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PubMed2003

Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques.

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.

Bacteria↗

PubMed2002

Prolonged antifungal effects of clotrimazole-containing mucoadhesive thermosensitive gels on vaginitis.

To develop more effective treatment for vaginal candidasis, clotrimazole (CT) was formulated in mucoadhesive thermosensitive gels (MTG). Several MTG formulations composed of poloxamers (P) 407, 188, and polycarbophil (PC) were prepared. P188 and PC increased the mucoadhesiveness but reduced the syringebility of liquid forms of the gels. Based on the balance between the mucoadhesiveness and syringebility, MTG composed of P407/P188/PC (15/15/0.2 or 15/20/0.2) were further studied. Of the two MTG, the formulation with 15% of P188 gelled at higher temperature and revealed lower elastic modulus. In vitro, sustained release of CT from MTG was observed. In vivo antifungal activity of CT, tested against Candida albicans vaginitis in female rats, was significantly prolonged after vaginal delivery using MTG. At 10 days post-dose, the c.f.u. of C. albicans was more than 10(4)-fold decreased in MTG-treated groups. Moreover, the vaginal delivery of CT in MTG enhanced the viability of epithelial cells without affecting the morphology of vaginal mucosa. These results indicate that CT-containing vaginal MTG might be further developed for safe, convenient, and effective treatment of vaginal candidasis with reduced dosing interval.

Adhesives↗

PubMed1992

A comparison of the different methods available for determining BCG-macrophage interactions in vitro, including a new method of colony counting in broth.

Different methods of determining BCG viability based on colony forming unit (CFU) counting and radio-isotope labelling were comparatively assessed. These included radio-isotope labelling with [3H]uracil, [3H]uridine, [3H]glycerol, and CFU counting, by both agar plate dilution, and microcolony counting in broth. The sensitivity ranges of the different techniques were determined in both macrophage-free and macrophage-treated systems and used to assess the anti-mycobacterial potential of human monocyte-derived macrophages following BCG infection.

Colony Count, Microbial↗

PubMed1989

Mechanism of penicillin killing in the absence of bacterial lysis.

Exposure of group A streptococci (a nonlytic-death phenotype) to benzylpenicillin (penicillin G) produced a dose-dependent, rapid, and extensive hydrolysis of total cellular RNA, with the subsequent loss of hydrolysis products from the cell. This loss of RNA correlated well with loss of viability and was not accompanied by solubilization of the cell wall or comparable losses of either protein or DNA. Simultaneous treatment with penicillin G and either chloramphenicol or rifampin resulted in reduced levels of killing and the complete inhibition of RNA loss. These findings define a new mechanism of penicillin G-induced killing in the absence of cell wall disruption and suggest a basis for drug-induced antagonism of penicillin G-mediated nonlytic death.

Bacterial Proteins↗

PubMed1982

Ca2+-tolerant adult canine myocytes: preparation and response to anoxia/acidosis.

A procedure is described for the production of large numbers of Ca2+-tolerant adult canine ventricular myocytes. Gentle tissue dissociation is achieved in Spinner flasks using 320 mosM enzyme buffer containing collagenase hyaluronidase, and trypsin in the presence of millimolar levels of Ca2+. The technique allows for the complete removal of erythrocytes, the gentle removal of closely adhering nonmuscle cells (less than 3% contamination), and the selective removal of damaged from nondamaged myocytes. Total myocyte yields averaged 4-6 x 10(6)/g wet wt; 61.0 +/- 6.5% of the cells have rod-shaped morphology and are "viable" based on trypan blue exclusion. Only 3.9 +/- 1.1% of the rod-shaped cells beat spontaneously when challenged with 2 mM Ca2+. Exposure to 2 mM Ca2+ at 37 degrees C results in minimal loss of viability (2.1 +/- 1.3%/h) based on both trypan blue uptake and creatine kinase release. Simulation of cellular (i.e., membrane) injury in vitro is performed by the separate application of the perturbations of anoxia, acidosis, and low glucose to the canine myocyte suspensions; the data suggest that these myocytes are affected independently by these perturbations and are in good agreement with the results obtained by others using the isolated rat myocyte system.

Acidosis↗

PubMed2004

Use of carvacrol and cymene to control growth and viability of Listeria monocytogenes cells and predictions of survivors using frequency distribution functions.

