period, with 68.7% variation in S-REITs’ returns explained by the model. In the long run, the market factor has less explanatory power than the size and value factors; the positive long-term multiplier of the size factor indicates that small S-REIT companies have higher returns, along with higher risk, while the negative multiplier of the value indicator suggests that S-REITs portfolios prefer to allocate growth REITs with low book-to-market ratios. The empirical findings from a modified FF3 model, which additionally incorporates Spain’s gross domestic product (GDP) growth rate, two consumer price index (CPI) macro-factors and three dummy variables, indicates that GDP growth rate and CPI also affect S-REITs’ yields, while investment funds with capital calls have a small influence on S-REITs’ returns. Practical implications The regression results of the standard and extended FF3 model can help researchers understand S-REITs’ risk and return through a general stock pattern. Potential investors are given more information to consider the new Spanish investment vehicle before making a decision. Originality/value The paper uses standard techniques but applies them for the first time to the S-REIT market.

scholarly journals Loss of Function inEscherichia coliExposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Sarah Forbes ◽  
Nicola Morgan ◽  
Gavin J. Humphreys ◽  
Alejandro Amézquita ◽  
Hitesh Mistry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Biofilm Formation ◽  
Benzalkonium Chloride ◽  
Loss Of Function ◽  
Free Medium ◽  
Content Type ◽  
Expression Of Genes ◽  
Repeated Passage

ABSTRACTAssessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues,Escherichia coliMG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/liter and 1,000 ng/liter (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility, and biofilm formation were assessed, and differentially expressed genes were determined via transcriptome sequencing. Planktonic growth rate and biofilm formation were significantly reduced (P< 0.001) following BAC adaptation, while BAC minimum bactericidal concentration increased 2-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation compared with the respective control organism passaged in BAC-free medium. When the BAC-adapted bacterium was grown in BAC-free medium, 1,052 genes were upregulated and 753 were downregulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared with unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced the expression of genes associated with outer-membrane porins, motility, and chemotaxis. This was manifested phenotypically through the loss of function (motility). Repeated passage in a BAC-free environment resulted in the upregulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure ofE. colito BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth rate and biofilm formation, and loss of motility.IMPORTANCEExposure to very low concentrations of biocides in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide benzalkonium chloride (BAC) resulted in loss of function (motility) and a general reduction in bacterial fitness but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in theEscherichia colitranscriptome. These results demonstrate the importance of including phenotypic characterization in studies designed to assess the risks of biocide exposure.

scholarly journals Bacterial Community Shift and Coexisting/Coexcluding Patterns Revealed by Network Analysis in a Uranium-Contaminated Site after Bioreduction Followed by Reoxidation

2018 ◽  
Vol 84 (9) ◽  
Author(s):  
Bing Li ◽  
Wei-Min Wu ◽  
David B. Watson ◽  
Erick Cardenas ◽  
Yuanqing Chao ◽  
...  
Keyword(s):  
Network Analysis ◽  
High Throughput ◽  
Bacterial Communities ◽  
High Throughput Sequencing ◽  
Ecological Niches ◽  
Content Type ◽  
Oak Ridge ◽  
Α Diversity ◽  
Before And After

