<Emphasis Type="Italic">In Vitro</Emphasis> antibacterial effects of antilipopolysaccharide DNA aptamer-C1qrs complexes

DNA aptamers were developed against lipopolysaccharide (LPS) from E. coli O111:B4 and shown to bind both LPS and E. coli by a colorimetric enzyme-based microplate assay. The polyclonal aptamers were coupled to human C1qrs protein either directly using a bifunctional linker or indirectly using biotinylated aptamers and a streptavidin-C1qrs complex. Both systems significantly reduced colony counts when applied to E. coli O111:B4 and K12 strains across a series of 10x dilutions of the bacteria in the presence of human serum; it was diluted 1: 10(3) in order to avoid significant bacterial lysis by the competing alternate pathway of complement activation. A number of candidate DNA aptamer sequences were cloned and sequenced from the anti-LPS aptamer library for future screening of antibacterial or "antibiotic" potential and to aid in eventual development of an alternative therapy for antibiotic-resistant bacterial infections.     

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine''s sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.     

Counting viable Azotobacter in vertisols

In counting Azotobacter in vertisols by the soil dilution and spread-plating method, mean colony counts/plate did not decrease in proportion to the dilution factor and consequently derived counts of Azobacter cells/g soil decreased with increasing dilution of the soil suspension. This non-proportionality phenomenon was analysed in several experiments with six soils. Conformity to the Poisson distribution for counts on parallel plates was measured by Fisher's index of dispersion (χ²), which proved too high (P<.05) at lower dilutions, indicating inaccurately low mean colony counts. At highest dilutions, proportional errors increased resulting in less precise estimates of means, because with a Poisson distribution the standard deviation is equal to the square root of the mean, and the multiplication factor for derived counts/g soil is greatest at highest dilutions. By varying both plate surface area and dilution factor, results indicated that the non-proportionality phenomenon is caused by crowding at lower dilutions increasing the probability of colony coincidence on the plates. Graphical analysis of results of several dilution series indicated that derived counts are best based on dilutions giving 10–40 colonies/9 cm diameter plate, which is best achieved from two-fold dilution series.     

A PRELIMINARY BACTERIOLOGICAL SCREENING TEST FOR QUATERNARY AMMONIUM COMPOUNDS AND FORMULATIONS

SUMMARY: The test is based on a colony count method to determine the survival of a given test organism, Bacterium coli , in the presence of sterile homogenized whole milk added with the bacterial suspension to the quaternary ammonium compound dilutions, for an exposure time of two min.
Attention is drawn to the special cleaning of the glassware used. The preparation and use of the Q.A.C. inhibitor, lecithin in 'Lissapol N', is described, with details of the testing procedure.
Inconsistencies in survivor counts occurring when Q.A.C. solutions were tested against saline suspensions of Bact. coli were reduced by carrying out the test in the presence of whole milk solids.
Examples quoted show how this test indicated a difference in bactericidal efficiency between (a) two different Q.A.C. solutions, and (b) the same Q.A.C. included in two powder formulations of differing pH value.     

ROLL TUBES AND AGAR STRIP TUBES FOR THE BACTERIAL COLONY COUNT OF MILK

SUMMARY: Roll-tube colony counts, using the Astell equipment, were lower than the corresponding Petri dish counts with 27 out of 31 raw milks (87%). The difference between the counts by the two methods was greater than 25% of the plate count for 12 (39%) of the samples.
When the same dilution of milk was used for both strip-tube and plate colony counts, about equal numbers of samples gave counts from the strip tubes above and below about the colony count from plates. When, in order to obtain a more reasonable strip-tube count, the plates and strip tubes were prepared from different dilutions of the milk, the counts from the latter were, with only 3 exceptions out of 35 milks, below those from the former. The difference between the counts was greater than 25% of the plate count for 15 (43%) of the milks, a figure similar to that obtained in comparing roll-tube and plate colony counts.     