The antibacterial action of carvacrol and cymene on two Listeria monocytogenes strains (STCC4031 and NCTN4032) was studied. Carvacrol or cymene showed inhibitory effect on the growth of L. monocytogenes during lag and exponential growth phases and was more evident with increasing concentrations in brain heart infusion broth at 30 degrees C. Carvacrol or cymene also decreased the survival of mid-exponential-growth-phase L. monocytogenes STCC4031 cells in potassium-N-2-hydroxy-ethylpiperazine-N-ethanesulfo nic acid, at 30 degrees C. The combination of carvacrol and cymene resulted in an increased antibacterial effect on the growth and a synergistic effect on the viability of L. monocytogenes compared with the natural compounds applied separately. The analysis of survival curves by the Weibull frequency distribution function allowed an accurate prediction of the level of inactivation achieved. Interestingly, an important bactericidal effect (4.7-log reduction) of low concentrations of both antimicrobials combined (0.75 mM) was observed on L. monocytogenes in carrot juice. This study indicates the potential use of carvacrol and cymene applied simultaneously for preservation of minimally processed foods.

Anti-Bacterial Agents↗

PubMed1991

[Comparative studies on the cultivability and morphology of rat hepatocytes isolated by trypsin or collagenase].

In the present study rat hepatocytes isolated either by trypsin or by collagenase were investigated concerning its cultivability and its morphological properties. The cultivation was carried out as monolayer for 24 h. The yields of cells prepared by trypsin or collagenase amounted to 8 x 10(7) cells per liver and 15-30 x 10(7) cells per liver, respectively, with viabilities measured by trypan blue exclusion test of 70-80 and 90-95%, respectively. Nonhepatocytes were not taken into consideration. Using the electron microscopy it could be established that both freshly isolated and 24 h cultured hepatocytes were intact. There were no morphological differences between cells isolated by trypsin and cells isolated by collagenase. After 24 h cultivation hepatocytes prepared by trypsin showed a little tendency in forming a slightly flattened appearance and in forming intercellular contacts.

Animals↗

PubMed1998

Giardia lamblia: in-vitro sensitivity to some chemotherapeutic agents.

The susceptibility of Giardia lamblia to eight chemotherapeutic agents was studied in vitro. The criteria of viability of the parasite was the ability of the parasite to attach to the coverslip surface in situ, and to multiply in drug free media on subcultures. The giardicidal and 50% inhibitory concentrations of each drug were measured. The activity of metronidazole in vitro was found to be similar to that of mepacrine, whilst that of chloroquine was inferior. The sensitivity of G. lamblia to mepacrine and chloroquine is markedly greater than that reported for Entamoeba histolytica.

Animals↗

PubMed2025

Absolute quantification of the living skin microbiome overcomes relic-DNA bias and reveals specific patterns across volunteers.

BACKGROUND: As the first line of defense against external pathogens, the skin and its resident microbiota are responsible for protection and eubiosis. Innovations in DNA sequencing have significantly increased our knowledge of the skin microbiome. However, current characterizations do not discriminate between DNA from live cells and remnant DNA from dead organisms (relic DNA), resulting in a combined readout of all microorganisms that were and are currently present on the skin rather than the actual living population of the microbiome. Additionally, most methods lack the capability for absolute quantification of the microbial load on the skin, complicating the extrapolation of clinically relevant information. RESULTS: Here, we integrated relic-DNA depletion with shotgun metagenomics and bacterial load determination to quantify live bacterial cell abundances across different skin sites. Though we discovered up to 90% of microbial DNA from the skin to be relic DNA, we saw no significant effect of this on the relative abundances of taxa determined by shotgun sequencing. Relic-DNA depletion prior to sequencing strengthened underlying patterns between microbiomes across volunteers and reduced intraindividual similarity. We determined the absolute abundance and the fraction of population alive for several common skin taxa across body sites and found taxa-specific differential abundance of live bacteria across regions to be different from estimates generated by total DNA (live + dead) sequencing. CONCLUSIONS: Our results reveal the significant bias relic DNA has on the quantification of low biomass samples like the skin. The reduced intraindividual similarity across samples following relic-DNA depletion highlights the bias introduced by traditional (total DNA) sequencing in diversity comparisons across samples. The divergent levels of cell viability measured across different skin sites, along with the inconsistencies in taxa differential abundance determined by total vs live cell DNA sequencing, suggest an important hypothesis for certain sites being susceptible to pathogen infection. Overall, our study demonstrates a characterization of the skin microbiome that overcomes relic-DNA bias to provide a baseline for live microbiota that will further improve mechanistic studies of infection, disease progression, and the design of therapies for the skin. Video Abstract.

