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PubMed1995

Simple method for quantifying viable bacterial numbers in sputum.

AIMS: To establish a simple method of quantitative culture for determining the viable bacterial numbers present in expectorated sputum samples. METHODS: Sputum samples were homogenised with dithiothreitol, sterile saline or glass beads to determine which method recovered the greatest number of viable bacteria. Culture broths were also incubated with dithiothreitol and sampled over time to determine its effect on bacterial viability. Sputum samples homogenised with dithiothreitol were diluted in sterile saline and sampled using either standard bacteriological loops or a precision pipette to determine which method resulted in the least variation. RESULTS: Homogenisation of sputum using dithiothreitol increased the recovery of viable bacteria compared with sterile glass beads and/or saline, with no apparent effect on bacterial viability when incubated with culture broths. By inoculating agar plates with 10(-3), 10(-4) and 10(-5) dilutions of the homogenised sputum sample, all potential pathogens could easily be identified. A 10 microliter sample volume dispensed by precision pipette and spread with a "hockey stick" resulted in the least variation between plates (less than 16%) and an even distribution of bacterial colonies. Numbers of viable bacteria recovered from different aliquots of individual sputum samples were generally of the same order of magnitude. CONCLUSIONS: This method represents a relatively quick and simple technique for accurately quantifying viable bacteria present in sputum samples. The use of a small portion appears to be representative of the sample as a whole.

Colony Count, Microbial↗

PubMed1999

Caffeine can override the S-M checkpoint in fission yeast.

The replication checkpoint (or 'S-M checkpoint') control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control.

Amino Acid Sequence↗

PubMed1994

The Tn5 bleomycin resistance gene confers improved survival and growth advantage on Escherichia coli.

The bleomycin resistance gene (ble) of transposon Tn5 is known to decrease the death rate of Escherichia coli during stationary phase. Bleomycin is a DNA-damaging agent and bleomycin resistance is produced by improved DNA repair which also requires the host genes aidC and polA coding, respectively, for an alkylation-inducible gene product and DNA polymerase I. In the absence of the drug, this DNA repair system is believed to cause the slower death rate of bleomycin-resistant bacteria. In this study, the effect of ble and aidC genes on the viability of bacteria and their growth rate in chemostat competitions was studied. The results indicate, that bleomycin-resistant bacteria display greater fitness under these conditions. Another beneficial effect of transposon Tn5 had been previously attributed to the insertion sequence IS 50 R. We were not able to reproduce this result with IS 50 R, however, the complete transposon was beneficial under similar conditions. Moreover, we showed the Tn5 fitness effect to be aidC-dependent. The ble gene was discovered after the fitness effect of IS 50 R had been established; it has not previously been considered to mediate the beneficial effect of Tn5. This possibility is discussed based on the molecular mechanism of bleomycin resistance.

Bleomycin↗

PubMed2003

The effect of increasing copper content in phosphate-based glasses on biofilms of Streptococcus sanguis.

This paper reports the effect of a series of phosphate-based glasses based on the Na(2)O-CaO-P(2)O(5) system doped with increasing amounts of copper and the effect of this increasing copper content on the viability of an in vitro biofilm of Streptococcus sanguis over an 8 day period in a constant depth film fermenter. The addition of copper to the glass caused the solubility to change, so the glasses were adjusted in order that their solubility in artificial saliva was nominally the same (0.3062 +/- 0.07 mg cm(-2) h(-1)). Initial experiments on glasses with 1.5% and 10% copper showed that after 6 h there was no statistical difference between the copper containing glasses and the non-copper containing glass and HA in terms of the viability of the biofilms. However, at 24 h there was an approximately 0.8-0.9 log reduction in viability of the biofilms grown on the 5% copper glass and an approximately 1.0-1.3 log reduction for the 10% copper containing glass. Further experiments on the glass with 10% copper and another glass with 15% copper were carried out in a time dependent study. For both glasses a clear decrease in viable counts at 24 h was found but for both glasses these returned to levels similar to those of controls. The initial decrease in viability during the first 24 h is likely to be due to the antibacterial effect of the copper and this could be correlated with copper content. The recovery after 24 h is probably due to the dead cells forming a barrier, making diffusion of the antibacterial ions, increasingly difficult. This study has shown that phosphate-based glasses could potentially be used to deliver antibacterial ions to help combat oral infections. Copper, which has been shown to have antibacterial properties, could be incorporated but some development of the glasses used in this investigation may be required. Further work is needed to determine the effectiveness of copper containing glasses on oral bacterial communities.

