Inter-species differences in the growth of bifidobacteria cultured on human milk oligosaccharides

Human milk (HM) contains as the third most abundant component around 200 different structures of human milk oligosaccharides (HMOs). HMOs are the first and irreplaceable prebiotics for infants, supporting bifidobacteria as the most important bacterial group in an infant intestine. The aim of our study was to test the growth of bifidobacteria in HM and on HMOs. Bifidobacteria were isolated from two groups of infants. The first one (eight strains) were isolated from infants who had bifidobacteria in their feces but, after a short period of time (4 to 24 days), bifidobacteria were no longer detected in their feces (disappeared bifidobacteria [DB]). The second group of bifidobacteria (eight strains) originated from infants with continual presence of bifidobacteria in their feces (persistent bifidobacteria [PB]). There were significant differences (p?Bifidobacterium bifidum and B. breve species were able to utilize HMOs, while B. adolescentis and B. longum subsp. longum species did not. The ability to grow in HM and to utilize HMOs seem to be important properties of bifidobacteria which are able to colonize infant intestinal tract.     

Specific growth rate of bifidobacteria cultured on different sugars

The ability of six bifidobacterial strains (3 of human origin and 3 isolates from fermented milk products) to utilize glucose, lactose, melezitose, sucrose, raffinose, and stachyose was determined. Dairy-related bifidobacterial strains were identified asBifidobacterium animalis (2 strains) or asB. pseudolongum (1 strain). Human strains includedB. longum (2 strains) andB. breve (1 strain). All strains fermented lactose, sucrose, raffinose, and stachyose. Melezitose was utilized only byB. longum. B. pseudolongum did not ferment either glucose or melezitose. All isolates had a higher specific growth rate on raffinose and stachyose than on glucose. Dairy strains grew slowly on glucose compared to human strains.     

A simple model to describe transient differences between cell number and biomass growth rates of Escherichia coli

Information on the response of a microbial culture to dynamic environmental conditions is necessary for the design of transient operation processes. However, most attempts at modelling culture response have been directed at describing the steady-state behavior. Thus, there is a need for adequate dynamic models for process design. Simulations of nutrient shifts were completed using a "single-cell" model for Escherichia coli. It was discovered that the specific mass growth rate and the specific number of cells growth rate were different under transient conditions, whereas at steady state (balanced growth) these rates are equivalent. Using these observations, a simple delay model to describe the transient behavior of the two growth rates is formulated and tested. The model contains as state variables only the readily measurable macroscopic quantities (biomass, cell number, and limiting nutrient). This model agreed well with the predictions of the single-cell model.     

Regulatory effects of bifidobacteria on the growth of other colonic bacteria

In the human large intestine bifidobacteria are a numerically important group of micro-organisms which are considered to exert a range of biological activities related to host health. One aspect is the inhibitory effect of these bacteria on other species, possibly excluding long term colonization by invasive pathogens. It has been suggested that the mechanism of inhibition carried out by bifidobacteria is related to the fermentative production of acids such as acetate and lactate. Experiments reported in this paper attemptedto address this theory. Co-culture experiments whereby Bifidobacterium infantis was incubated with Escherichia coli and Clostridium perfringens, in a varietyof fermentation systems, indicated that the bifidobacterium was able to exert an inhibitory effect not necessarily related to acid production. Further studies showed that eight species of bifidobacteria could variously excrete an anti-microbial substance with a broadspectrum of activity. Species belonging to the genera Salmonella, Listeria, Campylobacter and Shigella, as well as Vibrio cholerae, were all affected. These results show that bifidobacteria are able to exert more than one mechanism of inhibition, which may be of some importance with regard to protection against gastroenteritis.     

