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PubMed1988

The collagenase technique: how to isolate and examine adult Onchocerca volvulus for the evaluation of drug effects.

The collagenase technique developed more than twelve years ago has successfully been applied to isolate living and intact O. volvulus from excised onchocercomata for various biological, morphological, biochemical, immunological and in vitro studies. The experience gained with this technique and its modifications is critically reviewed giving practical advice for the selection, preservation and treatment of nodules for different purposes, especially for the evaluation of antifilarial drug effects. The composition of the worm load, the morphology of adult worms found in untreated patients, signs of degeneration and ageing of worms are described to show the broad morphological spectrum of the worm population in endemic areas to avoid false conclusions in drug trials. When these parameters are carefully considered, the collagenase technique provides a reliable method for examination of onchocercomata. An immediate answer can be obtained concerning the viability of the parasite. The qualitative and quantitative assessment of reproduction (embryogram) can give further valuable information on any changes in the reproductive dynamics and provide a sensitive indicator of any disturbances in the adult worms which may explain the early or late reinvasion of microfilariae into the skin after treatment with microfilaricidal drugs.

Animals↗

PubMed1995

Correlation of teichoic acid D-alanyl esterification with the expression of methicillin resistance in Staphylococcus aureus.

Methicillin-susceptible and -resistant isolates of Staphylococcus aureus, including a teichoic acid-deficient mutant, were examined under varying physiological conditions for their degree of teichoic acid and lipoteichoic acid D-alanyl esterification. Methicillin-resistant strains grown in the presence of methicillin and NaCl possessed significantly decreased amounts of D-alanyl ester when compared with methicillin-susceptible isolates. These strains also exhibited reduced autolysis. An autoradiographic procedure was used to detect mutants, isolated by Tn917 and nitrosoguanidine mutagenesis of methicillin-susceptible strains, which were defective in D-alanyl ester formation. The quantitative uptake of D-[14C]alanine in these mutants was determined and the effect of methicillin on the growth and viability of each mutant was compared with the wild-type strain. S. aureus mutant strains, defective in the uptake and incorporation of D-alanine, were shown to exhibit slightly reduced autolysis and an enhanced expression of methicillin resistance.

Alanine↗

PubMed1998

[Biological features and problems in detection of extrapulmonary tuberculosis pathogen].

The viability, enzymatic activity and Mycobacterium tuberculosis drug resistance was compared in pulmonary and extrapulmonary tuberculosis. To decrease oxygen in the culture medium baseline values and to use various oxidants as protectors were shown to be promising in enhancing the efficiency of cultural diagnosis of extrapulmonary tuberculosis.

Antitubercular Agents↗

PubMed2002

Microbial contamination of cord blood stem cells.

BACKGROUND AND OBJECTIVES: After storage, low levels of contaminating bacteria in standard blood components can reach bacteraemic levels, causing severe transfusion-associated sepsis. For cord blood (CB), the significance of low levels of contaminating bacteria and the optimal detection method is unknown and not supported by available guidelines. MATERIALS AND METHODS: Spiking experiments and testing of various subfractions of CB units were used to determine the behaviour of bacteria during centrifugation, freezing and thawing. For routine testing of CB, different volumes were compared for the detection of potential pathogens and micro-organisms of low pathogenicity. RESULTS: Centrifugation, as applied to CB fractionation, does not show concentration of bacteria in any particular fraction and supports the possibility of culture of waste fractions. Dimethylsulphoxide (DMSO) and freezing does not affect the viability of bacteria under the conditions used in this study. Owing to the low contamination level, a large sample volume of 20 ml was more sensitive than a 10-ml sample volume. Eighty five per cent of the isolated strains can be considered to be of low pathogenicity. CONCLUSION: When an optimal waste fraction sample volume of 20 ml was cultured, the contamination rate of CB was found to be approximately 13%, with low levels of < 1 colony-forming unit (CFU)/ml. Such levels of bacteria of low pathogenicity are expected to be of clinical importance only when CB is expanded in vitro for a prolonged period of time.