Humans↗

PubMed2001

[Viability and activity of the lactic bacteria (Streptococcus salivarius ssp thermophilus y Lactobacillus delbrueckii ssp bulgaricus) del yogurt en Venezuela].

National and international legislations have agreed that the population of lactic bacteria in yogurt must be viable and not less than 10(6) ufc/g. In Venezuela, during last years, observations indicate that the number of viable cells in some commercial samples show high variations, as low levels. This research attempted to find the origin of this problem in the local industry. For this purpose 105 commercial samples were analyzed during their shelf life and 32 samples of yogurt prepared in the laboratory following the flow diagram of the local industry. The different conditions of freeze dried lactic culture, were also analyzed. These samples were evaluated for viable cell count of lactic bacteria and possible variations of pH and acidity. The absence or low number of lactic bacteria detected in some commercial samples is due to the use of inadequate working cultures that show imbalanced proportions of the two microorganisms, besides a low count below 106 ufc/g. The succesive propagation and storage time of mother culture, and the overacidification of the product, produce subletal injury to the microbial cells of the yogurt starter culture. The data indicate that manufacturing practices significantly affect the survival of the lactic flora.

Colony Count, Microbial↗

PubMed1996

5-Azacytidine treatment of the fission yeast leads to cytotoxicity and cell cycle arrest.

A fission yeast gene which shares considerable sequence homology with cytosine-specific DNA methyltransferases has recently been identified. This discovery has led us to investigate the effects of the treatment of fission yeast with the nucleoside analogue 5-azacytidine (5-azaC). 5-AzaC is known to inhibit cytosine methylation as a result of the formation of stable covalent complexes between DNA (cytosine-5) methyltransferases (C5 Mtases) and 5-azaC containing DNA. Here we demonstrate that 5-azaC treatment of Schizosaccharomyces pombe leads to reversible cell cycle arrest at the G2/M transition. This reversible arrest is dependent on the cell cycle checkpoint mechanisms which act to prevent the onset of mitosis in the presence of either damaged or unreplicated DNA. Treatment of S. pombe cell division cycle and checkpoint mutants indicates that 5-azaC causes DNA damage and is likely to inhibit a late stage in DNA replication. The data show that viability in the presence of the drug requires both the DNA damage and the replication checkpoint pathways to be functional. 5-AzaC also elicits a transcriptional response which is associated with DNA damage and the inhibition of DNA replication in fission yeast, and this response is absent in cells carrying G2 checkpoint mutations. The implications of these observations for both the use of 5-azaC in cancer chemotherapy and the existence of cytosine methylation in fission yeast are discussed.

Azacitidine↗

PubMedDate not supplied

Comparative in vitro activity of lomefloxacin, a difluoro-quinolone.

Lomefloxacin is a new difluoro-quinolone. In this study, we have determined the in vitro activity of lomefloxacin against a wide range of clinical bacterial isolates and compared it with that of other fluoro-quinolones and some unrelated antimicrobials. Lomefloxacin was very active against Enterobacteriaceae (MIC90, 0.5 micrograms/ml) with activity comparable to that of ofloxacin (MIC90, 0.25 micrograms/ml). Lomefloxacin was moderately active against isolates of Pseudomonas aeruginosa (MIC90, 4 micrograms/ml), and again the activity was comparable to ofloxacin (MIC90, 4 micrograms/ml) but was eightfold less than ciprofloxacin (MIC90, 0.5 micrograms/ml). Lomefloxacin was also active against isolates of Staphylococcus aureus (MIC90, 1 micrograms/ml), irrespective of methicillin susceptibility, and this activity was most comparable to ofloxacin (MIC90, 0.5 micrograms/ml) and ciprofloxacin (MIC90, 0.5 micrograms/ml). Lomefloxacin was fourfold less active than either ofloxacin or ciprofloxacin against isolates of Enterococcus faecalis (MIC90, 8 micrograms/ml) and Streptococcus pneumoniae (MIC90, 8 micrograms/ml). In common with ofloxacin and ciprofloxacin, lomefloxacin was very active against isolates of Neisseria spp. (MIC90, less than or equal to 0.06 micrograms/ml), Haemophilus spp. (MIC90, less than or equal to 0.06 micrograms/ml), Legionella spp. (MIC90, less than or equal to 0.06 micrograms/ml), Vibrio spp. (MIC90, less than or equal to 0.06 micrograms/ml), and Campylobacter jejuni (MIC90, 1 microgram/ml). Lomefloxacin showed poor activity against isolates of Bacteroides spp. (MIC90, 16 micrograms/ml) or Clostridium difficile MIC90, 32 micrograms/ml) and was only moderately active against isolates of Clostridium perfringens (MIC90, 2 micrograms/ml), Peptostreptococcus spp. (MIC90, 4 micrograms/ml), Chlamydia trachomatis (MIC90, 4 micrograms/ml), Mycoplasma hominis (MIC90, 2 micrograms/ml), and Urea-plasma urealyticum (MIC90, 8 micrograms/ml). Lomefloxacin was found to be bactericidal at concentrations generally close to the MIC with greater than 3 log10 reduction in viability of exponentially dividing cultures of Escherichia coli and S. aureus within 5 hr of exposure to concentrations at eight times the MIC. These results indicate a potential clinical role for lomefloxacin in the treatment of genitourinary tract infections caused by Gram-positive and Gram-negative bacteria, respiratory tract infections caused by susceptible organisms, and soft tissue infections caused by S. aureus.

