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PubMed1996

Frequency distribution of Candida albicans blastospores adhered to mucosal epithelial cells in-vitro.

Although several methods are available for examination of microbial adherence to epithelial cells, these do not distinguish between adherence of viable and non-viable micro-organisms. This study reports the use of acridine orange-stained blastospores of Candida albicans in conjunction with direct epifluorescence microscopy to determine viable (orange-fluorescing) and non-viable (green-fluorescing) blastospore adherence to buccal epithelial cells. The method was also employed to examine the effects of chlorhexidine treatment at subminimum inhibitory concentrations on the adherence of viable and non-viable blastospores. There was good correlation in the assessment of blastospore viability between the direct epifluorescence microscopy technique and the standard serial dilution and plating method for viable counting, confirming the reliability of direct epifluorescence microscopy. Chlorhexidine treatment before acridine orange staining did not alter this assessment of viability. Blastospore adherence to buccal epithelial cells resulted in a similarly skewed distribution whether examined using a crystal violet stain in conjunction with light microscopy or using direct epifluorescence microscopy, therefore validating the direct epifluorescence microscopy technique for the enumeration of blastospore adherence. Chlorhexidine treatment (0.0005% v/v, 30 min) of either blastospores or buccal epithelial cells altered the distribution of adherent blastospores per epithelial cell by increasing the number of epithelial cells having no adherent blastospores. No differences in adherence were, however, observed between blastospore or epithelial cells after treatment with this agent. Examination of the adherence of viable and non-viable blastospores to buccal epithelial cells using direct epifluorescence microscopy revealed a greater adherence capacity of non-viable than viable blastospores for buccal epithelial cells. Treatment of blastospores with chlorhexidine altered the frequency distributions of viable and non-viable blastospores with lower numbers of blastospores adherent per epithelial cell. The larger reduction in adherent viable blastospores in comparison with their non-viable counterparts is, however, an important observation which might have clinical relevance. Microbial cells adhere to epithelial cells resulting in a skewed distribution; study of this distribution gives useful information about the adherence process. Viable and non-viable components of a microbial population have different adherence capabilities and treatment of such populations with an antimicrobial agent exerting anti-adherent activity at sub-minimum inhibitory concentrations reduces the amount of adherence of these viable/non-viable components to different extents.

Adhesiveness↗

PubMedDate not supplied

Asymmetric ketone reduction with immobilized yeast in hexane: biocatalyst deactivation and regeneration.

There is a need to develop methods for producing enantiomerically pure pharmaceuticals because the racemic mixtures made today will probably not be allowed in the future. Synthetic chiral catalysts are being developed for this purpose, as well as new product separation techniques. Another possible option is to use biocatalysts, such as purified enzymes or whole microbial cells, since these can result in the production of mostly a single enantiomer. This study emphasizes the use of alginate-entrapped yeast cells to catalyze the reduction of ketones as a model system. The emphasis is on the factors that might limit the reactivity of such cells, such as equilibrium conditions, substrate or product inhibition, solvent toxicity, loss of cell viability, or the degradation of intracellular levels of enzymes or cofactors. It was found that there was a progressive loss of catalytic activity of the immobilized yeast cells, which appeared to be mainly associated with a loss of cell viability and a decline of intracellular NAD(H) levels during the reaction. The other factors investigated did not have a large effect. A regeneration scheme was developed in order to replenish the intracellular NAD(H) lost during the reaction, which involved removing the biocatalyst from the reaction and supplying the cells with a nutrient source. This resulted in an increase in the NAD(H) to initial levels and also resulted in a maintenance of the ketone reduction rate over time.

Alcohol Dehydrogenase↗

PubMed1994

Penetrating keratoplasty in the West Bank and Gaza.

Four hundred and sixteen penetrating keratoplasties performed at St John Ophthalmic Hospital, Jerusalem, between January 1988 and July 1992 were reviewed retrospectively. The leading indications were keratoconus, microbial keratitis, trachomatous corneal scarring and herpes simplex keratitis. After a mean follow-up of 12.4 months (> 1-48 months) 79.8% of the grafts remained clear. The results in terms of graft clarity and visual improvement varied among the different diagnostic groups. No relationship was found between donor age, cadaver time or storage time and graft survival. This series demonstrates the viability of penetrating keratoplasty in this area of low economic development but highlights the need for strict patient selection to ensure optimal use of scarce donor material.

