Exponential Growth Phase Research Articles

In Vitro Infection of Human Macrophages with Porphyromonas gingivalis W83

This study aimed to investigate the innate immune response of human macrophages to Porphyromonas gingivalis W83 using a novel in vitro infection model. The growth kinetics of P. gingivalis W83 were analyzed, revealing an exponential growth phase at 8 h (optical density = 0.70). To establish a reliable macrophage model, the differentiation of THP-1 monocytes into macrophages was optimized using low concentrations of phorbol 12-myristate 13-acetate (PMA). This approach induced enhanced adherence and morphological changes, with full differentiation achieved after 48 h of PMA treatment followed by 24 h of rest. Polarization towards the pro-inflammatory M1 phenotype was successfully induced with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), as confirmed using cytokine profiling. Cytokine analysis using Luminex® technology demonstrated significant increases in interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6, indicating the effective activation of macrophages towards a pro-inflammatory phenotype. Building upon this macrophage model, this study investigated the interactions between macrophages and P. gingivalis W83 during its exponential growth phase. After a one-hour infection period, bacterial DNA quantification in supernatants and lysed macrophages revealed minimal levels of internalized or adherent bacteria, supporting the hypothesis that P. gingivalis effectively evades immune detection. These findings emphasize the utility of this model in uncovering the sophisticated immune evasion strategies employed by P. gingivalis, with significant implications for the development of targeted therapeutic interventions.

Influence of Cell Physiological Status on the Intensified Fed-Batch Cultures at Ultra-High Seeding Density.

Recent years, intensified fed-batch culture with ultra-high seeding density (uHSD-IFB) is coming to the forefront of manufacturers' choice for its enhanced productivity. However, the effects of seed cell physiological state and aeration strategies on these processes remain underexplored due to the ultra-high seeding density. Currently, the pre-production seeding inoculum (N-1) crucial for the uHSD-IFB cultures relies heavily upon case-by-case empirical experiences. To develop a rational seeding approach as a guideline, we here explored the impact of perfusion rates and cell growth states on the subsequent uHSD-IFB processes. It was found that seed cells in the exponential growth phase with high perfusion rates in the N-1 perfusion stage allowed for higher viable cell density and titer in the production stage. In particular, lower levels of reactive oxygen species, higher proportions of G1 and S phase, and higher specific cell oxygen uptake rates (OURs) were exhibited in these cells, resulting in higher cell specific growth rates and integral of viable cell concentration (IVCC) throughout the production cultures. Further investigation into the effect of aeration strategies was carried out in the benchtop bioreactors. A final yield of 4.5g/L, an increase of nearly 110%, was achieved by a sophisticated dual sparger system compared to the other two processes with either one l-shaped or micro-sparger. These results provide a direction for the design and establishment of high-titer processes in intensified fed-batch cultures at ultra-high seeding density. Synopsis: In this work, we first explored the impact of perfusion rates and cell growth states on the subsequent uHSD-IFB processes. Further investigation into the effect of aeration strategies of intensified fed-batch process was carried out in the benchtop bioreactors. These results provide a direction for the design and establishment of high-titer processes in intensified fed-batch cultures at ultra-high seeding density.

Sensitivity of bulk electrical impedance spectroscopy (bio-capacitance) probes to cell and culture properties: Study on CHO cell cultures.

Bulk electrical impedance spectroscopy (bio-capacitance) probes, hold significant promise for real-time cell monitoring in bioprocesses. Focusing on Chinese hamster ovary (CHO) cells, we present a sensitivity analysis framework to assess the impact of cell and culture properties on the complex permittivity spectrum, εmix, and its associated parameters, permittivity increment, Δε, critical frequency, fc, and Cole-Cole parameter, α, measured by bio-capacitance probes. Our sensitivity analysis showed that Δε is highly sensitive to cell size and concentration, making it suitable for estimating biovolume during the exponential growth phase, whereas fc provides information about cumulative changes in cell size, membrane permittivity, and cytoplasm conductivity during the transition to death phase. The analysis indicated that specific information about cell membrane permittivity or internal conductivity cannot be extracted from εmix spectrum. Based on the sensitivity analysis, we proposed two alternative parameters for monitoring cells in bioprocesses: Δε1 MHz and Δε1 MHz/Δε0.3 MHz, using measurements at 300 kHz, 1 MHz, and 10 MHz. Δε1 MHz is suitable for estimating viable cell density during the exponential growth phase due to its lower sensitivity to cell size. Δε1 MHz/Δε0.3 MHz can replace fc due to similar sensitivities to cell size and dielectric properties. These frequencies are within most bio-capacitance probes' optimal operation range, eliminating the need for low-frequency electrode polarization and high-frequency stray capacitances corrections. Experimental measurements on CHO cells confirmed the results of sensitivity analysis.