ABSTRACTA site in Oak Ridge, TN, USA, has sediments that contain >3% iron oxides and is contaminated with uranium (U). The U(VI) was bioreduced to U(IV) and immobilizedin situthrough intermittent injections of ethanol. It then was allowed to reoxidize via the invasion of low-pH (3.6 to 4.0), high-nitrate (up to 200 mM) groundwater back into the reduced zone for 1,383 days. To examine the biogeochemical response, high-throughput sequencing and network analysis were applied to characterize bacterial population shifts, as well as cooccurrence and coexclusion patterns among microbial communities. A pairedttest indicated no significant changes of α-diversity for the bioactive wells. However, both nonmetric multidimensional scaling and analysis of similarity confirmed a significant distinction in the overall composition of the bacterial communities between the bioreduced and the reoxidized sediments. The top 20 major genera accounted for >70% of the cumulative contribution to the dissimilarity in the bacterial communities before and after the groundwater invasion.Castellaniellahad the largest dissimilarity contribution (17.7%). For the bioactive wells, the abundance of the U(VI)-reducing generaGeothrix,Desulfovibrio,Ferribacterium, andGeobacterdecreased significantly, whereas the denitrifyingAcidovoraxabundance increased significantly after groundwater invasion. Additionally, seven genera, i.e.,Castellaniella,Ignavibacterium,Simplicispira,Rhizomicrobium,AcidobacteriaGp1,AcidobacteriaGp14, andAcidobacteriaGp23, were significant indicators of bioactive wells in the reoxidation stage. Canonical correspondence analysis indicated that nitrate, manganese, and pH affected mostly the U(VI)-reducing genera and indicator genera. Cooccurrence patterns among microbial taxa suggested the presence of taxa sharing similar ecological niches or mutualism/commensalism/synergism interactions.IMPORTANCEHigh-throughput sequencing technology in combination with a network analysis approach were used to investigate the stabilization of uranium and the corresponding dynamics of bacterial communities under field conditions with regard to the heterogeneity and complexity of the subsurface over the long term. The study also examined diversity and microbial community composition shift, the common genera, and indicator genera before and after long-term contaminated-groundwater invasion and the relationship between the target functional community structure and environmental factors. Additionally, deciphering cooccurrence and coexclusion patterns among microbial taxa and environmental parameters could help predict potential biotic interactions (cooperation/competition), shared physiologies, or habitat affinities, thus, improving our understanding of ecological niches occupied by certain specific species. These findings offer new insights into compositions of and associations among bacterial communities and serve as a foundation for future bioreduction implementation and monitoring efforts applied to uranium-contaminated sites.

Osteosarcoma Developed in the Period of Maximal Growth Rate Have Inferior Prognosis

2008 ◽  
Vol 30 (6) ◽  
pp. 419-424 ◽  
Author(s):  
Jun Ah Lee ◽  
Min Suk Kim ◽  
Dong Ho Kim ◽  
Jung Sub Lim ◽  
Kyung Duk Park ◽  
...  
Keyword(s):  
Growth Rate ◽  
Maximal Growth ◽  
Maximal Growth Rate

scholarly journals High-Throughput Sequencing Reveals the Incomplete, Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic Treatment with Ampicillin and Gentamicin

2012 ◽  
Vol 56 (11) ◽  
pp. 5811-5820 ◽  
Author(s):  
Fiona Fouhy ◽  
Caitriona M. Guinane ◽  
Seamus Hussey ◽  
Rebecca Wall ◽  
C. Anthony Ryan ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Antibiotic Treatment ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Short Term ◽  
Parenteral Antibiotic ◽  
Content Type ◽  
Combined Use ◽  
Cessation Of Treatment

ABSTRACTThe infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA andrpoB-specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions ofProteobacteria(P= 0.0049) and significantly lower proportions ofActinobacteria(P= 0.00001) (and the associated genusBifidobacterium[P= 0.0132]) as well as the genusLactobacillus(P= 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, theProteobacterialevels remained significantly higher in the treated infants (P= 0.0049), but theActinobacteria,Bifidobacterium, andLactobacilluslevels had recovered and were similar to those in the control samples. Despite this recovery of totalBifidobacteriumnumbers,rpoB-targeted pyrosequencing revealed that the number of differentBifidobacteriumspecies present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.

Estimating Bacterial Growth Parameters by Means of Detection Times

1999 ◽  
Vol 65 (2) ◽  
pp. 732-736 ◽  
Author(s):  
József Baranyi ◽  
Carmen Pin
Keyword(s):  
Growth Rate ◽  
Specific Growth Rate ◽  
Bacterial Growth ◽  
Specific Growth ◽  
Growth Parameters ◽  
Physiological State ◽  
Cell Number ◽  
Maximum Specific Growth Rate ◽  
Specific Rate ◽  
The Individual