Anti-Bacterial Agents↗

PubMed1989

Phagocytic activity of polymorphonuclear leukocytes on Escherichia coli previously exposed to metronidazole.

A study was made of the action of different concentrations of metronidazole of the viability of Escherichia coli under aerobic and anaerobic conditions. The viability of E. coli was reduced by 60 to 99% after 24 hours of anaerobic incubation, according to the concentration of metronidazole tested. In addition, there were significant morphological changes in the bacteria. Exposure of antibiotic-induced filaments of E. coli LP 136 to phagocytosis caused the cfu/ml value to drop by 60% after 120 minutes. Under identical conditions, using the mutant strain E. coli RYC 819, which did not become filamented by metronidazole although it did present similar ultrastructural changes, this reduction reached 83%. These results may explain the therapeutic success of metronidazole in polymicrobial infections.

Adult↗

PubMed1999

In vitro antibacterial effect of RC-Prep components on Streptococcus sobrinus.

The aim of this study was to examine the bacteriostatic and bactericidal effects of the components of RC-Prep: EDTA, urea peroxide and glycol. The minimal inhibitory concentration and the minimal bactericidic concentration of EDTA, urea peroxide and glycol were tested on the viability of Streptococcus sobrinus (S. sobrinus). The combined antibacterial effect of these components was also examined on S. sobrinus. The minimal inhibitory concentration of EDTA was found to be 0.125%, of urea peroxide 0.25%, and of glycol 30%. The minimal bactericidic concentration of EDTA was 0.25%, of urea peroxide 0.5% and of glycol 50%. An antibacterial synergistic effect was found between specific combinations of urea peroxide, EDTA and glycol. No antagonistic effects were found in the various combinations between the components. The antimicrobial effect of the components of RC-Prep was enhanced in specific combinations of the ingredients. This effect might contribute to the intracanal cleansing properties of this medication.

Anti-Infective Agents, Local↗

PubMed1998

Use of an autobioluminescent Salmonella hadar to monitor the effects of acid and temperature treatments on cell survival and viability on lactic acid-treated poultry carcasses.

To determine the long-term effects of a lactic acid rinse on viability and recovery of pathogens, Salmonella Hadar was isolated from poultry and bioluminescent constructs obtained by transformation with the lux (CDABE) gene cassette from Photobacterium phosphoreum. Results indicated that the transformed Salmonella Hadar lux was otherwise phenotypically similar to the wild-type strain. Viability studies were performed by measuring luminescence following lactic acid treatment of turkey breast and subsequent storage at -12, 0, 5, and 10 degrees C. The ability of the S. Hadar lux strain to recover was determined by monitoring light output after incubation at 22 degrees C for 10 h. The results showed that metabolic activity was significantly (P < 0.05) affected by lactic acid and by storage temperatures of -12, 0, and 5 degrees C. The lowest recovery rate was observed after rinsing with lactic acid and storing at 5 degrees C. The study demonstrated that bacterial bioluminescence is an effective way of monitoring in "real time" the ability of bacteria to recover from stress.

Abattoirs↗

PubMed1978

[Properties of a Candida albicans strain possessing an increased sensitivity to polyene antibiotics].

Characteristics of a mutant of the yeast-like fungus Candida albicans with increased sensitivity to amphotericin B, levorin and nystatin is presented. Increased sensitivity to the polyens in the isolate must be associated with changes in the cell wall and/or with decreased total viability of the strain. The following factors were in favour of this: presence of significant amounts of pseudomycelial cells with thinner cell walls as compared to the yeast cells in the population of the mutant strain; a higher rate of potassium liberation from the cells of the mutant strain in the presence of amphotericin B; a lower growth rate of the mutant strain on solid and liquid media; an increased death rate of the cells in the intact culture of the mutant strain as compared to the initial strain; absence of changes in the absorption spectrum of sterols isolated from the cells of the mutant strain as compared to the initial culture.

Amphotericin B↗

PubMed2000

Evaluation of ChemChrome V6 for bacterial viability assessment in waters.

The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared. Both dyes are fluorogenic esters converted to free fluorescein by esterase activity. The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters. Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non-labelled with the CB. In contrast, CV6-stained bacteria were easily detected by both flow cytometry and solid-phase cytometry. As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters. Viable counts determined by CV6 staining were always higher than cfu counts. In contrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts.