Synergistic Interaction of Spirulina Sp. and Folic Acid- Producing Bacteria for Folate Production

Folate, an essential nutrient crucial for DNA synthesis, cell division, and fetal neural tubedevelopment, remains a global health concern due to deficiencies in certain populations. Toaddress this issue, we investigated the synergistic interaction between Spirulina, a nutrient-rich microalga, and two strains of folic acid-producing bacteria, Bacillus subtilis-1 and Bacillus subtilis-2, to enhance folate production. Spirulinahas high nutritional content, combined with the folate production capabilities of the selected bacteria, offered a promising opportunity for sustainable folate synthesis. In this study, Spirulinaand the two strains of Bacillus subtilis were cultured separately to optimize growth conditions for each organism. Co-culture experiments were then conducted, combining Spirulina with Bacillus subtilis-1 and also Bacillus subtilis-2, to investigate their collective potential for folate production. The specific growth rates of both Spirulinaand the bacteria were measured individually and in combination using spectrophotometric methods, and their dry weights were determined to assess biomass productivity. Folate quantification in the microalgal-bacterial cultures was performed using a spectrophotometric analysis based on the phosphate buffer extraction method. This method facilitated the measurement of folate content investigated the impact of the symbiotic relationship between Spirulina and bacteria, particularly in terms of enhancing vitamin B12 acquisition and its impact on folate synthesis. Our results revealed a synergistic enhancement in folate production at the exponential growth phase of Bacillus subtilis-1 and Bacillus subtilis-2 co-culture. The synergistic relationship between Spirulina and Bacillus subtilis-1, as well as Bacillus subtilis-2, is manifested in elevated folate synthesis, highlighting the significant impact of microbial cooperation on nutrient production. Additionally, we observed fluctuations in folate production at the stationary phase, highlighting the subtle equilibrium achieved through symbiotic interactions. These findings shed light on the potential of harnessing the synergistic potential of microorganisms for sustainable folate synthesis. This research advances co-culturing methods to optimize folate production, tackling global folate deficiency challenges and fostering innovative, sustainable nutritional solutions.

Biological activity of biomass, phycocyanin and exopolysaccharides from a native strain of Spirulina subsalsa, grown in low cost saline medium

To evaluate the biological activity of biomass, phycocyanin extracts, and exopolysaccharides from S. subsalsa, axenic cultures were conducted at 9 ‰ salinity by adding seawater and using a low-cost saline medium: [N]=14 mM. The cultivation was performed in an external environment and was manually stirred. The cyanobacteria were centrifuged when they reached the exponential and stationary growth phases. The filtrate was used to obtain the exopolysaccharides by acetone precipitation, and the biomass was used to obtain the extracts in the different solvents. Phycocyanin was extracted in water. The activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Micrococcus luteus and Staphylococcus aureus, using the agar diffusion technique was evaluated. The results showed that only the ethanolic biomass extracts, harvested in stationary phase, had mild to moderate antibacterial activity against Bacillus cereus and Staphylococcus aureus. The antifungal effect of the extracts was also evaluated. The aqueous extracts (exopolysaccharides and phycobiliproteins) and ethanolic, chloroformic and hexane extracts of S. subsalsa, did not inhibit the growth of Rhizopus orizae and Aspergillus niger. The toxicity of the extracts was tested using the crustaceans Artemia franciscana and Daphnia magna. The extracts of exopolysaccharides and biomass were found to be innocuous, but the aqueous extracts of phycocyanin showed significant cytotoxic activity against A. franciscana (LC50 exponential phase= 1.69 μg mL-1; LC50 stationary phase= 2.59 μg mL-1). The results suggest that the native strain of S. subsalsa, cultivated in a seawater enriched medium and in a low-cost saline medium has potential to produce bioactive compounds with antimicrobial properties.