MODELING OF SWEET, BITTER AND IRRITANT SENSATIONS AND THEIR INTERACTIONS ELICITED BY MODEL ICE WINES

Interactions between taste and irritant sensations elicited by model ice wine solutions were investigated, including the use of U and Γ′ models for predicting the perceived intensity of these sensory interactions. Fifteen solutions of varying ethanol and sugar concentrations representative of commercial ice wine values were evaluated in two trials by a trained sensory panel (n = 12) for perceived sweetness, bitterness and heat intensities. Sweetness perception of lower sugar‐concentration level in ice wine model solution was affected by ethanol concentration. The sweetness intensities of the sugar and ethanol mixtures are higher than the sweetness intensities of sugar solutions. The Γ′ index indicates a slight synergy between ethanol and sugar on sweetness perception. The bitterness intensities elicited by ethanol–sugar mixtures are lower than those elicited by unmixed ethanol solutions. The Γ′ index indicates inhibition of ethanol and sugar perception on bitterness perception. Suppression of heat sensation was found in model base wine solutions across sugar and ethanol concentrations.     

Antibody-quantum dot conjugates exhibit enhanced antibacterial effect<Emphasis Type="Italic">vs.</Emphasis> unconjugated quantum dots

The effect of a 20-min exposure to antibody-quantum dot (Ab-QD) conjugates on colony counts of Escherichia coli was assessed by the spread-plate method and compared with exposure to unconjugated QDs having only amine or carboxyl groups on their surfaces. Under these conditions, Ab-QD conjugates generally exhibited >90% reduction in colony-forming units as compared to untreated E. coli and E. coli treated with unconjugated QDs after incubation for as long as 41 h. The antibacterial effect of Ab-QD conjugates vs. unconjugated QDs on Salmonella enterica subsp. enterica serovar Typhimurium was also assessed by means of a disk-diffusion technique which demonstrated greater growth inhibition (approximately 3 mm greater) by Ab-QD conjugate-impregnated disks than by unconjugated-QD-only-impregnated disks at a 10-microg disk load. At a 25-microg disk load, both treatment groups exhibited nearly equal growth inhibition.     

FURTHER OBSERVATIONS ON GROWTH OF SINGLE CELLS OF COCCOID BLUE-GREEN ALGAE

Initiation of growth (lag) and subsequent growth to visible colonies was examined for single cells of several coccoid blue-nreen algae under controlled incuba-tion conditions. At 30 or 39 C, with tungsten or fluorescent illumination, organisms such as Agmenel-lum quadruplicatum, strains PR-6 and strain BG-J, showed no evidence of lag associated with initiation of growth. The final colony count was within 20% of the expected number derived from cell counts and serial dilutions. For Anacystis nidulnns, Tx 20, a new medium C, 10 with EDTA as chelator was developed. In this medium groiuth of single cells at 39 C was excellent with quantitative recovery and no evidence of lag. At 30 C, however, Tx 20 showed anomalous behavior, lag and nonquantitative cell recovery. This behavior at 30 C is not yet understood.     

Rapid Detection of Gram-Negative Bacteriuria by Use of the Limulus Endotoxin Assay

The Limulus in vitro endotoxin assay was evaluated as a possible method for the prompt detection of significant gram-negative bacteriuria in children. This assay is capable of detecting endotoxin associated with intact cell walls of viable gram-negative bacteria as well as free endotoxin. Quantitative results are obtained following a 1-h incubation of Limulus lysate and 10-fold dilutions of otherwise untreated urine. A standard curve of Limulus activity and viable cell counts of Escherichia coli and Klebsiella pneumoniae in urine demonstrated that a positive Limulus reaction at a dilution of 1:100 or 1:1,000 indicated a colony count of at least 100,000 bacteria/ml. A positive Limulus reaction only from undiluted urine or at a dilution of 1:10 indicated less than 100,000 cells/ml. These experimental observations were confirmed by comparing the Limulus test with quantitative plate counts on 209 urine specimens from a mixed pediatric population. These results indicate that the Limulus assay is a simple, accurate method for rapid presumptive detection of gram-negative bacteriuria in patients where an immediate diagnosis is needed. This test would also seem promising for screening large patient populations for bacteriuria or for monitoring the effectiveness of treatment of urinary tract infections.     