Comparative growth rates and yields of ciliates and heterotrophic dinoflagellates

Growth rates, ingestion rates and grazer yields (grazer volumeproduced/prey volume consumed) were measured for six protozoanspecies (ciliates: Favella sp., Strombidinopsis acuminatum,Uronema sp.; heterotrophic dinoflagellates: Amphidinium sp.,Gymnodinium sp., Noctiluca scintillans) in laboratory batchculture experiments. Comparative growth data indicate that theprymnesiophyte Isochrysis galbana, the prasinophyte Mantoniellasquamata, two cryptophyte species and several autotrophic dinoflagellatespecies were suitable foods for these grazers. When grown onoptimized diets at 13C, maximum ciliate growth rates (range0.77–1.01 day^–1 uniformly exceeded maximum heterotrophicdioflagellate growth rates (range 0.41–0.48 day^–1).A compilation of published data demonstrates that this growthrate difference persists across a range of ciliate and dinoflagellatetaxa and cell sizes. Comparison of volume-specific ingestionrates and yields for the six species studied here showed thatthere was no single explanation for this growth rate disparity.Heterotrophic dinoflagellates exhibited both low ingestion ratesand, in one case, low yields; ciliates were able to achievehigher growth rates via either higher ingestion rates or higheryields, depending on ciliate species. Volume yield increasedover time throughout the exponential growth phase in nearlyall experiments, suggesting variation in response to changingfood concentrations or long-term acclimation to culture conditions.Higher maximum ciliate growth rates mean that these grazershave the potential to exercise tighter control over incipientblooms of their prey than do heterotrophic dinoflagellates.     

Effect of specific growth rates on productivity in continuous open and partial cell retention animal cell bioreactors.

A clonal derivative of a transfectant of the SP2/0 myeloma cell line producing a chimeric monoclonal antibody was cultivated in both continuous open and continuous partially-closed bioreactors. Using an open system for the determination of kinetic parameters, we showed that the production of this chimeric mAb was growth associated. As such, the volumetric productivity increased linearly with increasing dilution rate up to the maximum dilution rate. Three continuous cultivations employing partial cell retention were conducted. In agreement with mathematical predictions, the product titer and volumetric productivity were independent of the degree of cell retention when the total dilution was held constant. When cells were maintained at a low specific growth rate, the product titer was independent of dilution rate and the volumetric productivity increased with increasing dilution rate, again in agreement with mathematical predictions. Since the partially-closed bioreactor could be operated at dilution rates in excess of the maximum specific cellular growth rate, volumetric productivities were greater than those achievable in the open bioreactor. However, when cells were maintained at a high specific growth rate, cell accumulation was limited and product titers decreased at high dilution rates. Therefore, the volumetric productivity in this latter case did not increase at higher dilution rates.     

Interrelationship among specific rates of cell growth,substrate consumption,and metabolite formation in some simple microbial reactions producing primary metabolites

Applying the carbon balance principle, the interrelationship between ν = μ/Y + m (μ is the specific growth rate of microorganism, v is the specific substrate consumption rate) and π = Aμ B (Luedeking–Piret eqyuation, π is the specific metabolite formation rate) has been established for three types of simple microbial reactions. Equations for the kinetic parameters A and B have been proposed for each of the three types of microbial reactions, Expresses in terms of γ_x, γ_s and γ_p (carbon contents of dry cell, mass, major carbon energy source, and metabolite) as well as the parameters Y and m. Values of both A and B calculated from the proposed equations were compared with their experimental data for lactic acid fragmentation, aerobic SCP production, and alcohol fermentation. The estimated values agreed with the observed ones with reasonably small deviations.     