Architecture of the Sap S-layer of Bacillus anthracis revealed by integrative structural biology

Bacillus anthracis is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer.

The Impact of Using Laser and Milling Techniques to Create Zirconia Patterns on Streptococcus oralis Biofilm Formation

This study aimed to evaluate zirconia dental implant surfaces patterned using Nd:YAG laser or conventional milling techniques against Streptococcus oralis adhesion and biofilm formation. Zirconia dental implant discs were subjected to surface patterning treatments and categorized into four groups: groove texturing by conventional milling (GM), pore texturing by conventional milling (PM), groove texturing by Nd:YAG laser (GL), and pore texturing by Nd: YAG laser (PL). Streptococcus oralis CECT 907T was cultivated on enriched blood agar plates and then transferred to a brain–heart infusion modified medium and incubated at 37 °C under anaerobic conditions until reaching the exponential growth phase. The bacterial suspension was then seeded on 24-well plates containing the treated discs. The viability of bacteria within the biofilm was determined based on colony-forming unit (CFU) counts, while the total biofilm was quantified by measuring its biomass. A qualitative analysis was conducted using scanning electron microscopy (SEM) images to evaluate the bacterial morphology. The statistical analysis of multigroup comparisons was performed using Kruskal–Wallis test with post hoc pairwise comparison, as well as Mann Whiney U test, with significance set at p < 0.05. After both 1 h and 24 h of incubation of Streptococcus oralis on the discs, all groups showed similar results, with no statistically significant differences (p > 0.05). A comparison of the Nd: YAG laser-treated surfaces with conventionally milled surfaces, as well as grooves versus pores for CFU counts, also revealed no statistically significant differences (p > 0.05) for both 1 h and 24 h of culture. Biomass quantification at both the 1 h and 24-h time points showed similar results across the groups, without statistical differences. When comparing the conventionally machined surfaces to Nd: YAG laser-treated surfaces in terms of biomass, no significant differences were observed (p > 0.05). Similarly, the comparison between groove-patterned surfaces and pore-patterned surfaces showed no statistically significant difference. The groove and pore patterns on zirconia surfaces with Nd: YAG laser or conventional milling did not change the Streptococcus oralis adhesion and biofilm formation behavior. Additional studies are recommended to expand our knowledge in this area.

Sustainable production of a highly pure (R,R)-2,3-butanediol from crude glycerol using metabolically engineered Klebsiella pneumoniae GEM167 strain

BackgroundAmong 2,3-butanediol (2,3-BDO) stereoisomers, (R,R)-2,3-BDO is particularly noteworthy for its application in the agricultural industry. It is an eco-friendly plant immune system stimulant, promoting plant growth and enhancing resistance to biotic and abiotic stresses.ResultsThis study aimed to address the limitations of a previous study, which produced (R,R)-2,3-BDO with only 98% purity despite Kp-dhaD overexpression. First, BLi-gldA demonstrated significantly higher activity and selectivity in converting racemic acetoin to (R,R)-2,3-BDO compared to others among 2,3-BDO dehydrogenases (Kp-dhaD and Kp-gldA from Klebsiella pneumoniae, and BLi-gldA from Bacillus licheniformis). The K. pneumoniae GEM167 ΔadhEΔldhAΔbudC-BLi-gldA/pETM6 strain produced the highest (R,R)-2,3-BDO amount, with 99% purity (73.51 ± 1.69 g/L at 48 h), by isopropyl β-D-1-thiogalactopyranoside addition at the early exponential growth phase (6 h) compared to other cell growth phases. The availability of crude glycerol was investigated, and crude glycerol promoted cell growth resulting in efficient (R,R)-2,3-BDO in the early stage of culture [90.32 ± 1.12 g/L (R,R)-2,3-BDO with 99.0% purity after 60 h]. The productivity and yield remained comparable for crude glycerol (1.51 g/L/h, 0.41 g/g) and pure glycerol (1.53 g/L/h, 0.43 g/g).ConclusionsThis study successfully produced 99% enantiopure (R,R)-2,3-BDO from crude glycerol for the first time using the K. pneumoniae GEM167 ΔadhEΔldhAΔbudC-BLi-gldA/pETM6 strain. (R,R)-2,3-BDO production from crude glycerol, a biodiesel process byproduct, is expected to contribute to a sustainable and circular biomass supply chain and biodiesel production system by positively influencing the stable cultivation of biodiesel crops even under unpredictable climate conditions.Graphical abstract