4-Quinolones↗

PubMed2005

A novel and convenient self-drying system for bacterial preservation.

An effective microbial preservation technology for the long-term storage of viable prokaryotic cells is described. The method combines an almost instantaneous drying step with minimal stress to the cells during drying to maximize survival on rehydration. This is achieved by contact of a microlitre aliquot of a bacterial suspension with a novel, pre-dried activated charcoal cloth based matrix contained within a re-sealable system that can then be stored. The simple methodology completely circumvents the requirement for further drying or preparation. Using this method, a standard laboratory Escherichia coli strain was successfully revived following 390 days storage at 4, 20 and 30 degrees C. Data obtained yielded approximately 20%, 6% and 0.1% viable organisms at the aforementioned temperatures, respectively, following initial inoculations of 1.1 x 10(8)/microl cells. While these figures represent a significant viability loss, there is sufficient recovery of microorganisms required for maintaining culture collections.

Aliivibrio fischeri↗

PubMed2000

Lack of antimicrobial effect on periodontopathic bacteria by ultrasonic and sonic scalers in vitro.

BACKGROUND: The purpose of this study was to assess the antimicrobial effects of a sonic and ultrasonic scaler generally used for subgingival scaling on gram-negative and gram-positive periodontopathic bacteria. METHOD: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, or Peptostreptococcus micros were suspended in Schaedler's broth medium and treated by a sonic or a magnetostrictive ultrasonic scaler for 30 s and 150 s in vitro. Bacterial suspensions treated by an ultrasonic cell disruptor served as a positive control and untreated bacterial suspensions served as a negative control. Following sonication, samples were serially diluted, streaked on blood agar plates and incubated for 2-5 days at 37 degrees C. RESULTS: Treatment by the sonic or ultrasonic scaler for up to 150 s did not reduce the viability of any of the tested periodontal pathogens. Compared to untreated controls, the viability of A. actinomycetemcomitans and P. gingivalis was significantly (p<0.05) reduced only following ultrasonication with the cell disruptor after 30 s (0.72 and 0.54 log CFU/ml, respectively) and of A. actinomycetemcomitans, P. gingivalis, C. rectus, and P. micros after 150 s (1.98, 1.34, 1.95 and 1.98 log CFU/ml, respectively). CONCLUSION: The data of the study may indicate that the assessed sonic and ultrasonic scaler used for subgingival debridement do not result in killing of the tested periodontal pathogens.

Colony Count, Microbial↗

PubMed1990

kem mutations affect nuclear fusion in Saccharomyces cerevisiae.

We have identified mutations in three genes of Saccharomyces cerevisiae, KEM1, KEM2 and KEM3, that enhance the nuclear fusion defect of kar1-1 yeast during conjugation. The KEM1 and KEM3 genes are located on the left arm of chromosome VII. Kem mutations reduce nuclear fusion whether the kem and the kar1-1 mutations are in the same or in different parents (i.e., in both kem kar1-1 X wild-type and kem X kar 1-1 crosses). kem 1 X kem 1 crosses show a defect in nuclear fusion, but kem 1 X wild-type crosses do not. Mutant kem 1 strains are hypersensitive to benomyl, lose chromosomes at a rate 10-20-fold higher than KEM+ strains, and lose viability upon nitrogen starvation. In addition, kem 1/kem 1 diploids are unable to sporulate. Cells containing a kem 1 null allele grow very poorly, have an elongated rod-shape and are defective in spindle pole body duplication and/or separation. The KEM 1 gene, which is expressed as a 5.5-kb mRNA transcript, contains a 4.6-kb open reading frame encoding a 175-kD protein.