Growth of infant faecal bifidobacteria and clostridia on prebiotic oligosaccharides in in vitro conditions

Our aim was to isolate bifidobacteria and clostridia from infant faeces and to test the growth of bifidobacteria and clostridia on prebiotic oligosaccharides. Seventy breast-fed infants aged between 3 and 253 days were tested for the presence of bifidobacteria and clostridia in their faeces. Ten strains of clostridia and 10 strains of bifidobacteria were isolated from infant faecal samples. Four strains of bifidobacteria originated from culture collections and 1 strain from fermented milk product were also tested. Subsequently, bacterial isolates were tested for their growth on prebiotic oligosaccharides in, in vitro conditions. Forty-six infants exhibited high numbers of bifidobacteria (usually higher than 9 logCFU/g) in their faeces. There were undetectable amounts of bifidobacteria in faecal samples in 24 of the studied infants (34%), these babies on the other hand possessed significant amounts of clostridia in their faecal flora. Both bifidobacteria and clostridia utilized all substrates tested. Bifidobacteria grew significantly better in the medium with galactooligosaccharides. Higher growth of clostridia was observed on raffinose and lactulose. Conversely, bifidobacteria grew slightly better in the medium with stachyose, inulin, Raftilose P85 and P95. However, these differences were not significant. Our results suggest that commercially available prebiotics support the growth of infant faecal clostridia. It is therefore questionable if bifidobacteria-deficient infants should be supplemented with prebiotics.     

Life-history correlates of maximum population growth rates in marine fishes

Theory predicts that populations of animals with late maturity, low fecundity, large body size and low body growth rates will have low potential rates of population increase at low abundance. If this is true, then these traits may be used to predict the intrinsic rate of increase for species or populations, as well as extinction risks. We used life-history and population data for 63 stocks of commercially exploited fish species from the northeast Atlantic to test relationships between life-history parameters and the rate of population increase at low abundance. We used cross-taxonomic analyses among stocks and among species, and analyses that accounted for phylogenetic relationships. These analyses confirmed that large-bodied, slow-growing stocks and species had significantly lower rates of recruitment and adult production per spawning adult at low abundance. Furthermore, high ages at maturity were significantly correlated with low maximum recruit production. Contrary to expectation, fecundity was significantly negatively related to recruit production, due to its positive relationship with maximum body size. Our results support theoretical predictions, and suggest that a simply measured life-history parameter can provide a useful tool for predicting rates of recovery from low population abundance.     

Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis

Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.     

Clonal variation in maximum specific growth rate and susceptibility towards antimicrobials

AIMS: To examine associations between growth rate within bacterial populations and survival patterns following treatment with antimicrobial agents. METHODS AND RESULTS: Time survival data were generated for the inactivation of Escherichia coli populations, grown as batch and continuous cultures, exposed to ciprofloxacin, benzalkonium chloride and tetracycline. Time-survivor plots were biphasic. Surviving cells were collected and immediately re-exposed to agent or were regrown and then re-exposed. Survivors were resistant to immediate challenge with any of the treatment agents. This resistance was lost on regrowth suggesting that survival reflects an expressed phenotype within a subset of the culture (persisters) rather than individual resistant clones or nonspecific quenching of the test agent. The fraction of persisters increased with decreasing growth rate when cultures were prepared in continuous culture. CONCLUSIONS: Clonal growth rates within populations were determined by culture of individual cells within microtitre plate wells. The fraction of clones, in batch cultures, growing maximally at rates below the apparent threshold for susceptibility to the test agents was sufficient to explain the results of continuous culture experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of persisters in populations of bacteria relate to small subset of cells that are growing only slowly or are metabolically quiescent.     

Comparison of maximum specific growth rates and lag times estimated from absorbance and viable count data by different mathematical models

Maximum specific growth rate (mu(max)) and lag time (lambda) were estimated from viable count and absorbance data and compared for different microorganisms, incubation systems and growth conditions. Data from 176 growth curves and 120 absorbance detection times of serially diluted cultures were evaluated using different mathematical growth models. Accurate estimates of mu(max) and lambda were obtained from individual absorbance growth curves by using the Richard model, with values of the parameter m fixed to 0.5, 1.0 or 2.0 to describing different degrees of growth dampening, as well as from absorbance detection times of serially diluted cultures. It is suggested to apply the two techniques complementarily for accurate, rapid and inexpensive estimation of microbial growth parameter values from absorbance data. In contrast, considerable limitations were demonstrated for the ability of the Exponential, the Gompertz and the Logistic models to estimate mu(max) and lambda values accurately from absorbance data. Limitations of these models were revealed due the wide range of growth conditions studies.     

