scholarly journals Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
C. Trunet ◽  
N. Mtimet ◽  
A.-G. Mathot ◽  
F. Postollec ◽  
I. Leguerinel ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Flow Cytometry ◽  
Bacillus Licheniformis ◽  
Model Parameters ◽  
Vegetative Cells ◽  
Content Type ◽  
Bacillus Weihenstephanensis ◽  
Spore Outgrowth ◽  
Growth Limits ◽  
Kinetics Of

ABSTRACT Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis. Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH. IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

scholarly journals Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

2014 ◽  
Vol 81 (2) ◽  
pp. 562-568 ◽  
Author(s):  
C. Trunet ◽  
N. Mtimet ◽  
A.-G. Mathot ◽  
F. Postollec ◽  
I. Leguerinel ◽  
...  
Keyword(s):  
Heat Resistance ◽  
Bacillus Licheniformis ◽  
Content Type ◽  
Bacillus Weihenstephanensis ◽  
Heat Treated ◽  
Wide Range ◽  
Growth Limits ◽  
Cardinal Temperature ◽  
Resistance Data ◽  
Recovery Temperature

ABSTRACTThe apparent heat resistance of spores ofBacillus weihenstephanensisandBacillus licheniformiswas measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores ofB. weihenstephanensiswere produced at 30°C and 12°C, and spores ofB. licheniformiswere produced at 45°C and 20°C.B. weihenstephanensisspores were then heat treated at 85°C, 90°C, and 95°C, andB. licheniformisspores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C forB. weihenstephanensisand 15°C to 60°C forB. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

scholarly journals Germination, Outgrowth, and Vegetative-Growth Kinetics of Dry-Heat-Treated Individual Spores ofBacillusSpecies

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Lin He ◽  
Zhan Chen ◽  
Shiwei Wang ◽  
Muying Wu ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Spore Germination ◽  
Differential Interference Contrast Microscopy ◽  
Content Type ◽  
Dry Heat ◽  
Heat Treated ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Dry Heat Treatment ◽  
Kinetics Of

ABSTRACTDNA damage kills dry-heated spores ofBacillus subtilis, but dry-heat-treatment effects on spore germination and outgrowth have not been studied. This is important, since if dry-heat-killed spores germinate and undergo outgrowth, toxic proteins could be synthesized. Here, Raman spectroscopy and differential interference contrast microscopy were used to study germination and outgrowth of individual dry-heat-treatedB. subtilisandBacillus megateriumspores. The major findings in this work were as follows: (i) spores dry-heat-treated at 140°C for 20 min lost nearly all viability but retained their Ca2+-dipicolinic acid (CaDPA) depot; (ii) in most cases, dry-heat treatment increased the average times and variability of all major germination events inB. subtilisspore germination with nutrient germinants or CaDPA, and in one nutrient germination event withB. megateriumspores; (iii)B. subtilisspore germination with dodecylamine, which activates the spore CaDPA release channel, was unaffected by dry-heat treatment; (iv) these results indicate that dry-heat treatment likely damages spore proteins important in nutrient germinant recognition and cortex peptidoglycan hydrolysis, but not CaDPA release itself; and (v) analysis of single spores incubated on nutrient-rich agar showed that while dry-heat-treated spores that are dead can complete germination, they cannot proceed into outgrowth and thus not to vegetative growth. The results of this study provide new information on the effects of dry heat on bacterial spores and indicate that dry-heat sterilization regimens should produce spores that cannot outgrow and thus cannot synthesize potentially dangerous proteins.IMPORTANCEMuch research has shown that high-temperature dry heat is a promising means for the inactivation of spores on medical devices and spacecraft decontamination. Dry heat is known to killBacillus subtilisspores by DNA damage. However, knowledge about the effects of dry-heat treatment on spore germination and outgrowth is limited, especially at the single spore level. In the current work, Raman spectroscopy and differential interference contrast microscopy were used to analyze CaDPA levels in and kinetics of nutrient- and non-nutrient germination of multiple individual dry-heat-treatedB. subtilisandBacillus megateriumspores that were largely dead. The outgrowth and subsequent cell division of these germinated but dead dry-heat-treated spores were also examined. The knowledge obtained in this study will help understand the effects of dry heat on spores both on Earth and in space, and indicates that dry heat can be safely used for sterilization purposes.

scholarly journals Kinetics of Germination of Wet-Heat-Treated Individual Spores of Bacillus Species, Monitored by Raman Spectroscopy and Differential Interference Contrast Microscopy

2011 ◽  
Vol 77 (10) ◽  
pp. 3368-3379 ◽  
Author(s):  
Guiwen Wang ◽  
Pengfei Zhang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Heat Treatment ◽  
Raman Spectroscopy ◽  
Spore Germination ◽  
Great Majority ◽  
Lytic Enzyme ◽  
Differential Interference Contrast ◽  
Content Type ◽  
Heat Treated ◽  
Wet Heat ◽  
Kinetics Of