Adolescent↗

PubMed1995

A kinetic model for comparing proteolytic processing activity and inhibitor resistance potential of mutant HIV-1 proteases.

A kinetic model is presented for the comparison of potential proteolytic processing activities of wild-type and mutant human immunodeficiency virus proteases in the presence and absence of protease inhibitors. The protease processing of gag substrate in the immature virions is assessed by the kinetic parameters, kcat, Km and Ki. The relationship of the estimated potential processing activities to the viability of the HIV mutants and their tendencies to resist inhibitors also are discussed. A fully developed model has the potential to stimulate the results of inhibitor resistance either in vivo or in cell culture.

Drug Resistance, Microbial↗

PubMed1989

Culture of seminal fluid in infertile men and relationship to semen evaluation.

Bacterial flora of the seminal fluid and its influence on semen quality, was examined in 225 asymptomatic unselected men. Each semen sample was cultured aerobically, anaerobically, for genital mycoplasmas, and for Chlamydia trachomatis. Semen analysis was made according to standard methods recommended by the W.H.O. All 225 semen samples had microbial isolates. All isolates had colony counts of 10(2) colony forming units (cfu/ml). Thirty-three cases had greater than 10(2) cfu/ml, 85 cases had greater than 10(3) cfu/ml and 78 cases greater than 10(5) cfu/ml. The most common organisms isolated were Ureaplasma urealyticum in 86 samples and C. trachomatis in 26 samples. The most frequent abnormal parameters were viability (117 of 212, 52%), motility (85 of 212, 40%) and number of sperm cells (74 of 225, 32.8%). No significant correlation was found between abnormal semen parameters and presence of U. urealyticum, and C. trachomatis. We concluded that asymptomatic bacteriospermia (infection) in the semen did not significantly affect the count, motility or morphologic features of the specimen.

Bacteria↗

PubMed2005

Simultaneous enumeration of viable Enterobacteriaceae and Pseudomonas spp. within three hours by multicolor fluorescence in situ hybridization with vital staining.

A new means of rapidly and simultaneously counting viable phylogenetically different bacteria was developed. The cyanine dimer dye, BOBO-3 that selectively stains bacteria with damaged membranes were used to evaluate bacterial viability based on membrane integrity. Viable Enterobacteriaceae and Pseudomonas spp. could be selectively detected within three hours using multicolor fluorescence in situ hybridization (FISH) following BOBO-3 staining (BOBO3-FISH).

Bacteriological Techniques↗

PubMed2003

Liquid formulation of the biocontrol agent Candida sake by modifying water activity or adding protectants.

AIMS: To evaluate the effect of modification of water activity (aw) and the addition of protective substances in the preservation medium of liquid formulations of the biocontrol agent Candida sake stored at 4 and 20 degrees C. METHODS AND RESULTS: The aw of the preservation medium of C. sake was modified from 0.72 to 0.95 by adding glycerol or polyethylene glycol (PEG). Moreover, several protectant substances at different concentrations were evaluated. Modification of lower aw-levels (0.721-0.901) with glycerol did not maintain the viability of the yeast cells. Higher aw-levels (0.93-0.95) with either glycerol or PEG improved the viability but not at acceptable viability levels. C. sake cells maintained viabilities >60% when sugars, such as trehalose, and polyols, such as glycerol and PEG were used as protectants in liquid formulations. Moreover, liquid formulations of C. sake stored at 4 degrees C showed higher number of viable counts than at 20 degrees C. When different sugars were tested, all of them, except 10% fructose, resulted in a viability higher than 50% of the C. sake formulations. Biocontrol of liquid formulation treatments was similar to fresh cells in controlling Penicillium expansum on wounded apples. CONCLUSIONS: Sugars such as lactose and trehalose could be considered as good protectants in order to obtain liquid formulations of C. sake cells as they maintain the viability >70% for 4 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF STUDY: This study shows that a suitable liquid formulation for commercial application can be produced with high viability and conservation of biocontrol efficacy. Moreover, if 10% lactose is the protectant used in the formulation, the economic costs would not be limiting for industrial production.

Candida↗

PubMed2005

mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state.

