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PubMed2000

National Research Council report on potable reuse.

In 1998, the United States National Research Council prepared a report assessing the viability of potable reuse. The report concludes that planned indirect potable reuse is a viable application of reclaimed water but should be considered for implementation only after other alternative measures, such as development of new water sources, nonpotable reuse, and water conservation, have been evaluated and determined not to be economically or technically feasible. Although health-effects research has not indicated that there have been any adverse health effects resulting from drinking highly treated reclaimed water, those data are sparse. There are uncertainties associated with assessing the potential health risks of drinking reclaimed water. The report acknowledges that several issues remain unresolved regarding reclaimed water treatment and reliability, microbial and chemical constituents of concern, and water quality and health effects monitoring. Research needs include methods to detect emerging pathogens, identification and quantification of trace organic compounds, development of suitable in vivo toxicological methods, and evaluation of the effectiveness and reliability of multiple treatment barriers. The report also concludes that direct potable reuse is not a viable option to consider at this time.

Conservation of Natural Resources↗

PubMed2005

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

Bacteriocins↗

PubMed1994

A sensitive assay system screening antiviral compounds against herpes simplex virus type 1 and type 2.

A highly sensitive and accurate assay system was developed for in vitro evaluation of anti-herpes simplex virus (anti-HSV) agents using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and human embryonic lung fibroblast (MRC-5) cells. This assay system was found to be highly sensitive for both HSV-1 and -2. Confluent MRC-5 cells were infected with either HSV-1 KOS strain or HSV-2 G strain of 25 TCID50 in the presence of various concentrations of test compounds. The optical density of formazan was used to determine cell viability. The EC50 values of acyclovir and several other anti-HSV agents were found to be similar to those obtained by the plaque reduction method. These results indicate that this MTT assay is useful for screening anti-HSV-1 and -2 agents.

Acyclovir↗

PubMed1995

The influence of pressure and temperature of compressed CO2 on the survival of yeast cells.

In order to study the potential use of supercritical CO2 extraction for the recovery of products from yeast cell cultures, the effects of CO2 on different yeast strains over a range of pressures and temperatures have been examined. Viability was shown to be dependent on temperature and dissolved CO2 concentration, and can be described by a sigmoidal (S-shaped) curve. Cell death was mainly due to an 'anaesthesia effect' rather than cell rupture. Important differences in sensitivity were observed for the strains studied, with the following order of resistance: Kluyveromyces fragilis > Saccharomyces cerevisiae > Candida utilis.

Candida↗

PubMed2004

Phyllanthus piscatorum, ethnopharmacological studies on a women's medicinal plant of the Yanomamï Amerindians.

The shrub Phyllanthus piscatorum Kunth (Euphorbiaceae) is cultivated by various ethnic groups of the Amazon because of its piscicidal properties. During ethnobotanical fieldwork among the Yanomamï Amerindians in Venezuela we observed that Phyllanthus piscatorum was exclusively cultivated and used by the women. Aerial parts of this herbaceous shrub are employed as fish poison and medicine to treat wounds and fungal infections. In addition, the leaves are used as tobacco substitute. Ethnobotanical data regarding the context of the use of this plant are presented. To validate ethnobotanical information related to its medicinal indications, antimicrobial, and antiprotozoal properties of water, methanol (MeOH) and dichloromethane (DCM) extracts were studied. No activity against Gram-positive bacterial strains but significant activity against the fungi Aspergillus fumigatus, Aspergillus flavus and the yeast Candida albicans were found. All extracts showed weak in vitro activity against Plasmodium falciparum and Trypanosoma brucei rhodesiense. The extracts were further investigated for cytotoxic effects in an in vitro test system with leukemia Jurkat T, HeLa, and human peripheral mononuclear blood cells (PBMCs). During the first 48 h the extracts did not exhibit any cytotoxicity. After 72 h the DCM extract potently inhibited viability of HeLa cells. Although in several communities along the upper Orinoco the cultivation and use of Phyllanthus piscatorum is being lost because of the ongoing acculturation, the traditional medicinal use of Phyllanthus piscatorum might provide an effective and cheap remedy against dermatological diseases linked with Candida albicans infections.