Bacteria↗

PubMed1982

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion.

Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals.

Animals↗

PubMed1997

Antilisterial activity of three bacteriocins used at sub minimal inhibitory concentrations and cross-resistance of the survivors.

The kinetics of inhibition of Listeria innocua strain Lin11 in a nutrient broth containing either nisin, pediocin AcH, or enterococcin EFS2 at concentrations ensuring a 2-3 log reduction of the population were first investigated. The rate of inhibition differed considerably between the bacteriocins. At the time that maximum viability loss occurred in the cultures containing either nisin (12 AU ml(-1)) or pediocin AcH (50 AU ml(-1)) the survivors resumed growth, although the medium retained most of its initial inhibitory activity. The survivors displayed increased resistance not only toward the bacteriocin they were in contact with, but toward the two other bacteriocins under study.

Bacteriocins↗

PubMed1989

Pathogenesis and chemotherapy of experimental Legionella pneumophila infection in the chick embryo.

The pathogenicity of Legionella pneumophila, serogroup 1, strain Nottingham N7, was assessed in terms of LD50 data and the ability of the organism to induce pathological lesions in the fertile hen's egg. Histopathological examination of embryo organs after inoculation with 1, 10, 100 and 1000 times the yolk sac LD50 revealed a disseminated infection. Systemic spread of the organism resulted in widespread necrosis and evidence of consolidation together with generation of copious amounts of oedema fluid. These were particularly severe in the liver, heart, spleen and kidney. The infection elicited a massive inflammatory response typified by infiltration with polymorphonuclear leucocytes and lymphocytes. Selected antimicrobial chemotherapeutic agents were investigated in protection studies for their capacity to ameliorate or control disease processes in this test system. Of those examined ciprofloxacin was most effective in reducing the incidence of lesions in these tissues and for prolonging embryo viability. Rifampicin, and to a lesser degree, erythromycin and doxycycline, also showed antimicrobial activity in these in vivo trials. These results indicate that the fertile hen's egg may be a useful alternative to other animal systems for the in vivo testing of clinically putative antimicrobial agents in the treatment of Legionnaires' disease.

Animals↗

PubMed2003

Pre-harvest factors influencing the acid resistance of Escherichia coli and E. coli O157:H7.

The effects of pH, acetate, propionate, or butyrate concentration, and diet on acid resistance of fecal Escherichia coli and E. coli O157:H7 were determined by in vitro and in vivo experiments. The pH tested was from 4.0 to 8.0, and the VFA concentrations tested were 0 to 100 mM. The E. coli O157:H7 used was strain 505B. In an in vivo study, cattle were fed a grain-based diet, then either not switched or switched to a grain-based diet with 3% added calcium carbonate or two fiber-based diets (soybean hulls or hay). Acid resistance was expressed as viability after acid-shock at pH 2.0 for 1 h and 4 h for fecal E. coli and E. coli O157:H7, respectively. Enumeration methods used were multitube fermentation, agar plate, and petri-film methods. The E. coli O157:H7 was not found in continuous culture inocula or in vivo samples. The viability of fecal E. coli decreased linearly (P < 0.01) as the culture pH increased, and viability of E. coli O157:H7 was highest (P < 0.01) when cultivated at pH 6.0. The viability of fecal E. coli and E. coli O157:H7 showed quadratic responses (P < 0.05) as acetate and butyrate concentrations increased at pH 7.2, with maximal acid resistance at 20 and 12 mM, respectively. As propionate concentration increased, the acid resistance was not different (P > 0.05) for fecal E. coli. Acid resistance of E. coli was induced by acetate and butyrate, even though the environmental pH was near neutral. Similar results were measured in the in vivo study, where viability after acid shock was more dependent on VFA concentration than on pH. Increasing the dietary calcium carbonate concentration also increased (P < 0.05) acid resistance of fecal E. coli. Results from these studies demonstrated that culture pH and VFA affect acid resistance of E. coli.

Animals↗

PubMed2000

Probiotic bacteria: selective enumeration and survival in dairy foods.