AIMS: This work investigates the maintenance of viability and potential virulence of Vibrio parahaemolyticus in a viable but nonculturable population (VBNC) state by reverse transcription-polymerase chain reaction (RT-PCR). METHODS AND RESULTS: Housekeeping genes, 16S-23S rDNA and rpoS, as well as virulence genes, tdh1 and tdh2, were selected and detected by PCR in a pathogenic strain of V. parahaemolyticus (Vp4). Their expression was then studied by RT-PCR in V. parahaemolyticus Vp4 cultivated in rich medium at 37 degrees C. The 16S-23S rDNA and rpoS, tdh1, tdh2 genes were transcripted at the mid-logarithmic, stationary and late stationary phases, corresponding to various physiological states. The expression of these genes was also studied by RT-PCR in a VBNC population of V. parahaemolyticus Vp4 in artificial seawater (ASW). The effect of temperature (washing of bacterial culture and microcosms) on the attaining VBNC bacteria was first considered. Washing of V. parahaemolyticus Vp4, collected at the mid-logarithmic phase, at 10 or 4 degrees C before inoculation in ASW at 4 degrees C allowed bacteria entered the VBNC state between 22 and 31 days. The 16S-23S rDNA and rpoS gene were expressed in the VBNC bacteria whereas no expression of the tdh1 and tdh2 genes was observed in the same populations. CONCLUSION: The two selected housekeeping genes, 16S-23S rDNA and rpoS, proved to be good viability markers for V. parahaemolyticus Vp4 in culturable and VBNC states. These first data indicated that the pathogenic strain Vp4 would not maintain the expression of the virulence genes, tdh1 and tdh2, in VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of RT-PCR for investigating the maintenance or not of viability and potential virulence in VBNC V. parahaemolyticus will facilitate further study to evaluate the potential risk presented by this pathogen in the environment.

Bacterial Proteins↗

PubMed1997

Loss of intrinsic aminoglycoside resistance in Acinetobacter haemolyticus as a result of three distinct types of alterations in the aac(6')-Ig gene, including insertion of IS17.

The distribution of the aac(6')-Ig gene, encoding aminoglycoside 6'-N-acetyltransferase-Ig [AAC(6')-Ig], was studied in 96 Acinetobacter haemolyticus strains and 12 proteolytic Acinetobacter strains, including Acinetobacter genomospecies 6, 13, and 14 and 3 unnamed species assigned to this genomic group by DNA-DNA hybridization. This gene was detected by DNA-DNA hybridization in all 96 A. haemolyticus strains and by PCR in 95 strains but was not detected in strains of other species, indicating that it may be used to identify A. haemolyticus. Three A. haemolyticus strains were susceptible to tobramycin and did not produce an aminoglycoside 6'-N-acetylating activity, although they contained aac(6')-Ig-related sequences. An analysis of three susceptible A. haemolyticus strains indicated that aminoglycoside resistance was abolished by the following three distinct mechanisms: (i) a point mutation in aac(6')-Ig that led to a Met56-->Arg substitution, which was shown by analysis of a revertant to be responsible for the loss of resistance; (ii) a polythymine insertion that altered the reading frame; and (iii) insertion of IS17, a new member of the IS903 family. These observations indicated that AAC(6')-Ig is not essential for the viability of A. haemolyticus, although the aac(6')-Ig gene was detected in all members of this species.

Acetyltransferases↗

PubMed2003

The recX gene product is involved in the SOS response in Herbaspirillum seropedicae.

The recA and the recX genes of Herbaspirillum seropedicae were sequenced. The recX is located 359 bp downstream from recA. Sequence analysis indicated the presence of a putative operator site overlapping a probable sigma70-dependent promoter upstream of recA and a transcription terminator downstream from recX, with no apparent promoter sequence in the intergenic region. Transcriptional analysis using lacZ promoter fusions indicated that recA expression increased three- to fourfold in the presence of methyl methanesulfonate (MMS). The roles of recA and recX genes in the SOS response were determined from studies of chromosomal mutants. The recA mutant showed the highest sensitivity to MMS and UV, and the recX mutant had an intermediate sensitivity, compared with the wild type (SMR1), confirming the essential role of the RecA protein in cell viability in the presence of mutagenic agents and also indicating a role for RecX in the SOS response.