Chemostat modeling of Escherichia coli persistence in conventionalized mono-associated and streptomycin-treated mice

We have previously shown that Escherichia coli BJ4 has similar doubling time in mice that are mono-associated (having only the inoculated E. coli BJ4) or streptomycin-treated (having mainly gram-positive bacteria plus the inoculated E. coli BJ4). We also showed that when the mice were conventionalized (fed cecum homogenate from conventional mice or ones with a complete microbial flora), the introduction of complete flora in both cases increased the in vivo doubling time, while decreasing the colony counts in fecal samples. To determine whether the increase in doubling time could explain the decrease in colony counts, we analyzed our previous results by a chemostat model. The analysis shows that the increasing doubling time alone is sufficient to explain the decrease in colony counts in mono-associated mice, but not in the streptomycin-treated mice. The observed decreasing rate in colony counts in streptomycin-treated mice is slower than predicted. Furthermore, whereas the model predicted a decrease to extinction in both mice, the E. coli persist at a frequency 10-80 times higher in streptomycin-treated mice than in mono-associated mice. Thus, while a chemostat model is able to explain some of the population dynamics of intestinal bacteria in mice, additional factors not included in the model are stabilizing the system. Because we find that E. coli declines more slowly and to a higher stabilization frequency in streptomycin-treated mice, which have a more diverse flora before conventionalization, we take these results to suggest that the persistence of E. coli populations is promoted by species diversity. We propose that a mechanism for the persistence may be the presence of new E. coli niches created by keystone species in the more diverse flora.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Bismuth-inhibitory effects on bacteria and stimulation of fungal growth in vitro

Bismuth salicylate was found to inhibit the growth of a range of bacteria and yeast, “Candida albicans”. In general the growth of bacteria did not result in increase in bismuth solubilisation, in contrast, bismuth solubilisation increased following the growth of C. albicans. A significant increase in the biomass (dry weight) of Aspergillus niger and Aspergillus oryzae occurred in vitro when these fungi were grown in the presence of bismuth salicylate. Biomass increase occurred over a range of bismuth compound additions, which in the case of A. oryzae was associated with increase in the solubilisation of the insoluble bismuth compounds.     

Metabolism of bismuth subsalicylate and intracellular accumulation of bismuth by Fusarium sp. strain BI

Enrichment cultures were conducted using bismuth subsalicylate as the sole source of carbon and activated sludge as the inoculum. A pure culture was obtained and identified as a Fusarium sp. based on spore morphology and partial sequences of 18S rRNA, translation elongation factor 1-alpha, and beta-tubulin genes. The isolate, named Fusarium sp. strain BI, grew to equivalent densities when using salicylate or bismuth subsalicylate as carbon sources. Bismuth nitrate at concentrations of up to 200 muM did not limit growth of this organism on glucose. The concentration of soluble bismuth in suspensions of bismuth subsalicylate decreased during growth of Fusarium sp. strain BI. Transmission electron microscopy and energy-dispersive spectroscopy revealed that the accumulated bismuth was localized in phosphorus-rich granules distributed in the cytoplasm and vacuoles. Long-chain polyphosphates were extracted from fresh biomass grown on bismuth subsalicylate, and inductively coupled plasma optical emission spectrometry showed that these fractions also contained high concentrations of bismuth. Enzyme activity assays of crude extracts of Fusarium sp. strain BI showed that salicylate hydroxylase and catechol 1,2-dioxygenase were induced during growth on salicylate, indicating that this organism degrades salicylate by conversion of salicylate to catechol, followed by ortho cleavage of the aromatic ring. Catechol 2,3-dioxygenase activity was not detected. Fusarium sp. strain BI grew with several other aromatic acids as carbon sources: benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, gentisate, d-mandelate, l-phenylalanine, l-tyrosine, phenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, and phenylpropionate.     