An object-oriented model of the invasion and eradication of Poa annua L. on King George Island, Antarctica

We implemented a custom object-oriented model to predict the spread of annual bluegrass population at Point Thomas Oasis and to evaluate potential eradication outcome at different eradication success rates. We modeled unobstructed population growth without eradication, sole elimination of plants and removal of plants together with soil within 10cm around each specimen. Both eradication scenarios considered removal efficiency of 50 – 100%. The population with no elimination campaign applied, was still in the exponential phase of growth after 45 simulated years and almost fulfilled the study area. The removal efficiency of 50% sufficed to control the species, maintaining the population number at a stable and low level. Once the removal action ceased, the population number increased dramatically. 100% elimination efficiency for 20 years did not guarantee eradication, as leaving even five or less seeds in the soil could initiate population reestablishment. Soil removal within 10cm around each plant speeded up the elimination. The predicted duration of eradication process to reach 30 or less seeds in the soil, was around 20 years. The removal of annual bluegrass is obstructed by the soil seed bank. The most important parameter driving eradication success is eradication efficiency. Low removal efficiency simulation indicates that poor management, even for a few years, may be amended if the eradication efficiency increases. Our model is transferable for other potential invasive species at Point Thomas Oasis by changing dispersal and germination parameters. Its transferability for other locations would require changing the modeled space.

The Diatom Nanofrustulum shiloi as a Promising Species in Modern Biotechnology

The article presents the results of studies of intensive culture of a new species of bentoplanktonic diatom N. shiloi (Lee, Reimer et McEnery) Round, Hallsteinsen et Paasche 1999 for the Black Sea. The features of the process of isolating the species into an algologically pure culture, as well as the morphological and taxonomic characteristics of the strain in light and electron scanning microscopes are described in detail. The biochemical and production characteristics of the strain were studied, as well as the ability to accumulate fucoxanthin (Fx) and polyunsaturated fatty acids (PUFA) in laboratory conditions. In the exponential growth phase, the specific culture growth rate was µ=0.8 1/day, and the maximum productivity P = 0.46 g dry weight /(L day). The accumulation of PUFAs in the biomass of N. shiloi reached 67.39 mg/g dry weight of algae. The Fx concentration in the biomass at the beginning of the stationary growth phase was 10 mg/g dry weight. The fairly high rate of Fx biosynthesis in microalgae cells, as well as the composition of fatty acids of the Black Sea strain, make it possible to classify N. shiloi as a promising object in biotechnology.

The effect of silicate on polyamine oxidase genes in Skeletonema dohrnii

By using Skeletonema dohrnii as the experimental algal species, we investigated how silicate concentration impacts the polyamine metabolism of diatoms in our experiment. Three different silicate concentrations were set for cultivation, and Skeletonema dohrnii at the exponential growth phase was selected to analyze basic physiological parameters, polyamine content, and Polyamine oxidase (PAO) gene expression under varying silicate concentrations. Results showed that low silicate concentrations led to reduced growth rate and polyamine content, with down-regulation of PAO gene expression. Conversely, high silicate concentrations did not significantly increase growth rate but did increase polyamine content and up-regulate the PAO gene. These findings suggested that changes in silicate concentration impact the growth rate and polyamine content of Skeletonema dohrnii, with the PAO gene potentially playing a role in regulating the algal response to silicate concentration variations. This study provides a foundation for further exploration of the PAO gene function in Skeletonema dohrnii.

In Vivo Quantification of Surfactin Nonribosomal Peptide Synthetase Complexes in Bacillus subtilis.