Bacterial Proteins↗

PubMed2001

Growth and viability of commercial Bifidobacterium spp. in honey-sweetened skim milk.

Two commercial Bifidobacterium bifidum (Bf-1 and Bf-6) were cultured in 12% (wt/vol) reconstituted nonfat dry milk (NDM) containing 5% (wt/wt) honey, sucrose, fructose, or glucose. Inoculated samples were incubated anaerobically at 37 degrees C for 48 h. Samples were collected at 12-h intervals and examined for (i) specific growth rate, (ii) pH, and (iii) levels of fermentation end products (lactic and acetic acids) as measured by high-performance liquid chromatography (HPLC). Viability of the organisms during 28 days of refrigerated storage at 4 degrees C was also assessed at 7-day intervals. Growth promotion and acid production were greatest when Bf-1 and Bf-6 were grown in the presence of honey. For both Bf-1 and Bf-6, retention of viability was greatest up to 14 days of refrigerated storageat 4 degrees C when they were grown and stored in the presence of honey compared to other sweeteners.

Acetic Acid↗

PubMed2002

Antibacterial effect of a magnetic field on Serratia marcescens and related virulence to Hordeum vulgare and Rubus fruticosus callus cells.

The exposure to a static magnetic field of 80+/-20 Gauss (8+/-2 mT) resulted in the inhibition of Serratia marcescens growth. Callus cell suspensions from Hordeum vulgare and Rubus fruticosus were also examined and only the former was found to be affected by the magnetic field, which induced a decreased viability. S. marcescens was shown to be virulent only toward H. vulgare and this virulence was reduced by the presence of the magnetic field. The modification of glutathione peroxidase activity under the different experimental conditions allowed us to speculate on the possibility of an oxidative-stress response of H. vulgare both to S. marcescens infection and magnetic field exposure. Since the control of microbial growth by physical agents is of interest for agriculture, medicine and food sciences, the investigation presented herein could serve as a starting point for future studies on the efficacy of static magnetic field as low-cost/easy-handling preservative agent.

Cell Division↗

PubMed1976

Aflatoxin B1 metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri) liver.

Aflatoxicol, R0, was isolated from Mt. Shasta strain rainbow trout (Salmo gairdneri), and liver homogenates were incubated with aflatoxin B1. Its identity was confirmed by mass, infrared, and ultraviolet spectrometry. The structure was identical to one of the diastereomers prepared by chemical reduction of aflatoxin B1. Aflatoxicol was apparently formed by a reduced nicotinamide adenine dinucleotide phosphate-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout. Aflatoxicol was not lethal in phosphate buffer to Bacillus subtilis GSY 1057 (metB4, hisA1, uvr-1) nor were aflatoxins B1, Q1, and B2. In the presence of reduced nicotinamide adenine dinucleotide phosphate and trout liver microsomes, aflatoxicol reduced the viability of B. subtilis. Aflatoxin B2, which lacks the vinyl ether present in the other compounds, could not be activated. The product of aflatoxin B1 activation by trout liver microsomes was sought after incubation of 14C-labeled aflatoxin B1. The radioactivity was found in unaltered aflatoxin B1 and in three extremely polar metabolites. The quantity of the new metabolites and the level of microbial lethality was reduced by addition of cytosine and cysteine to the incubation medium. The vinyl ether configuration was a structural requirement for activation, and this finding and the nature of the enzymatic reaction were consistent with the hypothesis that the compounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B. subtilis. The formation of aflatoxicol as the major product of trout liver metabolism is of great significance considering that it could be activated to a lethal compound and that rainbow trout are one of the most sensitive species to aflatoxin B1-induced carcinoma.

Aflatoxins↗

PubMed1981

Role of ribosome recycling in uptake of dihydrostreptomycin by sensitive and resistant Escherichia coli.