A number of health benefits have been claimed for probiotic bacteria such as Lactobacillus acidophilus, Bifidobacterium spp., and Lactobacillus casei. Because of the potential health benefits, these organisms are increasingly incorporated into dairy foods. However, studies have shown low viability of probiotics in market preparations. In order to assess viability of probiotic bacteria, it is important to have a working method for selective enumeration of these probiotic bacteria. Viability of probiotic bacteria is important in order to provide health benefits. Viability of probiotic bacteria can be improved by appropriate selection of acid and bile resistant strains, use of oxygen impermeable containers, two-step fermentation, micro-encapsulation, stress adaptation, incorporation of micronutrients such as peptides and amino acids and by sonication of yogurt bacteria. This review will cover selective enumeration and survival of probiotic bacteria in dairy foods.

Bifidobacterium↗

PubMed1990

Survival of Acanthamoeba in contact lens rinse solutions.

Acanthamoeba may cause a severe keratitis in contact lens wearers. Since most sterilization techniques require rinsing the lenses prior to insertion, contaminated solutions represent a potential vector for transmission of Acanthamoeba. The ability of rinse solutions to sustain an inoculum of Acanthamoeba polyphaga was investigated. A. polyphaga was exposed to 0.1% benzalkonium chloride, 0.001% thimerosal/0.1% edetate disodium, 0.1% edetate disodium, saline, tap water, and distilled water. The status of the organism was evaluated with direct microscopic counts and cultures to confirm viability. Incubation with 0.1% edetate disodium, saline, tap water, and distilled water resulted in the maintenance of reduced populations of viable organisms for 7 days. Benzalkonium chloride preserved saline and solutions containing thimerosal with edetate rendered the Acanthamoeba nonviable.

Acanthamoeba↗

PubMed2000

Potential of macrolide antibiotics to inhibit protein synthesis of Pseudomonas aeruginosa: suppression of virulence factors and stress response.

Recently we have reported that sub-minimum inhibitory concentrations (MICs) of macrolide antibiotics, such as erythromycin, clarithromycin, and azithromycin, induce loss of viability of Pseudomonas aeruginosa with longer incubation periods. In the present study we examined the effects of sub-MICs of macrolide antibiotics on protein synthesis and the expression of heat shock proteins (Gro-EL) in P. aeruginosa and the association of these factors with the viability of P. aeruginosa. In seven strains of P. aeruginosa clinical isolates, inhibition of protein synthesis was generally observed in bacteria grown on agar with sub-MIC azithromycin (8 microg/ml) at 24 h, and this was followed by loss of viability after an additional 24-h incubation. The inhibition of protein synthesis was shown in bacteria treated with sub-MICs of erythromycin and clarithromycin, but not with sub-MICs of other antibiotics examined (josamycin, tobramycin, ofloxacin, clindamycin, and ceftazidime) even at relatively high sub-MICs. In the heat shock condition (45 degrees C), strong expression of the heat shock protein Gro-EL was induced in bacteria grown on antibiotic-free medium, whereas there was a delay of such a response in bacteria exposed to 4 microg/ml of azithromycin. Reflecting these results, an abrupt reduction of viability in azithromycin-treated bacteria was observed within 3 h in the heat shock condition. Western blot analysis, using specific antibody for Gro-EL, demonstrated that erythromycin, clarithromycin, and azithromycin, at concentrations of 0.5-2 microg/ml, inhibited the expression of lower-molecular weight Gro-EL bands in the constitutive state. These results indicated that macrolides, at concentrations far below the MICs, suppressed protein synthesis in P. aeruginosa, an effect which may be associated with the inhibition of P. aeruginosa virulence and its loss of viability with longer incubation. Moreover, it is likely that the macrolides may sensitize bacteria to stresses, as these antibiotics induced alterations in a major stress protein, Gro-EL, in constitutive and inducible states.

Anti-Bacterial Agents↗

PubMed1991

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining.

In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white.

Colony Count, Microbial↗

PubMed1985

Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector.

Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. beijerinckii::Tn76 isolates at a high frequency. Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.