ABSTRACT We developed a new numerical method to estimate bacterial growth parameters by means of detection times generated by different initial counts. The observed detection times are subjected to a transformation involving the (unknown) maximum specific growth rate and the (known) ratios between the different inoculum sizes and the constant detectable level of counts. We present an analysis of variance (ANOVA) protocol based on a theoretical result according to which, if the specific rate used for the transformation is correct, the transformed values are scattered around the same mean irrespective of the original inoculum sizes. That mean, termed the physiological state of the inoculum,α̂, and the maximum specific growth rate, μ, can be estimated by minimizing the variance ratio of the ANOVA procedure. The lag time of the population can be calculated as λ = −ln α̂/μ; i.e. the lag is inversely proportional to the maximum specific growth rate and depends on the initial physiological state of the population. The more accurately the cell number at the detection level is known, the better the estimate for the variance of the lag times of the individual cells.

scholarly journals A Roadblock-and-Kill Mechanism of Action Model for the DNA-Targeting Antibiotic Ciprofloxacin

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Nikola Ojkic ◽  
Elin Lilja ◽  
Susana Direito ◽  
Angela Dawson ◽  
Rosalind J. Allen ◽  
...  
Keyword(s):  
Growth Rate ◽  
Dna Damage ◽  
Production Rate ◽  
Mechanism Of Action ◽  
Sos Response ◽  
Dynamical Response ◽  
Bacterial Cells ◽  
Content Type ◽  
Dna Production

ABSTRACT Fluoroquinolones, antibiotics that cause DNA damage by inhibiting DNA topoisomerases, are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here, we investigated the growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low concentrations. We measured the long-term and short-term dynamical response of the growth rate and DNA production rate to ciprofloxacin at both the population and single-cell levels. We show that, despite the molecular complexity of DNA metabolism, a simple roadblock-and-kill model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates in the presence of ciprofloxacin. The model also predicts dynamical changes in the DNA production rate in wild-type E. coli and in a recombination-deficient mutant following a step-up of ciprofloxacin. Our work highlights that bacterial cells show a delayed growth rate response following fluoroquinolone exposure. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. We also show that the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response, challenging the accepted view. Our work highlights the importance of including detailed biophysical processes in biochemical-systems models to quantitatively predict the bacterial response to antibiotics.

scholarly journals Effect of Salt Stress on Mutation and Genetic Architecture for Fitness Components in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (10) ◽  
pp. 3831-3842
Author(s):  
Christopher Kozela ◽  
Mark O. Johnston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Salt Stress ◽  
Mutation Rate ◽  
Genetic Architecture ◽  
Maximal Growth ◽  
Fitness Components ◽  
Environmental Variance ◽  
Maximal Growth Rate ◽  
Mutational Variance

Mutations shape genetic architecture and thus influence the evolvability, adaptation and diversification of populations. Mutations may have different and even opposite effects on separate fitness components, and their rate of origin, distribution of effects and variance-covariance structure may depend on environmental quality. We performed an approximately 1,500-generation mutation-accumulation (MA) study in diploids of the yeast Saccharomyces cerevisiae in stressful (high-salt) and normal environments (50 lines each) to investigate the rate of input of mutational variation (Vm) as well as the mutation rate and distribution of effects on diploid and haploid fitness components, assayed in the normal environment. All four fitness components in both MA treatments exhibited statistically significant mutational variance and mutational heritability. Compared to normal-MA, salt stress increased the mutational variance in growth rate by more than sevenfold in haploids derived from the MA lines. This increase was not detected in diploid growth rate, suggesting masking of mutations in the heterozygous state. The genetic architecture arising from mutation (M-matrix) differed between normal and salt conditions. Salt stress also increased environmental variance in three fitness components, consistent with a reduction in canalization. Maximum-likelihood analysis indicated that stress increased the genomic mutation rate by approximately twofold for maximal growth rate and sporulation rate in diploids and for viability in haploids, and by tenfold for maximal growth rate in haploids, but large confidence intervals precluded distinguishing these values between MA environments. We discuss correlations between fitness components in diploids and haploids and compare the correlations between the two MA environmental treatments.

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