ABSTRACTRaman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores ofBacillus cereusandBacillus megaterium, as well as of several isogenicBacillus subtilisstrains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca2+and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time,Tlag, at which spores began release of the great majority of their CaDPA during the germination ofB. subtilisspores with different nutrient germinants and also increased the variability ofTlagvalues. (iii) The time period, ΔTrelease, betweenTlagand the time,Trelease, at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values ofIrelease, the intensity of a spore's DIC image atTrelease, than did untreated spores and had much longer time periods, ΔTlys, for the reduction inIreleaseintensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases inTlagand ΔTreleasewere also observed when wet-heat-treatedB. subtilisspores were germinated with the nonnutrient dodecylamine, while the change inIreleasewas less significant. (vi) The effects of wet-heat treatment on nutrient germination ofB. cereusandB. megateriumspores were generally similar to those onB. subtilisspores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

scholarly journals Differentiation of Vegetative Cells into Spores: a Kinetic Model Applied toBacillus subtilis

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Emilie Gauvry ◽  
Anne-Gabrielle Mathot ◽  
Olivier Couvert ◽  
Ivan Leguérinel ◽  
Matthieu Jules ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Vegetative Cell ◽  
Goodness Of Fit ◽  
Fluorescent Protein ◽  
Raw Materials ◽  
Spore Formation ◽  
Vegetative Cells ◽  
Content Type ◽  
Kinetics Of ◽  
Over Time

ABSTRACTSpore-forming bacteria are natural contaminants of food raw materials, and sporulation can occur in many environments from farm to fork. In order to characterize and to predict spore formation over time, we developed a model that describes both the kinetics of growth and the differentiation of vegetative cells into spores. The model is based on a classical growth model and enables description of the kinetics of sporulation with the addition of three parameters specific to sporulation. Two parameters are related to the probability of each vegetative cell to commit to sporulation and to form a spore, and the last one is related to the time needed to form a spore once the cell is committed to sporulation. The goodness of fit of this growth-sporulation model was assessed using growth-sporulation kinetics at various temperatures in laboratory medium or in whey forBacillus subtilis,Bacillus cereus, andBacillus licheniformis. The model accurately describes the kinetics in these different conditions, with a mean error lower than 0.78 log10CFU/ml for the growth and 1.08 log10CFU/ml for the sporulation. The biological meaning of the parameters was validated with a derivative strain ofBacillus subtilis168 which produces green fluorescent protein at the initiation of sporulation. This model provides physiological information on the spore formation and on the temporal abilities of vegetative cells to differentiate into spores and reveals the heterogeneity of spore formation during and after growth.IMPORTANCEThe growth-sporulation model describes the progressive transition from vegetative cells to spores with sporulation parameters describing the sporulation potential of each vegetative cell. Consequently, the model constitutes an interesting tool to assess the sporulation potential of a bacterial population over time with accurate parameters such as the time needed to obtain one resistant spore and the probability of sporulation. Further, this model can be used to assess these data under various environmental conditions in order to better identify the conditions favorable for sporulation regarding the time to obtain the first spore and/or the concentrations of spores which could be reached during a food process.

Inactivation kinetics of Bacillus cereus vegetative cells and spores from different sources by antimicrobial photodynamic treatment (aPDT)

LWT ◽  
2021 ◽  
Vol 142 ◽  
pp. 111037
Author(s):  
Leonardo do Prado-Silva ◽  
Verônica O. Alvarenga ◽  
Gilberto Ú.L. Braga ◽  
Anderson S. Sant’Ana
Keyword(s):  
Bacillus Cereus ◽  
Inactivation Kinetics ◽  
Photodynamic Treatment ◽  
Vegetative Cells ◽  
Kinetics Of ◽  
Different Sources

A dynamic simulator for the ergonomics evaluation of powered torque tools for human assembly

2017 ◽  
Vol 37 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Haluk Ay ◽  
Anthony Luscher ◽  
Carolyn Sommerich
Keyword(s):  
Risk Factors ◽  
Joint Torque ◽  
Human Hand ◽  
Model Parameters ◽  
Test Rig ◽  
Musculoskeletal Discomfort ◽  
Data Set ◽  
Content Type ◽  
Working Posture ◽  
Displacement Measurements