Amino Acid Sequence↗

PubMed2004

Selective and effective bactericidal activity of the cobalt (II) cation against Helicobacter pylori.

BACKGROUND: Although the anti-Helicobacter pylori activity of bismuth is well established, the therapeutic potential of other metal ions against the organism is not known. MATERIALS AND METHODS: We measured the minimum inhibitory concentrations of a series of metal ions, including several cobalt (II) compounds against four type strains and seven clinical isolates of H. pylori using three standard broth culture media and a defined medium. Other intestinal bacteria were also investigated for specificity of action. RESULTS: Cobalt chloride had marked activity against H. pylori (minimum inhibitory concentration range was 0.03-1.0 mg/l). The effect was specific because other transition metals had no effect and other intestinal bacteria were not affected by cobalt chloride. Activity was attributable to free cobalt ions as ligands inhibited activity in proportion to their affinity for the ions. Inhibition of cobalt activity was also observed in the presence of nickel, in a dose dependent fashion. However, cobalt activity was not directed towards the nickel-dependent urease enzyme because its effect was similar in wild-type and urease negative mutant strains of H. pylori. Finally, the viability of H. pylori was reduced at the same rate with 2 mg/l cobalt as with 1 mg/l amoxicillin. CONCLUSIONS: Cobalt competes for nickel in its acquisition by H. pylori, but mediates toxicity in a nonurease dependent fashion. As cobalt MIC is similar to some antibiotics and 10 to a hundred times lower than for bismuth, cobalt may represent an effective form of therapy for H. pylori infection.

Cations, Divalent↗

PubMed2002

Characterization of a novel, broad-based fungicidal activity for the antiarrhythmic drug amiodarone.

Fungal infections are common in patients with acquired immunodeficiency syndrome and pose a major health management problem. There is a need for identification of new antifungals to complement the limited current repertoire and to combat newly arising resistant fungal strains. We have identified a novel antifungal activity for the antiarrhythmic drug amiodarone. Extensive characterization of this activity shows that amiodarone exhibits a growth inhibition for several diverse fungi, including species of Cryptococcus, Saccharomyces, Aspergillus, Candida, and Fusarium. The antifungal activity was shown to be fungicidal; Cryptococcus neoformans treated with amiodarone lost viability within hours of drug exposure. Growth inhibition could be suppressed by addition of very high concentrations (10 mM) of calcium to the medium, suggesting that disruption of calcium homeostasis was involved in the antifungal activity. Direct measurement of radiolabeled calcium efflux showed that addition of amiodarone resulted in an immediate efflux of cellular calcium. In conclusion, amiodarone displays broad-based fungicidal activity and may be acting in part by perturbing the calcium balance.

Amiodarone↗

PubMed1998

Plaque mineralisation in vitro.

Dental calculus is plaque mineralised by deposition of calcium and phosphate resulting from interactions between the oral microbial plaque flora and components of oral fluids. An artificial-mouth microcosm dental plaque culture system has been developed to study aspects of plaque mineralisation, including pH control. Five plaques were grown from saliva under simulated oral conditions in a mucin-containing medium, and sucrose was applied to mimic meals. The plaques were mineralised with a urea-based, calcium-phosphate-monofluorophosphate-urea (CPMU) mineralising solution. Alkaline pH oscillations were generated by the plaques in response to CPMU applications, and an acidic oscillation followed sucrose applications. Plaque mineralisation by the CPMU procedure was almost totally dependent on the urea present in the mineralising solution, but total mineralisation also increased as the resting pH increased as a result of urea in the medium. Following four CPMU applications with a sucrose application every 12 hours improved plaque viability and mineralisation. The plaque mineral formed resembled a carbonated hydroxyapatite; other potential calcium phosphate minerals were undetectable except for calcium carbonate. A wide range of mineral deposition patterns in plaque were seen by electron microscopy.

Acids↗