Azotobacter↗

PubMed1998

Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.

The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.

Animals↗

PubMed2005

Solute-specific effects of osmotic stress on Staphylococcus aureus.

AIM: To determine if cell death from osmotic stress is because of lack of sufficient energy to maintain cell metabolism. Additionally, the solute-specific effect of five humectants on bacterial osmoregulation and cell survival was examined. METHODS AND RESULTS: Staphylococcus aureus was placed into 84% relative humidity (RH) broth (five humectants used individually). ATP, ADP and cell viability measurements were determined over time. The results indicate that ATP is not the limiting factor for cell survival under excessive osmotic stress. Although the same RH was achieved with various humectants, the rates of cell death varied greatly as did the sensitivities of the cell populations to osmotic stress. CONCLUSIONS: The results from this study provide strong evidence that mechanisms of osmotic inactivation depend on the solute. The molecular mobility of the system may be an important means to explain these differences. SIGNIFICANCE AND IMPACT OF THE STUDY: By bringing together an understanding of solute-specific effects, microbial physiology and genetics, the mechanisms of inactivation of micro-organisms by solute-specific osmotic stress may be elucidated, and this knowledge may then be exploited to ensure the production of high quality, safe foods.

Adenosine Diphosphate↗

PubMed2005

Evaluation by differential scanning calorimetry of the effect of acid, ethanol, and NaCl on Escherichia coli.

The influence of acid, ethanol, and NaCl on the cellular components and inactivation of Escherichia coli were evaluated using differential scanning calorimetry. Cell viability was assessed using plate counting. The thermal stability for ribosomal subunit denaturation and the total apparent enthalpy decreased with increasing ethanol, salt, and acid concentrations. The reduction of the ribosomal subunit denaturation peak was the primary contributor to the decrease in the total apparent enthalpy. Thermograms indicated that even at concentrations at which less than a 0.4-log reduction of cell viability with a concomitant minimal reduction of total apparent enthalpy occurred, a decrease in onset temperature of ribosomal transition was evident. Acid treatments at pH 3 induced by HCl and by 0.4 M acetic acid caused the DNA denaturation temperature in vivo to decrease. Application of chemical treatment prior to heat treatment noticeably reduced the viability of E. coli cells at all the heat treatment temperatures (60, 62.5, and 65 degrees C) compared with that of heat treatment alone, suggesting an increased sensitivity of bacteria to heat treatment. Differential scanning calorimetry in vivo can be used to assess the effectiveness of hurdles when thermal processing technologies with hurdles are designed.

Acetic Acid↗

PubMed1989

Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae.

A systematic study of the qualitative and quantitative effects of sterol on nystatin sensitivity has been made in a single organism. The use of a sterol auxotroph of Saccharomyces cerevisiae offered a convenient way to control the sterol content of the yeast cell. There was a correlation between the ergosterol content of the cell and sensitivity to nystatin, as monitored by both potassium leakage from the cell and viability. When the sterol auxotroph contained high levels of ergosterol, the cells were sensitive to the effects of nystatin. When the ergosterol content was low or when ergosterol was replaced by cholesterol or cholestanol, sensitivity to nystatin was markedly decreased. Although resistant to nystatin, cholestanol enriched cells showed an enhanced background of potassium ion loss.

Cholesterol↗

PubMed1982

[Isolation and characterization of epithelial cells of colon mucosa: preliminary results].

A procedure for the isolation of epithelial cells from rat and human large bowel is described; the minced tissue was carefully washed with saline and incubated for 45 min in a collagenase-jaluronidase solution; the dispersion of the epithelial cells was achieved by a subsequent treatment with the calcium chelator EDTA. The isolated cells were characterized by cytological and histochemical (PAS, alkaline phosphatase, N-acetyl-D-glucosaminidase) procedures; viability index was assessed by the trypan blue exclusion test; the ability to grow in culture on both liquid and semisolid media was also tested. This method can be successfully applied either to normal or neoplastic colonic mucosa, the resulting cell suspension being suitable for further characterization.

Animals↗