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PubMed1975

Bacillus subtilis GSY 1057 assay for aflatoxin B activation by rainbow trout (Salmo gairdneri).

A rapid and sensitive microbial assay was developed to detect lethal products of aflatoxin B metabolism by rainbow trout (Salmon gairdneri) Mt. Shasta strain. Bacillus subtilis GSY 1057 (hisA1, uvr-1, metB4), a DNA repair deficient strain, was incubated for 20 min in the 20,000 times g supernate from trout liver homogenates which had been preincubated for 10 min with various levels of aflatoxin B. Serial dilutions of the incubation mixture were plated in triplicate on tryptose blood agar base plates and colonies were counted after 12 hr at 37 degrees C. One mumole aflatoxin B in 3.2 ml incubation mixture reduced viability 60%.

Aflatoxins↗
PubMedDate not supplied

A replica plating technique for in vitro study of susceptibility of Candida albicans to miconazole, econazole and ketoconazole: some data for standardization.

We report our studies in vitro on the activity of ketoconazole (KZ), econazole and miconazole on 115 isolates of Candida albicans of clinical origin tested by an agar dilution method with three different culture media: yeast morphology agar, Sabouraud maltose agar and Casitone agar (CA). The antifungal effects of the imidazole compounds (IC) were strongest in CA. Eighty percent of the strains were inhibited by 0.1 micrograms/ml of KZ, the most active drug. The frequent appearance of "veil growth" at higher concentrations of the IC made the exact determination of the minimal inhibitory concentration (MIC) difficult. Microscopic examination of the growth on plates at the highest drug dilutions was performed and the results compared with a replica plating technique of IC plates on other plates with antibiotic-free media. The following were observed: a) a residual capacity of cell division of Candida strains even in the presence of high antifungal concentrations to which they are considered sensitive; and b) a certain viability (7% of the cases) of some fungal cells in the residual growth, particularly in those strains with higher conventional MIC. A standardized methodology is needed for the clinical evaluation of the susceptibility tests of Candida to the imidazolic compounds.

Candida albicans↗
PubMed1996

The yeast VRG4 gene is required for normal Golgi functions and defines a new family of related genes.

Sodium vanadate is an effective agent for the enrichment of yeast mutants with defects in glycosylation steps that occur in the Golgi complex (Ballou, L., Hitzeman, R. A., Lewis, M. S., and Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3209-3212). We isolated and screened vanadate-resistant glycosylation mutants in the budding yeast, Saccharomyces cerevisiae, to identify any that may be defective in the secretory pathway, since changes in normal glycosylation may reflect defects within the secretory pathway. We identified one such mutant, allelic to vrg4/van2, that is defective in processes that occur specifically in the Golgi complex. Protein secreted from vrg4 mutants lacks the outer chain glycosylation that is normally extended during passage through the Golgi. This mutant fails to retrieve soluble endoplasmic reticulum proteins from the Golgi and accumulates the Golgi-specific biosynthetic intermediate of the vacuolar protein, carboxypeptidase Y. Analyses of intracellular membranes by staining with the fluorescent lipophilic dye, DiOC6, and by electron microscopy reveals a dramatic alteration in the membrane morphology of vrg4 mutant cells. The VRG4 gene encodes a 36.9-kDa membrane protein that is essential for cell viability. A sequence homology search has identified five related genes, establishing that VRG4 is a founding member of a family of structurally similar genes. Taken together, these results suggest that the VRG4 gene plays an important role in regulating Golgi functions and in maintaining the normal organization of intracellular membranes.

Amino Acid Sequence↗
PubMed2004

Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori.

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

Amoxicillin↗
PubMed1989

[Effects of endotoxins from periodontopathic bacteria on the collagen metabolism of cultured normal human gingival fibroblasts].