Purpose The purpose of this study is to design and develop a testing device to simulate interaction between human hand–arm dynamics, right-angle (RA) computer-controlled power torque tools and joint-tightening task-related variables. Design/methodology/approach The testing rig can simulate a variety of tools, tasks and operator conditions. The device includes custom data-acquisition electronics and graphical user interface-based software. The simulation of the human hand–arm dynamics is based on the rig’s four-bar mechanism-based design and mechanical components that provide adjustable stiffness (via pneumatic cylinder) and mass (via plates) and non-adjustable damping. The stiffness and mass values used are based on an experimentally validated hand–arm model that includes a database of model parameters. This database is with respect to gender and working posture, corresponding to experienced tool operators from a prior study. Findings The rig measures tool handle force and displacement responses simultaneously. Peak force and displacement coefficients of determination (R2) between rig estimations and human testing measurements were 0.98 and 0.85, respectively, for the same set of tools, tasks and operator conditions. The rig also provides predicted tool operator acceptability ratings, using a data set from a prior study of discomfort in experienced operators during torque tool use. Research limitations/implications Deviations from linearity may influence handle force and displacement measurements. Stiction (Coulomb friction) in the overall rig, as well as in the air cylinder piston, is neglected. The rig’s mechanical damping is not adjustable, despite the fact that human hand–arm damping varies with respect to gender and working posture. Deviations from these assumptions may affect the correlation of the handle force and displacement measurements with those of human testing for the same tool, task and operator conditions. Practical implications This test rig will allow the rapid assessment of the ergonomic performance of DC torque tools, saving considerable time in lineside applications and reducing the risk of worker injury. DC torque tools are an extremely effective way of increasing production rate and improving torque accuracy. Being a complex dynamic system, however, the performance of DC torque tools varies in each application. Changes in worker mass, damping and stiffness, as well as joint stiffness and tool program, make each application unique. This test rig models all of these factors and allows quick assessment. Social implications The use of this tool test rig will help to identify and understand risk factors that contribute to musculoskeletal disorders (MSDs) associated with the use of torque tools. Tool operators are subjected to large impulsive handle reaction forces, as joint torque builds up while tightening a fastener. Repeated exposure to such forces is associated with muscle soreness, fatigue and physical stress which are also risk factors for upper extremity injuries (MSDs; e.g. tendinosis, myofascial pain). Eccentric exercise exertions are known to cause damage to muscle tissue in untrained individuals and affect subsequent performance. Originality/value The rig provides a novel means for quantitative, repeatable dynamic evaluation of RA powered torque tools and objective selection of tightening programs. Compared to current static tool assessment methods, dynamic testing provides a more realistic tool assessment relative to the tool operator’s experience. This may lead to improvements in tool or controller design and reduction in associated musculoskeletal discomfort in operators.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals In VitroAntibacterial Activity of NB-003 against Propionibacterium acnes

2011 ◽  
Vol 55 (9) ◽  
pp. 4211-4217 ◽  
Author(s):  
J. Pannu ◽  
A. McCarthy ◽  
A. Martin ◽  
T. Hamouda ◽  
S. Ciotti ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Propionibacterium Acnes ◽  
Resistant Mutant ◽  
Microtiter Plate ◽  
Content Type ◽  
Oil In Water ◽  
Complete Disruption ◽  
Kinetics Of ◽  
Gel Formulations

ABSTRACTNB-003 and NB-003 gel formulations are oil-in-water nanoemulsions designed for use in bacterial infections.In vitrosusceptibility ofPropionibacterium acnesto NB-003 formulations and comparator drugs was evaluated. Both NB-003 formulations were bactericidal against allP. acnesisolates, including those that were erythromycin, clindamycin, and/or tetracycline resistant. In the absence of sebum, the MIC90s/minimum bactericidal concentrations (MBC90s) for NB-003, NB-003 gel, salicylic acid (SA), and benzoyl peroxide (BPO) were 0.5/2.0, 1.0/2.0, 1,000/2,000, and 50/200 μg/ml, respectively. In the presence of 50% sebum, the MIC90s/MBC90s of NB003 and BPOs increased to 128/1,024 and 400/1,600 μg/ml, respectively. The MIC90s/MBC90s of SA were not significantly impacted by the presence of sebum. A reduction in the MBC90s for NB-003 and BPO was observed when 2% SA or 0.5% BPO was integrated into the formulation, resulting in MIC90s/MBC90s of 128/256 μg/ml for NB003 and 214/428 μg/ml for BPO. The addition of EDTA enhanced thein vitroefficacy of 0.5% NB-003 in the presence or absence of 25% sebum. The addition of 5 mM EDTA to each well of the microtiter plate resulted in a >16- and >256-fold decrease in MIC90and MBC90, yielding a more potent MIC90/MBC90of ≤1/<1 μg/ml. The kinetics of bactericidal activity of NB-003 againstP. acneswere compared to those of a commercially available product of BPO. Electron micrographs ofP. acnestreated with NB-003 showed complete disruption of bacteria. Assessment of spontaneous resistance ofP. acnesrevealed no stably resistant mutant strains.

scholarly journals Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Cytometry ◽  
1998 ◽  
Vol 32 (4) ◽  
pp. 280-285 ◽  
Author(s):  
Loris Zamai ◽  
Adriana R. Mariani ◽  
Giorgio Zauli ◽  
Luigi Rodella ◽  
Rita Rezzani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Natural Killer ◽  
Natural Killer Activity ◽  
K562 Cells ◽  
Killer Activity ◽  
Kinetics Of
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