Surfactin, a potent biosurfactant produced by Bacillus subtilis, is synthesized using a non-ribosomal peptide synthetase (NRPS) encoded by the srfAA-AD operon. Despite its association with quorum sensing via the ComX pheromone, the dynamic behavior and in vivo quantification of the NRPS complex remain underexplored. This study established an in vivo quantification system using fluorescence labeling to monitor the availability of surfactin-forming NRPS subunits (SrfAA, SrfAB, SrfAC, and SrfAD) during bioprocesses. Four Bacillus subtilis sensor strains were constructed by fusing these subunits with the megfp gene, resulting in strains BMV25, BMV26, BMV27, and BMV28. These strains displayed growth and surfactin productivity similar to those of the parental strain, BMV9. Fluorescence signals indicated varying NRPS availability, with BMV27 showing the highest and BMV25 showing the lowest relative fluorescence units (RFUs). RFUs were converted to the relative number of NRPS molecules using open-source FPCountR package. During bioprocesses, NRPS availability peaked at the end of the exponential growth phase and declined in the stationary phase, suggesting reduced NRPS productivity under nutrient-limited conditions and potential post-translational regulation. This study provides a quantitative framework for monitoring NRPS dynamics in vivo, offering insights into optimizing surfactin production. The established sensor strains and quantification system enable the real-time monitoring of NRPS availability, aiding bioprocess optimization for industrial applications of surfactin and potentially other non-ribosomal peptides.

Molecular Basis of Thioredoxin-Dependent Arsenic Transformation in Methanogenic Archaea.

Methanogenic archaea are known to play a crucial role in the biogeochemical cycling of arsenic (As); however, the molecular basis of As transformation mediated by methanogenic archaea remains poorly understood. Herein, the characterization of the redox transformation and methylation of As by Methanosarcina acetivorans, a model methanogenic archaeon, is reported. M. acetivorans was demonstrated to mediate As(V) reduction via a cytoplasmic As reductase (ArsC) in the exponential phase of methanogenic growth and to methylate As(III) via a cytoplasmic As(III) methyltransferase (ArsM) in the stationary phase. Characterization of the ArsC-catalyzed As(V) reduction and the ArsM-catalyzed As(III) methylation showed that a thioredoxin (Trx) encoded by MA4683 was preferentially utilized as a physiological electron donor for ArsC and ArsM, providing a redox link between methanogenesis and As transformation. The structures of ArsC and ArsM complexed with Trx were modeled using AlphaFold-Multimer. Site-directed mutagenesis of key cysteine residues at the interaction sites of the complexes indicated that the archaeal ArsC and ArsM employ evolutionarily distinct disulfide bonds for interacting with Trx compared to those used by bacterial ArsC or eukaryotic ArsM. The findings of this study present a major advance in our current understanding of the physiological roles and underlying mechanism of As transformation in methanogenic archaea.

Azotobacter vinelandii N2 fixation increases in co-culture with the PGPR Bacillus subtilis in a nitrogen concentration-dependent manner.

Reducing inputs of chemical N fertilizers is essential to develop a more sustainable agriculture. The stimulation of biological nitrogen fixation by N2 fixers in multispecies cultures, here the plant growth-promoting rhizobacteria Azotobacter vinelandii and Bacillus subtilis, opens opportunities for the formulation of biofertilizers consortia. While most research on N2 fixation historically focussed on the exponential growth phase of microorganisms, we observed that Bacillus subtilis stimulated Azotobacter vinelandii N2 fixation mostly during the stationary phase. This result highlights that more research on the factors controlling N2 fixation repression during the stationary growth phase, especially bacteria-bacteria interactions, is eagerly needed.

Proteome Profiling of S. cerevisiae Strains Lacking the Ubiquitin-Conjugating Enzymes Ubc4 and Ubc5 During Exponential Growth and After Heat Shock Treatment.