The present study was performed to investigate the effects of endotoxins from periodontopathic bacteria on collagen metabolism. Endotoxins were extracted from Bacteroides gingivalis 381 and Bacteroides intermedius ATCC 25611 using the hot-phenol method. A commercially available endotoxin from Escherichia coli 0111: B4 was also used as a control. Human gingival fibroblasts (Gin-1, ATCC CRL 1292) were maintained with DMEM containing 10% FBS. When the fibroblasts became confluent they were exposed to each of the endotoxins in various concentrations (0, 5, 10, 15, 20 micrograms/ml). The effects of these endotoxins on the collagen metabolism of the fibroblasts were assessed on the basis of collagen synthesis and collagenase activity. The former was assessed by 3H-proline incorporation and bacterial collagenase digestable protein. The latter was assessed by fibril assay. The results were as follows: There was no change in glucose consumption, cell viability or morphology under the light microscope when the concentration of endotoxin was 20 micrograms/ml or less. 3H-proline incorporation into protein and collagen synthesis were inclined to decrease. Endotoxin from B. gingivalis resulted in an acceleration of collagenase activity. There findings indicate that the endotoxins from these bacteria might affect collagen metabolism early in gingivitis in vivo.

Bacteroides↗
PubMed2004

The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.

Animals↗
PubMed1998

Quantification of microbial productivity via multi-angle light scattering and supervised learning.

This article describes the use of chemometric methods for prediction of biological parameters of cell suspensions on the basis of their light scattering profiles. Laser light is directed into a vial or flow cell containing media from the suspension. The intensity of the scattered light is recorded at 18 angles. Supervised learning methods are then used to calibrate a model relating the parameter of interest to the intensity values. Using such models opens up the possibility of estimating the biological properties of fermentor broths extremely rapidly (typically every 4 sec), and, using the flow cell, without user interaction. Our work has demonstrated the usefulness of this approach for estimation of yeast cell counts over a wide range of values (10(5)-10(9) cells mL-1), although it was less successful in predicting cell viability in such suspensions.

Biotechnology↗
PubMedDate not supplied

Bioluminescence measurements of the antilisterial activity of nisin: comparison with ampicillin and streptomycin.

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of amplicillin and streptomycin under similar conditions. In the presence of nisin (3-12 micrograms/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35 degrees C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3-12 micrograms/ml) and streptomycin (15-60 micrograms/ml) during the first hour. However, concentrations of > or = 3 micrograms/ml ampicillin and > or = 30 micrograms/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.

Adenosine Triphosphate↗
PubMed1986

Alterations in macrophage collagenase secretion induced by gold sodium thiomalate.

The effects of gold sodium thiomalate (GST) on the production of specific collagenase by thioglycolate-elicited macrophages was investigated. Our studies demonstrated that GST administration can significantly decrease collagenase production in a dose-dependent manner. These effects were observed with levels of GST attainable in serum or synovial tissue during routine chrysotherapy. In addition, GST altered lysozyme secretion by activated macrophages in a pattern distinct from that of collagenase alteration. These effects of enzyme secretion were not secondary effects of GST on viability, general protein secretion, or the specific assay procedures utilized, and were not attributable to the thiomalate moiety. Thus, GST may exert its therapeutic effect in rheumatoid arthritis through interference with the production of degradative proteolytic enzymes, which are important effector molecules mediating tissue destruction.

Animals↗
PubMed1993

Use of polymerase chain reaction to assess efficacy of leprosy chemotherapy.

The assessment of chemotherapy efficacy in leprosy is difficult, since the only reliable method for determining whether the causative organism, Mycobacterium leprae, is viable depends on its growth in mouse foot pads. In an attempt to replace this expensive, time-consuming test, methods based on the polymerase chain reaction (PCR) have been developed. These methods depend on detection of DNA, which is more susceptible to degradation on cell death than are other cell components, so should be a more accurate indicator of viability. We have used a specific PCR assay to detect M leprae DNA in skin biopsy samples from leprosy patients. By use of limiting dilution PCR (LD-PCR), the concentration of M leprae DNA in the original sample could be measured. The DNA concentration was more closely correlated with the morphological index (derived from a staining technique that distinguishes morphologically intact and damaged bacteria) than with the number of bacteria visible (bacterial index, BI, which counts both alive and dead bacteria). In a longitudinal study of multibacillary patients on multi-drug therapy, skin biopsy samples were collected before treatment and 3, 6, 12, and 24 months after the start of therapy. While the BI showed little or no change during treatment, the number of genomes detected by PCR fell sharply, in parallel with the MI. We propose that PCR can be used as a rapid measure of M leprae viability and that this approach can be used for monitoring individual leprosy patients and for assessment of existing and new regimens. The method may be applicable to other infectious diseases in which culture of the causative organism is slow or impossible.

Base Sequence↗
PubMed2004

Non-disruptive, real-time analyses of the metabolic status and viability of Streptococcus mutans cells in response to antimicrobial treatments.