Bacteria↗

PubMed1999

Action of inorganic tin and organotins on a hydrocarbon-using yeast, Candida maltosa.

Two inorganic tin compounds, as well as mono- and tri-substituted methyl-, butyl-, and phenyltins were examined for their interactions with a hydrocarbon-using strain of Candida maltosa. Neither of the inorganic tins (SnII and SnIV) inhibited growth at concentrations up to 0.8 mM while binding to the yeast cells occurred to levels of 0.3 and 0.23 mM Sn/g cells, respectively. Neither inorganic tin caused leakage of potassium from the yeast cells. Among the organotins, tributyl- and triphenyltins caused total viability loss and near maximum potassium loss at initial concentrations of 0.08 mM. For these compounds binding to the cells increased with increasing initial concentrations to maximum values of 0.51 and 0.65 mM Sn/g cells respectively. The other organotin compounds were not inhibitory and did not cause potassium leakage from the cells. Tin from them became cell associated only in the cases of monobutyl- and monophenyl tin, which were bound at markedly lower levels (<0.1 mM Sn/g cells). These results are consistent with the conclusion that the inhibitory compounds can act on the yeast cell membrane but, although binding to the cells is a prerequisite for growth inhibition and cell leakage, no simple correlation was established between binding levels and toxicity effects.

Candida↗

PubMed2000

Determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method.

BACKGROUND: Recent research has indicated that bacteria within a biofilm may undergo changes in susceptibility to antimicrobial agents when compared to planktonic forms. This study was conducted to determine the bactericidal effect of an essential oil-containing mouthrinse (Listerine Antiseptic) on dental plaque bacteria in situ. METHODS: 1-day-old plaque in 17 subjects was sampled at baseline from the buccal surfaces of diagonally contralateral maxillary and mandibular bicuspids and 1st molars. Subjects were then randomly assigned either an essential oil mouthrinse or a sterile saline negative control and rinsed under supervision with 20 ml for 30 s. 30 min later, plaque was sampled from the remaining contralateral posterior teeth. Subjects repeated these procedures with their respective alternate rinse after 1 week. Pooled plaque samples from each subject at each sampling period were stained with a commercially-available fluorescent stain which fluoresces live and dead bacteria green and red, respectively. The stained plaque specimens were analyzed using computerized image analysis. A separate in vitro study was conducted to determine the relationship between the % red stain per sample and bacterial viability. RESULTS: Analysis of vital stained plaque specimens indicated that following rinsing with the essential oil mouthrinse, 78.7% of bacteria were dead compared to 27.9% following rinsing with the negative control (p<0.001). The in vitro findings demonstrated that the % red stain per sample is reflective of actual bacterial kill. CONCLUSIONS: This study confirms the findings of previous in vitro and in vivo studies which demonstrated the essential oil mouthrinse to have significant biocidal activity against oral micro-organisms. These studies all support the primacy of a bactericidal mechanism in producing the plaque and gingivitis reductions observed in numerous clinical trials conducted on the essential oil mouthrinse.

Anti-Infective Agents, Local↗

PubMed2000

Bactericidal action of high-power Nd:YAG laser light on Escherichia coli in saline suspension.

Infra-red light (1064 nm) from a high-power Nd:YAG laser caused more than 90% loss of viability of Escherichia coli during exposures that raised the temperature of PBS suspensions of the bacteria to 50 C in a thermocouple-equipped cuvette. In contrast, there was minimal loss of viability after heating the same suspensions to 50 degrees C in a water-bath, or in a PCR thermal cycler. The mechanism of laser killing at 50 degrees C was explored by differential scanning calorimetry, by laser treatment of transparent and turbid bacterial suspensions, and by optical absorbency studies of E. coli suspensions at 1064 nm. Taken together, the data suggested that the bactericidal action of Nd:YAG laser light at 50 degrees C was due partly to thermal heating and partly to an additional, as yet undefined, mechanism. Scanning electron microscopy revealed localized areas of surface damage on laser-exposed E. coli cells.

Colony Count, Microbial↗

PubMed2005

In vitro activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole compared with those of amphotericin B and fluconazole against clinical isolates of Candida spp. and fluconazole-resistant Candida albicans.