The Ubiquitin-Proteasome System (UPS) governs numerous cellular processes by modulating protein stability and activity via the conjugation of the small protein ubiquitin, either as a single molecule or as linkages with distinct functions. Dysregulation of the UPS has been associated with many diseases, including neurodegenerative and neurodevelopmental diseases, as well as cancer. Ubiquitin-conjugating enzymes (E2s) are important players of the UPS that work together with ubiquitin ligases (E3s) to promote substrate ubiquitylation. In this study, we conduct a comparative proteome-wide abundance profiling of S. cerevisiae cells during the exponential growth phase with and without heat shock treatment. We focus on cells with deletions of the two highly homologous E2s, UBC4 or UBC5, and use isobaric tag-based quantitative mass spectrometry to elucidate differences and similarities in their proteomic profiles. Our analysis revealed that the deletion of Ubc4 has a stronger effect on the proteome compared to the deletion of Ubc5, particularly in exponentially growing cells. In contrast, the effect on the proteome of deleting Ubc5 becomes evident only after heat shock, and even then, it remains minor compared to Ubc4. Furthermore, we identified proteins increasing in the absence of each enzyme, which may represent candidate substrates, potentially contributing to a better understanding of their cellular role.

The detection and utilization of volatile metabolomics in Klebsiella pneumoniae by gas chromatography-ion mobility spectrometry

This research aimed to analyze the volatile compounds emitted during the proliferation of Klebsiella pneumoniae (K. pneumoniae) in the laboratory setting using gas chromatography-ion mobility spectrometry (GC-IMS) and to investigate the potential of volatile metabolomics for detecting carbapenemase-producing strains of K. pneumoniae. The volatile metabolomics of K. pneumoniae were comprehensively analyzed using GC-IMS in tryptic soy broth (TSB) as the culture medium. Afterward, the growth stabilization period (T2) served as the primary time point for analysis, with the introduction of imipenem and carbapenemase inhibitors (avibactam sodium or EDTA) during the exponential growth phase (T0) to further investigate alterations in volatile molecules associated with K. pneumoniae. Standard strains were utilized as references, while clinical strains were employed for validation purposes. At T2, a total of 22 volatile organic compounds (VOCs) associated with K. pneumoniae were identified (3 VOCs found in both monomer and dimer forms). Significant differences in VOCs were observed between carbapenemase-negative and carbapenemase-positive strains, both standard and clinical, following the introduction of imipenem. Furthermore, the addition of avibactam sodium led to distinct changes in the VOC content of strains producing class A carbapenemase, while the addition of EDTA resulted in specific alterations in the volatile metabolic profiles of strains producing class B carbapenemase. GC-IMS demonstrated significant promise for analyzing bacterial volatile metabolomics, and its application in evaluating the volatolomics of K. pneumoniae may facilitate the timely detection of carbapenemase-producing strains.

Adaptive strategies of Listeria monocytogenes: An in-depth analysis of the virulent strain involved in an outbreak in Italy through quantitative proteomics

Despite the general classification of L. monocytogenes strains as equally virulent by global safety authorities, molecular epidemiology reveals diverse subtypes in food, processing environments, and clinical cases. This study focuses on a highly virulent strain associated with a listeriosis outbreak in Italy in 2022, providing insights through comprehensive foodomics approaches, with a specific emphasis on quantitative proteomics. In particular, the ST155 strain of L. monocytogenes strain was subjected in vitro to growth stress conditions (NaCl 2.4 %, pH 6.2, T 12 °C), mimicking the conditions present in the frankfurter, its original source. Then, the protein expression patterns were compared with those obtained in optimal growth conditions.Through quantitative proteomic analysis and bioinformatic assessment, different proteins associated with virulence during the exponential growth phase were identified. This study unveils unique proteins specific to each environment, providing insights into how L. monocytogenes adapts to conditions that are similar to those encountered in frankfurters. This investigation contributes valuable insights into the adaptive strategies of L. monocytogenes under stressful conditions, with implications for enhancing food safety practices.