Streptococcus mutans is one of a small number of recognized pathogens that lives among the hundreds of other bacterial species which comprise the oral flora. The virulence of this organism is intimately associated with its ability to live as an attached biofilm community on the tooth surface, and consequently, there is a great interest in its biofilm lifestyle. Currently, there are no established protocols that facilitate drug screening against this organism while it is entrenched in the biofilm. Furthermore, greater complications arise when attempting to perform these experiments in a multi-species setting. In an effort to circumvent these problems, we developed a quick, real-time, and non-disruptive method to probe the metabolic status of S. mutans growing as either a planktonic culture or a biofilm community. This assay takes advantage of the proven utility of luciferase measurements for drug screening. We placed the luciferase gene under the control of the S. mutans lactate dehydrogenase promoter (ldh) and integrated the construct onto its native position on the chromosome. We found this construct to be both highly expressed (<10000 cells easily detectable) and insensitive to many different growth parameters. When testing this reporter in both planktonic and biofilm cultures receiving either bacteriostatic or bactericidal antibiotics, we found the ldh-luc reporter to be a very accurate measurement of cell viability. Furthermore, we also demonstrated that this assay can generate useful information about the characteristics of intoxication caused by antibiotic activity. In addition, we modified the biofilm assay into the 96-well format and demonstrated the feasibility of high throughput drug screening of biofilm embedded S. mutans.

Anti-Bacterial Agents↗
PubMed2002

Vitality status of microorganisms in infected human root dentine.

AIM: This experimental study was initiated to establish a method for characterizing the vitality status of bacteria in infected human root dentine by differentiating between viable and dead microorganisms. METHODOLOGY: Twenty-four root segments of extracted human teeth were infected with either Streptococcus sanguinis or Enterococcus faecalis for 8 weeks. Baseline samples from root dentine (rd) were collected after 4 weeks. These were compared with samples taken at week 8 (control group: n = 12) and with samples collected at week 12 after calcium hydroxide treatment for four weeks (test group: n = 12). After marking viable and dead bacterial cells by two fluorescent dyes, the portion of viable bacteria (PVB) was determined, as well as the number of colony-forming units (CFU). RESULTS: Viable and dead bacteria were identified in all "rd" samples. PVBrd values were lower than PVB values of the bacterial suspension in the root canal lumen. In the control group, PVBrd and CFUrd did not markedly differ at week 4 and at week 8, regardless of the strain used. In the test group, viable but non-culturable sanguinis streptococci (mean PVBrd = 27%; CFUrd = 0) were detected, despite calcium hydroxide treatment. The viability of E. faecalis was not affected by calcium hydroxide. CONCLUSIONS: Fluorescence labelling of bacteria in human root dentine gives valuable additional information about their vitality status compared to the parameter CFU. The method may be suitable for following the fate of bacteria in dentinal tubules, for example in the presence of intracanal dressings.

Calcium Hydroxide↗
PubMed2003

A sensitive and rapid method to determine the viability of freeze-dried bacterial cells.

AIMS: To determine the potential use of flow cytometry for viability asssessment of freeze-dried bacterial cells. METHODS AND RESULTS: Escherichia coli CIP 54.8T and Vibrio metschnikovii CIP 104262 were analysed. The viability of freeze-dried cells resuspended in a nutrient broth was evaluated by culture whereas activity was determined by flow cytometry analysis of both esterase activity and cell death. Activity assessment by flow cytometry was found to be a rapid and good indicator of cell viability and was very efficient for quality control. For V. metschnikovii the fraction of active cells varies greatly depending on the freeze-drying procedure and within a given procedure. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial activity assessment by flow cytometry is very efficient for the control of freeze-dried cells.

Bacteriological Techniques↗
PubMed1992

Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.

Amino Acid Sequence↗
PubMed2001

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy.

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.

Animals↗
PubMed2005

Probiotic bacteria may become dormant during storage.

The determination of bacterial viability in probiotic products is of economic, technological, and clinical significance. We compared four methods to enumerate three Bifidobacterium strains in fermented oat products during storage. A subpopulation of nonculturable cells retained a functional cell membrane typical of viable cells, indicating that probiotic bacteria become dormant during storage.

Avena↗
PubMed2005

Novel sialic acid transporter of Haemophilus influenzae.

Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.

Biofilms↗