OBJECTIVES: The aim of this study was to investigate the in vitro antifungal activity of an isothiosemicarbazone cyclic analogue against isolates of Candida spp. including fluconazole-resistant Candida albicans. METHODS: We investigated the activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2) against 114 clinical isolates of Candida spp., representing five different species, by microdilution, according to the NCCLS method 27-A. The activity against C. albicans biofilms was also investigated. Toxicity in vitro was evaluated by MTT reduction assay. RESULTS: EM-01D2 demonstrated low toxicity, broad spectrum, fungicidal activity and was active against C. albicans and Candida krusei at concentrations lower than those shown by amphotericin B and fluconazole (P < 0.05). It maintained potent in vitro activity against fluconazole-resistant C. albicans isolates. Fungicidal activity occurred at concentrations 1-2 doubling dilutions greater than the corresponding MICs, and time-kill analysis indicated that a 99.9% loss of C. albicans viability occurred after 6 h of incubation in the presence of EM-01D2 at concentrations equal to four times the MIC. EM-01D2 was also active in inhibiting the growth of C. albicans ATCC 10231 biofilms, even though such inhibition occurred at concentrations higher than the MICs determined under planktonic growth conditions. However, when C. albicans biofilms were pre-exposed to subinhibitory concentrations of EM-01D2, a reduction of MIC50 of amphotericin B was observed. CONCLUSIONS: Based on these results, EM-01D2 could represent a template for the development of novel fungicidal agents.

Amphotericin B↗

PubMed2003

Coenzyme Q1-catalyzed luminol chemiluminescent assay for rapid antimicrobial susceptibility testing of Mycobacterium bovis.

Coenzyme Q1 is herein proposed as the best catalyst among coenzymes Q and vitamins K for quinone-catalyzed luminol chemiluminescent assays applied to rapid determination of viability or rapid antimicrobial susceptibility tests of Mycobacterium bovis. Luminol chemiluminescence intensity (LCI) was determined 10 min after the incubation of M. bovis with coenzyme Q1, and was proportional to CFU (colony-forming unit)/ml in the range of 9,000 to 2,250,000. LCI depended on the the production of the superoxide anion (O2-) rather than H2O2 during a 10-min incubation of M. bovis with coenzyme Q1, as superoxide dismutase reduced LCI more effectively than catalase. The minimal inhibitory concentrations (MICs) of 10 kinds of antituberculous agents estimated on the basis of decrease in LCI after one or two days' cultivation were in good agreement with MICs determined by turbidity analysis, which requires upwards of 1 week to complete.

Antibiotics, Antitubercular↗

PubMed2006

Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium.

AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.

Air↗

PubMed1974

In vivo studies of temperature-sensitive recB and recC mutants.

Some in vivo properties of Escherichia coli K-12 strains carrying recB270 (formerly recBts1) and recC271 (formerly recCts1) mutations have been determined. Single recB270 and recC271 mutants appear normal at 30 C with regard to ultraviolet and mitomycin C sensitivity, recombination proficiency, and viability. At 43 C these strains become sensitive to ultraviolet and mitomycin C, while showing only a slight decrease in recombination proficiency. The viable titers of the single mutants are somewhat reduced at 43 C. Double mutant strains carrying polA1 and recB270 or recC271 are inviable at 43 C. The double mutant strain (recB270 recC271) is sensitive to both UV and mitomycin C at 30 C, but shows only slightly reduced recombination proficiency. At 43 C the strain resembles absolute recB and recC mutants in all respects. In addition, the double mutant strain exhibits a temperature-induced drop in viable titer. The triple mutant polA1 recB270 recC271 is viable at 30 C. Two hypotheses are advanced to explain these results.

Cell Survival↗

PubMed1998

Aminoacyl tRNA synthetases as targets for new anti-infectives.

Because resistance has developed to mainline antibiotics, including vancomycin, new antibiotics are now being aggressively sought. For this purpose, aminoacyl tRNA synthetases are being pursued as targets for new drugs. These enzymes are universal and are essential for cell viability. The key to their usefulness lies in being able to find drugs that inhibit a pathogen synthetase but not its human cell counterpart. The possibility for species-specific inhibition was originally demonstrated with a natural product and has now been demonstrated with prototypical drugs that are based on the structure of an intermediate of the aminoacylation reaction. Efficacy of a rationally designed inhibitor has been shown in vivo with a pathogen infection established in an animal model. Although many challenges remain, these early results suggest that synthetases will continue to be of major interest for development of new anti-infectives.

Adenosine Monophosphate↗

PubMed1987

Solid tumor preparation for flow cytometry using a standard murine model.

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol-water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.

Animals↗