Rapid growth rate of Enterobacter sp. SM3 determined using several methods

BackgroundBacterial growth rate, commonly reported in terms of doubling time, is frequently determined by one of two techniques: either by measuring optical absorption of a growing culture or by taking samples at different times during their growth phase, diluting them, spreading them on agar plates, incubating them, and counting the colonies that form. Both techniques require measurements of multiple repeats, as well careful assessment of reproducibility and consistency. Existing literature using either technique gives a wide range of growth rate values for even the most extensively studied species of bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This work aims to apply several methods to reliably determine the growth rate of a recently identified species of Enterobacteriaceae, called Enterobacter sp. SM3, and to compare that rate with that of a well-known wildtype E. coli strain KP437.ResultsWe extend conventional optical density (OD) measurements to determine the growth rate of Enterobacter sp. SM3. To assess the reliability of this technique, we compare growth rates obtained by fitting the OD data to exponential growth, applying a relative density method, and measuring shifts in OD curves following set factors of dilution. The main source of error in applying the OD technique is due to the reliance on an exponential growth phase with a short span. With proper choice of parameter range, however, we show that these three methods yield consistent results. We also measured the SM3 division rate by counting colony-forming units (CFU) versus time, yielding results consistent with the OD measurements. In lysogeny broth at 37oC, SM3 divides every 21 ± 3 min, notably faster than the RP437 strain of E. coli, which divides every 29 ± 2 min.ConclusionThe main conclusion of this report is that conventional optical density (OD) measurements and the colony-forming units (CFU) method can yield consistent values of bacterial growth rate. However, to ensure the reproducibility and reliability of the measured growth rate of each bacterial strain, different methods ought to be applied in close comparison. The effort of checking for consistency among multiple techniques, as we have done in this study, is necessary to avoid reporting variable values of doubling time for particular species or strains of bacteria, as seen in the literature.

Modeling sub-exponential epidemic growth dynamics through unobserved individual heterogeneity: a frailty model approach.

Traditional compartmental models of epidemic transmission often predict an initial phase of exponential growth, assuming uniform susceptibility and interaction within the population. However, empirical outbreak data frequently show early stages of sub-exponential growth in case incidences, challenging these assumptions and indicating that traditional models may not fully encompass the complexity of epidemic dynamics. This discrepancy has been addressed through models that incorporate early behavioral changes or spatial constraints within contact networks. In this paper, we propose the concept of "frailty", which represents the variability in individual susceptibility and transmission, as a more accurate approach to understanding epidemic growth. This concept shifts our understanding from a purely exponential model to a more nuanced, generalized model, depending on the level of heterogeneity captured by the frailty parameter. By incorporating this type of heterogeneity, often overlooked in traditional models, we present a novel mathematical framework. This framework enhances our understanding of how individual differences affect key epidemic metrics, including reproduction numbers, epidemic size, likelihood of stochastic extinction, impact of public health interventions, and accuracy of disease forecasts. By accounting for individual heterogeneity, our approach suggests that a more complex and detailed understanding of disease spread is necessary to accurately predict and manage outbreaks.

Oxygen uptake rate analysis to evaluate the impact of hydrodynamic stress on the growth of the avian cell line DuckCelt®-T17

Scale-up of bioprocesses involving animal cell culture is hampered by the sensitivity of the cells to hydrodynamic stress, either from agitation or bubble bursting. Here, the hydrodynamic stress experienced by a recent cell line, the DuckCelt®-T17 avian cells, previously used for viral vaccine production, is investigated in shake flasks and in a 3 L bioreactor. Cell stress was assessed by monitoring the dissolved oxygen in the culture medium, which depends on Oxygen Transfer Rate (OTR) and Oxygen Uptake Rate (OUR) during cultivation. Classical parameters such as the maximum growth rate (µmax) and metabolite profiles were also determined. A dynamic model able to predict nutrient consumption, metabolic waste production, viable cell number and OUR was also developed and validated from the data measured in shake flasks. The experiments performed in the stirred tank bioreactor (STBR) show that OUR depended on both the cell growth phase and the stirring conditions. The oxygen consumption of the cells during the exponential growth phase (where there were no nutrient and O2 limitations) was significantly altered at average and maximum shear rates above 70 and 840 s−1, respectively, indicating highly shear-sensitive cells. OUR is a suitable tool to identify the hydrodynamic conditions for robust cell growth. The scale-up criteria to be favored for the DuckCelt®-T17 cell culture in STBRs would be the shear and/or the tip’s speed.