Lactose-Fermenting Salmonella from Dried Milk and Milk-Drying Plants

A study of 552 salmonella cultures revealed that 86 (15.6%) of the cultures fermented lactose. These had been isolated from dried milk products and milk-drying plants. Acid and gas were produced in lactose broth. Solid media containing lactose as the key ingredient for the differential reaction were not satisfactory for recognizing salmonella colonies. No problem was encountered in selecting salmonella colonies when bismuth sulfite agar was used.     

The Gastrointestinal Bacteria of Mink (Mustela vison L): Influence of Age and Diet

Total numbers of aerotolerant and anaerobic bacteria, and densities of Enterobacteriaceae, lactobacilli, staphylococci, salmonella and shigella, and campylobacteria were enumerated in the contents of the stomach, small intestine (and the associated mucosa), and colon of mink beginning at 2 weeks of age to adulthood, and in adults that were fed diets with different levels and types of fiber or food deprived. Highest densities of all bacterial groups were found in the colon at all ages (up to 108 cfu per g for total anaerobes), but were 2–4 orders of magnitude lower than those of other mammals. When all regions were pooled, significant age-related increases (p<0.05) were detected for anaerobes, aerobes, and staphylococci, and these coincided with the dietary shift at weaning. Enterobacteriaceae did not vary with age. Lactobacilli were never common isolates, but were detected more often after weaning, particularly in adults fed diets containing the 2 sources of fiber. Campylobacteria were detected only at 2 weeks of age, and salmonella and shigella were not isolated from any of the experimental mink. Total bacterial densities, the relative proportions of the bacterial groups, and age- and diet-related effects differ from those known for other mammals, which may be related to the carnivorous diet and rapid movement of digesta through the GIT.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Dose-related effects of red wine and alcohol on hemodynamics, sympathetic nerve activity, and arterial diameter

The cardiovascular benefits of light to moderate red wine consumption often have been attributed to its polyphenol constituents. However, the acute dose-related hemodynamic, vasodilator, and sympathetic neural effects of ethanol and red wine have not been characterized and compared in the same individual. We sought to test the hypotheses that responses to one and two alcoholic drinks differ and that red wine with high polyphenol content elicits a greater effect than ethanol alone. Thirteen volunteers (24-47 yr; 7 men, 6 women) drank wine, ethanol, and water in a randomized, single-blind trial on three occasions 2 wk apart. One drink of wine and ethanol increased blood alcohol to 38 +/- 2 and 39 +/- 2 mg/dl, respectively, and two drinks to 72 +/- 4 and 83 +/- 3 mg/dl, respectively. Wine quadrupled plasma resveratrol (P < 0.001) and increased catechin (P < 0.03). No intervention affected blood pressure. One drink had no heart rate effect, but two drinks of wine increased heart rate by 5.7 +/- 1.6 beats/min; P < 0.001). Cardiac output fell 0.8 +/- 0.3 l/min after one drink of ethanol and wine (both P < 0.02) but increased after two drinks of ethanol (+0.8 +/- 0.3 l/min) and wine (+1.2 +/- 0.3 l/min) (P < 0.01). One alcoholic drink did not alter muscle sympathetic nerve activity (MSNA), while two drinks increased MSNA by 9-10 bursts/min (P < 0.001). Brachial artery diameter increased after both one and two alcoholic drinks (P < 0.001). No beverage augmented, and the second wine dose attenuated (P = 0.02), flow-mediated vasodilation. One drink of ethanol dilates the brachial artery without activating sympathetic outflow, whereas two drinks increase MSNA, heart rate, and cardiac output. These acute effects, which exhibit a narrow dose response, are not modified by red wine polyphenols.     

Improved membrane filtration media for enumeration of total coliforms and Escherichia coli from sewage and surface waters.

Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35 degrees C. The new media were tested along with two standard media on 10 surface water and 7 sewage samples. The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification.     

Improved membrane filtration media for enumeration of total coliforms and Escherichia coli from sewage and surface waters

Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35 degrees C. The new media were tested along with two standard media on 10 surface water and 7 sewage samples. The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification.