Bacterial and fungal growth in total parenteral nutrition solutions

The most serious complication of prolonged intravenous infusion of hypertonic dextrose and amino acids is infection. Frequently, the etiology is fungal rather than bacterial. Previous authors have suggested that bacterial survival and growth in the solutions is suppressed by (a) high dextrose concentration, (b) high osmolality, or (c) low pH. This paper presents evidence that proposals (a) and (b) are untenable and (c) is only partly responsible. We call attention to the presence of a factor that is antibacterial but not antifungal; namely, a high concentration of glycine.     

Structure of a novel highly branched α-glucan enzymatically produced from maltodextrin

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility α-glucan containing a large amylase-resistant portion. This α-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility α-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The α-glucan was found to be a novel highly branched α-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.     

Immunological comparison of the b and c1 cytochromes from bovine heart mitochondria and the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Antibodies against cytochromes b and c1 of bovine heart mitochondria and the photosynthetic bacterium, Rhodopseudomonas sphaeroides R-26, were raised in rabbits. The purified antibodies showed high titers against their respective antigens in enzyme-linked immunosorbent assays. Less than 15% cross-reactivity between the mitochondrial and bacterial cytochromes was detected. Although antibodies against mitochondrial cytochrome b did not inhibit the mitochondrial cytochrome b-c1 complex, a 70% inhibition was obtained when these antibodies were incubated with delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids indicating that the catalytic site(s) of mitochondrial cytochrome b are masked by phospholipids. On the other hand, antibodies against bacterial cytochrome b showed significant inhibition of the intact bacterial cytochrome b-c1 complex, indicating that some of the catalytic site epitopes of bacterial cytochrome b are exposed to the hydrophilic environment. Similar to antibodies against mitochondrial cytochrome b, antibodies against bacterial cytochrome b inhibited 50% activity of the mitochondrial cytochrome b-c1 complex only when they were incubated with the delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids, indicating that the common epitopes between the cytochromes b are masked by phospholipids. Antibodies against mitochondrial and bacterial cytochromes c1 completely inhibited their respective cytochrome b-c1 complexes but no cross-immunoinhibition was observed. However, when antibodies against bacterial cytochrome c1 were incubated with the delipidated mitochondrial cytochrome b-c1 complex before reconstitution with phospholipids, a 65% inhibition was observed, indicating that the common epitopes between the cytochromes c1 were also somewhat masked by phospholipids. Antibodies against mitochondrial cytochrome c1 inhibited 70% of the succinate oxidase activity in the intact mitochondria preparation, but no inhibition was observed in submitochondrial particles, indicating that some mitochondrial cytochrome c1 epitopes are exposed to the cytoplasmic side.     

CD11c expression identifies a population of extrafollicular antigen-specific splenic plasmablasts responsible for CD4 T-independent antibody responses during intracellular bacterial infection

Although T-independent immunity is known to be generated against bacterial capsular and cell wall polysaccharides expressed by a number of bacterial pathogens, it has not been studied in depth during intracellular bacterial infections. Our previous study demonstrated that Ehrlichia muris, an obligate intracellular tick-borne pathogen, generates protective classical TI responses in CD4 T cell-deficient C57BL/6 mice. We found that E. muris T-independent immunity is accompanied by the expansion of a very large extrafollicular spleen population of CD11c(low)-expressing plasmablasts that exhibit characteristics of both B-1 and marginal zone B cells. The plasmablasts comprised up to 15% of the total spleen lymphocytes and approximately 70% of total spleen IgM(high)IgD(low) cells during peak infection in both wild-type and MHC class II-deficient mice. The CD11c(low) cells exhibited low surface expression of B220, CD19, and CD1d, high expression of CD11b, CD43, but did not express CD5. Approximately 50% of the CD11c(low) cells also expressed CD138. In addition to CD11b and CD11c, the plasmablasts expressed the beta(1) (CD29) and alpha4 (CD49d) integrins, as well as the chemokine receptor CXCR4, molecules which may play roles in localizing the B cells extrafollicular region of the spleen. During peak infection, the CD11c(low) cells accounted for the majority of the IgM-producing splenic B cells and nearly all of the E. muris outer membrane protein-specific IgM-secreting cells. Thus, during this intracellular bacterial infection, CD11c expression identifies a population of Ag-specific spleen plasmablasts responsible for T-independent Ab production.     