Exposure of streptomycin-resistant cells to puromycin results in uptake of dihydrostreptomycin comparable to that found with streptomycin-sensitive cells. This finding indicates that the enhanced phase of uptake, previously reported only in sensitive cells, may result from an increase in internal binding sites, presumably run-off ribosomes. The increased uptake of dihydrostreptomycin resulting from exposure to puromycin is greatest in both sensitive and resistant cells at concentrations below 100 microgram/ml. At 100 microgram/ml, exposure to puromycin in vivo results in significant, but not complete, polysome degradation and inhibition of protein synthesis. At 500 microgram/ml, where polysome degradation is complete in less than 2 min and where growth and protein synthesis are inhibited more than 90%, uptake of dihydrostreptomycin by both sensitive and resistant cells is inhibited. Puromycin has no effect on binding of dihydrostreptomycin to 70-S monosomes, as measured by equilibrium dialysis. The increased uptake of dihydrostreptomycin by resistant cells resulting from exposure to puromycin has no effect on viability. Addition of N-ethylmaleimide immediately and completely inhibits the puromycin-induced uptake of dihydrostreptomycin even when added after substantial polysome degradation has occurred.

Dihydrostreptomycin Sulfate↗

PubMed1994

PAD1 encodes phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in Saccharomyces cerevisiae.

The yeast enzyme phenylacrylic acid decarboxylase (PAD) confers resistance to phenylacrylic acids. Cinnamic acid (CA)-sensitive mutants lacking PAD activity were isolated and the PAD1 gene was cloned by phenotypic complementation. The nucleotide sequence of the smallest complementing fragment was determined. The predicted 242-amino-acid PAD polypeptide is 48.6% identical to the product of dedF of Escherichia coli. PAD activity and CA resistance, but not steady-state PAD1 mRNA levels, are influenced by mitochondrial genotype. PAD1 is a single-copy gene in the yeast genome and not essential for viability. The PAD1 locus was physically mapped to a position approx. 140 kb from the left end of chromosome IV.

Amino Acid Sequence↗

PubMed2004

Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea.

This work describes the purification and partial characterization of a novel antibacterial compound, here named marinocine, produced by Marinomonas mediterranea, a melanogenic marine bacterium with rich secondary metabolism. The antibacterial compound is a protein detected in the medium at death phase of growth. It has been purified to apparent homogeneity from the supernatants of cultures by means of ethanol precipitation followed by column chromatographies on DEAE-Sephadex and Sephacryl HR-200. The protein has an apparent molecular mass of 140-170 kDa according to gel permeation chromatography and non-denaturing SDS-PAGE, although in denaturing SDS-PAGE two mayor bands of 97 and 185 kDa appear. Marinocine is relatively heat-stable and shows a great resistance against many hydrolytic enzymes such as glycosidases, lipase, and proteases. The antibacterial range of the molecule includes Gram-positive and Gram-negative microorganisms, as well as some nosocomial isolates, Staphylococcus aureus and Pseudomonas sp., highly resistant to classical antibiotics. By contrast, marinocine did not show any effect on the eukaryotic microorganisms tested. Regarding eukaryotic CHO cells, the decrease on viability was much lower than the one observed on bacterial cells.

Amino Acids↗

PubMed2004

Dormancy, activation and viability of Rhizopus oligosporus sporangiospores.

Interruption of dormancy to improve viability of Rhizopus oligosporus sporangiospores is crucial for the application of stored starter cultures for fungal (tempe) production. We aimed to assess the extent of dormancy and factors that could result in activation. Whereas heat treatments were unsuccessful, Malt Extract Broth (MEB) showed to be a good activation medium, with 80% of dormant spores being activated as measured by fluorescence microscopy using a fluorescent marker, compared with 11% with the control. Peptone and yeast extract but not glucose played an important role in activating dormant spores. Metabolically active (fluorescent) and swollen spores, followed by germ tubes were obtained after activation in MEB for 25 min., 2 and 4 h, respectively, at 37 degrees C. Simultaneously, some interesting transitions took place. Dormant spores represent 85-90% of the total spores at harvest and after drying. Their number decreased to 21-32% after activation with MEB with a concomitant increase of metabolically active spores. As a result of storage, some dormancy was lost, yielding an increase of active spores from 11.2% at harvest to 28.8% after 3 months storage. Levels of active spores were well correlated with their viability. By activation of dormant spores, their viability increased; levels of viable and active spores were maximum in 1 month old starter (61.7% and 75.9% of total spores, respectively) but gradually decreased with concomitant increase of the number of dead spores.

Colony Count, Microbial↗

PubMed2003

A colorimetric assay for determination of cell viability in algal cultures.

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.

Adaptation, Physiological↗