Respiration rates predict differences in growth of coast redwood

The relation between growth rate traits (height, basal diameter, stem volume and branch diameter) and two measures of respiration rate [metabolic heat rate (q) and CO₂ production rate (Rco₂)] and their ratio (q/R_co2) was examined on a collection of 192 different genotypes of coast redwoods [Sequoia sempervirens (D.Don) Endl.]. Branch diameter was not correlated with any of the respiratory measures, but the other three growth traits gave highly significant (P < 0.001) correlations with positive slopes. Combining the four growth traits and the three respiratory variables (q, R_Co₂ and q/R_co2) to give two canonical variates, one representing growth and one representing respiration, gives an even stronger linear correlation (r= 0–85). These data suggest that simultaneous assay of multiple respiratory measures on juvenile trees can be used to predict their longer-term growth rates.     

Effects of myostatin and other growth factors on cultured human cells

A dedicated cell-based biological test system was used for studying specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.     

Sex-related differences in developmental rates of bovine embryos produced and cultured in vitro.

The classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex-determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex-related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.     

Control of cell division in the yeast Saccharomyces cerevisiae cultured at different growth rates

Saccharomyces cerevisiae has been grown with different generation times by alterations in media richness and by altering the flow rate of the limiting nutrient, glucose in a chemostat. Within the generation time range 2.89-approx. 8.0 h the time from the initiation of DNA synthesis to cell division was independent of generation time and was approx. 2 h. Thus the cell cycle of yeast can be divided into an expandable phase from cell division to the initiation of DNA synthesis, the length of which is dependent on growth rate and a constant phase from the initiation of DNA synthesis to cell division which takes a constant time independent of generation time. In cells growing with generation times longer than 8.6 h this constant phase expands somewhat in time. These results are reminiscent of the observation that in the bacterium Escherichia coliB/R the time from initiation of DNA synthesis to cell division is constant except at very slow growth rates.     

Quantitative dissection of clone-specific growth rates in cultured malaria parasites

Measurement of parasite proliferation in cultured red blood cells underpins many facets of malaria research, from drug sensitivity assays to assessing the impact of experimentally altered genes on parasite growth, virulence and fitness. Pioneering efforts to grow Plasmodium falciparum in cultured red blood cells revolutionised malaria research and spurred the development of semi-high-throughput growth assays using radio-labelled hypoxanthine (Hx), an essential nucleic acid precursor, as a reporter of whole-cycle proliferation [Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673–675; Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718]. The isotopic Hx assay remains the standard quantitative growth assay with which newer non-radioactive procedures based on fluorescent DNA dyes or ELISA are compared. All of these readouts are surrogate reporters of changes in bulk parasitemias, reflecting proliferation over entire asexual reproductive cycles. While quantitatively robust and amenable to semi-high-throughput applications, these methods are blind to the underlying developmental and cellular events of growth in human red blood cells. Modern whole-genome tools including gene knockouts, mutagenesis and small molecule screens promise to reveal much about basic parasite biology; however methods to precisely quantify the within-cycle growth process are needed. Here we elaborate on the classical growth index, i.e. changes in parasitemia, by quantifying sub-phenotypes of a rapid proliferator, the multi-drug resistant clone Dd2, and a standard wild-type clone, HB3. These data illustrate differences in cycle duration, merozoite production, and invasion rate and efficiency that underpin Dd2’s average 2-fold proliferation advantage over HB3 per erythrocytic cycle. The ability to refine growth phenotypes will inform the search for molecular determinants of differential parasite growth rates and broaden our understanding of killing mechanisms and cellular targets of antimalarial drugs.