Mini-review of the concentration variations found inthe alfresco atmospheric bacterial populations

Variations in the atmospheric surface layer's culturable, and to a lesser extent, total bacteria-associated atmospheric particlecharacteristics will be discussed in terms of (a)their temporal variation from 2 min resolutionthrough diurnal to annual periods, (b) the effect ofmeteorological conditions on their abundance andsize, (c) total to culturable bacterial ratio, and(d) the total number of bacteria per culturableparticle (e) bacterial survival in droplet/particles, and (f) the general particle size distribution including aerodynamic Count MedianDiameter (CMD).Meteorological and topographic conditions thatcontrol total and culturable bacteria-associatedatmospheric particle concentration will be presentedin terms of (a) precipitation, (b) wind direction,(c) time of day, (d) sky conditions (i.e., cloudy,sunny, rain, etc.), (e) season, and (f) atmosphericinversion conditions.Simulation models will be described that supporthypotheses of diurnal and annual concentrationcycles in the Earth's (and perhaps other planetaryatmospheres) atmospheric surface layer.     

Cytochromes inAzospirillum brasilense

Difference spectra of the crude cell-free extract ofAzospirillum brasilense sp. 7 indicate the presence of cytochrome b, cytochrome c, and one CO-binding pigment that exhibits the spectral characteristics of cytochrome o. All the pigments are present in varying concentrations at all stages of growth. With progress of the bacterial growth, there is a linear increase in the level of cytochrome b with a disproportionate increase in the level of cytochrome c. At the stationary phase, the amount of cytochrome b and c is increased by about sevenfold compared with that in the early log phase. The increase in the concentration of total cytochrome is not accompanied by an increase in the respiration rate of the cells. Both cytochrome b and cytochrome c are located in the particulate fraction of the cells and are not fully reducible by succinate alone.     

TNF-alpha-dependent and -independent maturation of dendritic cells and recruited CD11c(int)CD11b+ Cells during oral Salmonella infection

Maturation of dendritic cells (DC) is crucial for their ability to induce adaptive immunity. Although several mediators of DC maturation have been found, their contributions to DC maturation during infection are poorly understood. In this study we show that murine conventional (CD11c(high)) DC up-regulate costimulatory molecules in a subset-specific manner after oral Salmonella infection. Although both CD8alpha+ and CD8alpha- subsets increase CD86 expression, CD40 was preferentially up-regulated on CD8alpha+ DC, and CD80 was preferentially increased on CD8alpha- DC. In addition, high levels of CD80 and CD86 were found on CD11c(int)CD11b+ cells that accumulated in infected organs. Costimulatory molecules were simultaneously induced on CD11c(high) and CD11c(int)CD11b+ cells in Peyer's patches, mesenteric lymph nodes and spleen 5 days after infection despite different kinetics of peak bacterial burden in these organs. Up-regulation of costimulatory molecules occurred on all DC within the respective subset. Moreover, <1% of CD11c-expressing cells associated with Salmonella expressing enhanced GFP in vivo. Thus, DC maturation did not depend on bacterial uptake. Rather, infection-induced up-regulation of CD80, CD86, and CD40 on CD11c-expressing cells of mesenteric lymph nodes was dependent on TNFR type I (TNFRI) signaling. Although indirect up-regulation of costimulatory molecules on DC and CD11c(int)CD11b+ cells was TNFRI dependent, cells directly associated with Salmonella were able to mature independently of TNFRI signaling. Thus, Salmonella-induced TNF-alpha is an important mediator of indirect DC maturation during infection, whereas a TNF-alpha-independent maturation pathway contributes to direct maturation of bacteria-associated DC.     

Effects of dextrose, verapamil and magnesium during hypoxia in myocardial tissue

We studied the effects of superfusion of canine heart muscle tissue with a solution that mimicks hypoxia, acidosis and hyperkalemia (altered Tyrode's solution). Contracture (rise in resting tension) develops much sooner (5.2 +/- 0.8 vs. 30-40 min in 5 mM dextrose) in the absence of dextrose. High dextrose (55 mM) stabilizes the rise in tonic tension and protects against the action potential shortening during such superfusion. Presence of verapamil (1-1.5 microM) during altered Tyrode's superfusion considerably lessens the magnitude of the increase in tonic tension (31.7 +/- 8.6 vs. 129.5 +/- 32.6 mg in the control). Presence of high magnesium (5 mM) during altered Tyrode's superfusion also offers some protection against tonic tension increase (12.6 +/- 3.6 mg rise in tonic tension vs. 129.5 +/- 3.2 mg in the control), action potential shortening, and amplitude decrease. These results suggest that (a) magnesium and verapamil both have significant effects on the cellular calcium uptake, and (b) anaerobic metabolism utilizing either glycogen or exogenous glucose is capable of preventing contracture during ischemia.     

Mg2+ as an indicator of nutritional status in marine bacteria

Cells maintain an osmotic pressure essential for growth and division, using organic compatible solutes and inorganic ions. Mg²⁺, which is the most abundant divalent cation in living cells, has not been considered an osmotically important solute. Here we show that under carbon limitation or dormancy native marine bacterial communities have a high cellular concentration of Mg²⁺ (370–940 m) and a low cellular concentration of Na⁺ (50–170 m). With input of organic carbon, the average cellular concentration of Mg²⁺ decreased 6–12-fold, whereas that of Na⁺ increased ca 3–4-fold. The concentration of chlorine, which was in the range of 330–1200 m and was the only inorganic counterion of quantitative significance, balanced and followed changes in the concentration of Mg²⁺+Na⁺. In an osmotically stable environment, like seawater, any major shift in bacterial osmolyte composition should be related to shifts in growth conditions, and replacing organic compatible solutes with inorganic solutes is presumably a favorable strategy when growing in carbon-limited condition. A high concentration of Mg²⁺ in cells may also serve to protect and stabilize macromolecules during periods of non-growth and dormancy. Our results suggest that Mg²⁺ has a major role as osmolyte in marine bacteria, and that the [Mg²⁺]/[Na⁺] ratio is related to its physiological condition and nutritional status. Bacterial degradation is a main sink for dissolved organic carbon in the ocean, and understanding the mechanisms limiting bacterial activity is therefore essential for understanding the oceanic C-cycle. The [Mg²⁺]/[Na⁺]-ratio in cells may provide a physiological proxy for the transitions between C-limited and mineral nutrient-limited bacterial growth in the ocean''s surface layer.     

Biochemical and motile properties of Myo1b splice isoforms

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. Myo1b is alternatively spliced within the regulatory domain of the molecule, yielding isoforms with six (myo1b(a)), five (myo1b(b)), or four (myo1b(c)) non-identical IQ motifs. The calmodulin binding properties of the myo1b IQ motifs have not been investigated, and the mechanical and cell biological consequences of alternative splicing are not known. Therefore, we expressed the alternatively spliced myo1b isoforms truncated after the final IQ motif and included a sequence at their C termini that is a substrate for bacterial biotin ligase. Site-specific biotinylation allows us to specifically attach the myosin to motility surfaces via a biotin-streptavidin linkage. We measured the ATPase and motile properties of the recombinant myo1b splice isoforms, and we correlated these properties with calmodulin binding. We confirmed that calcium-dependent changes in the ATPase activity are due to calcium binding to the calmodulin closest to the motor. We found that calmodulin binds tightly to some of the IQ motifs (Kd < 0.2 microM) and very weakly to the others (Kd > 5 microM), suggesting that a subset of the IQ motifs are not calmodulin bound under physiological conditions. Finally, we found the in vitro motility rate to be dependent on the myo1b isoform and the calmodulin concentration and that the myo1b regulatory domain acts as a rigid lever arm upon calmodulin binding to the high affinity and low affinity IQ motifs.     

Modification of the Microtiter Technique for Antimicrobial Drug Susceptibility Testing by Incorporation of Indicators

A pH indicator and dextrose were incorporated into growth media as a modification of microbial microtiter methods for determining the minimal inhibitory concentration of antimicrobial drugs. This modified method was tested to evaluate the ease of reading end points by changes in the indicator color. Application of the procedure to two media, three indicators, and eight species of bacteria indicated that definitive end points could be reached as a result of indicator color change caused by acid production during bacterial growth. This method is accurate and reproducible. It is a modification which eliminates a need for plating and facilitates the reading of minimal inhibitory concentration end points.     

Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen

Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

Production of Eicosapentaenoic Acid (20:5 n-3) by Vibrio pelagius Isolated from Turbot (Scophthalmus maximus (L.)) Larvae

Fourteen bacterial strains isolated from turbot, Scophthalmus maximus (L.), larvae were screened for eicosapentaenoic acid (20:5 n-3) (EPA) production. Gas chromatography analysis revealed that one bacterial species, Vibrio pelagius, contained a high proportion of EPA in cellular lipid. This finding was confirmed by gas chromatography-mass spectrometry analysis. A higher concentration of EPA was detected when the bacterium was cultured at 4°C.     

Bacterial counts in canine duodenal fluid after exposure to saline, sodium bicarbonate and hypertonic dextrose solutions used to maintain patency of chronically implanted catheters

Flushing of intestinal vascular access ports (VAPs) is commonly performed to prevent the problems of blockage and infection, and in this study four different flushing solutions were compared. The growth of bacteria from canine duodenal contents was compared in: 0.9% saline, 50% dextrose, 8.4% sodium bicarbonate (NaHCO3) and 0.01 M phosphate buffered saline (PBS). Duodenal contents from three laboratory beagles were serially diluted in these four solutions, spread plated onto agar at 24 h periods for 7 days and bacterial counts were performed. Immediately after the duodenal juices were added, no significant differences could be seen in bacterial counts with any of the solutions. Over the 7 day period, bacterial numbers greatly increased in saline and phosphate buffered saline, but greatly decreased in dextrose and sodium bicarbonate solutions. Dextrose and sodium bicarbonate appeared to be the most promising flushing solutions tested to minimize infections of associated intestinal VAPs.     

Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

BackgroundCirculating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.MethodsWe measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.ResultsThe average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005).ConclusionsCirculating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.     

Bacterial Enrichment at the Gas-Water Interface of a Laboratory Apparatus

The gas-water interface (GWI) is likely to have important effects on bacterial adsorption and transport in unsaturated porous media. A glass apparatus that isolated GWIs in ports above a flowthrough suspension of a groundwater bacterial isolate was used to represent unsaturated porous media. The surface microlayer was collected by placing a polycarbonate filter on the GWI. The filter was stained, and the bacteria were enumerated by direct count. The significance of five independent variables on the surface density of cells (s, in cells per square millimeter) was determined by nonlinear multiple regression. Three of the variables were shown to be significant: surfactant concentration (d), time (t), and bulk bacterial concentration (B). The surface density decreased with increasing d and increased with increasing t and B. When surfactant was absent, the GWI became highly enriched in bacteria. For example, when d = 0, 48 h < t < 72 h, and 5,000 cells mm(sup-3) < B < 10,000 cells mm(sup-3), s averaged 3.0 x 10(sup4) cells mm(sup-2). This surface density occupied about 6.0% of the GWI, and the surface microlayer concentration of cells was 190 times the bulk concentration. The other two variables: pH (p) and ionic strength (I) were shown to be insignificant. The strong effect of d and the lack of effect of I and p support the hypothesis that hydrophobic interaction dominates bacterial adsorption to the GWI.     

High vectorial proton stoichiometry by cytochrome c oxidase from the thermophilic bacterium PS3 reconstituted in liposomes

The stoichiometry of vectorial H+ ejection, coupled to ferrocytochrome c oxidation by a three-subunit bacterial cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3, was measured. Three methods of measuring the H+/e- ratio were applied to proteoliposomes containing a relatively small amount of PS3 cytochrome oxidase, which showed a relatively low oxidation rate and a very low H+ leakage, as follows: (a) simultaneous measurements of H+ ejection and cytochrome c oxidation upon addition of a yeast ferrocytochrome c pulse, which enable us to calculate the H+/e- ratio as H+ ejected per cytochrome c oxidized; (b) computer simulations to find out the fit for the pH meter trace by changing the H+/e- ratio and the velocity constant of leakage; and (c) two successive measurements of initial rates of H+ movement in the absence and presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The H+/e- ratios obtained were 1.39, the 10-s value after ferrocytochrome c addition in (a), 1.35 in (b), and 1.33 in (c). This high H+/e- stoichiometry observed, exceeding 1 and as high as 1.4, is discussed with respect to the controversy of the H+/e- ratio at the cytochrome oxidase site.