Animals↗

PubMed1998

Cytochrome c biogenesis is involved in the transposon Tn5-mediated bleomycin resistance and the associated fitness effect in Escherichia coli.

The transposon Tn5 ble gene and the Escherichia coli alkylation-inducible aidC locus are co-operatively involved in the resistance to the anti-cancer drug and DNA-cleaving agent bleomycin and enhance fitness of bacteria in the absence of the drug. In this report, we demonstrate that the aidC locus is identical to nrfG, the last gene of the nrf operon involved in the periplasmic formate-dependent nitrite reduction. In the presence of Ble, NrfG expression is specifically induced and restores both bleomycin resistance and its associated beneficial growth effect in an aidC- strain. In vitro DNA protection assays reveal that purified Ble prevents bleomycin-mediated DNA breakage, as do bleomycin-binding proteins. Similarities between haems of the cytochrome c biogenesis nrf pathway and iron bleomycin suggest a DNA repair-independent molecular mechanism for both bleomycin resistance and increased viability. The Ble protein binds bleomycin and prevents DNA breakage. It also induces the nrf locus that may assimilate bleomycin into haem for extracellular transport or inactivate bleomycin. Inactivation of potent DNA oxidants confers a better fitness to the bacterium carrying the transposon, suggesting a symbiotic relationship between host and transposon.

Anti-Bacterial Agents↗

PubMed2006

Cationic lipids and surfactants as antifungal agents: mode of action.

OBJECTIVES: To determine the mechanism of antimicrobial action for cationic lipid dioctadecyldimethylammonium bromide (DODAB) and hexadecyltrimethylammonium bromide (CTAB) against Candida albicans. METHODS: Determination of DODAB or CTAB adsorption isotherms; cell viability; cell electrophoretic mobility (EM); and leakage of small phosphorylated compounds, proteins or DNA from fungus or haemoglobin from erythrocytes. RESULTS: High affinity isotherms for CTAB and DODAB adsorption onto fungus cells (10(8) cfu/mL) yield limiting adsorption at 7.8 and 3.7 x 10(9) molecules per cell, respectively. Negatively charged C. albicans cells (10(6) cfu/mL) remain viable whereas positively charged ones die. At 0.3 mM CTAB or 0.01 mM DODAB, EM is zero and fungus viability is 50%. Cells start to die at submicellar CTAB concentrations and fungus lysis does not play a significant role in the mechanism of antifungal action. Over 0.1-10 mM CTAB or DODAB, there is no leakage of tested compounds from C. albicans cells despite the low cell viability. In contrast to the fungus, under isotonic conditions, cationic amphiphiles induce haemolysis over a range of low DODAB (>0.01 mM) and CTAB (>0.001 mM) concentrations. CONCLUSIONS: The critical phenomenon determining antifungal effect of cationic surfactants and lipids is not cell lysis but rather the change of cell surface charge from negative to positive.

Antifungal Agents↗

PubMed1993

Some factors affecting airborne survival of Pseudomonas fluorescens indoors.

The effect of relative humidity (RH) on the airborne survival of a Pseudomonas fluorescens strain was studied at 20, 40, 60 and 80% RH indoors. The aero-stable spore of Bacillus subtilis subsp. niger, used as a tracer of physical losses was compared with a light scattering particle counter, as there were doubts about the reliability of the spore as tracer. The Rion counter was validated before use and found to give a good estimate of relative physical losses providing spray suspensions contained between 10(7) and 10(9) cfu ml-1 cells and that the humidity was not more than 80% RH. Pseudomonas fluorescens, strain P+S+, suspended in distilled water survived best at mid humidities and least at 80% RH. When suspended in 1% glycerol there was an apparent 'increase' in viability after an initial rapid reduction. This was thought to be due to delay in equilibration of glycerol in the cell membrane after concentration on dehydration. The cells were thought to be unstable and sensitive to the stress of rehydration before equilibration occurred. The findings are discussed in relation to Cox's theories of outer membrane damage on aerosolization.

Aerosols↗

PubMed1987

Effect of antibiotics on toxin production and viability of Clostridium perfringens.

We have recently reported (D.L. Stevens, K.A. Maier, B.M. Laine, and J.E. Mitten, J. Infect. Dis. 155:220-228, 1987) that clindamycin, rifampin, and tetracycline were more efficacious than penicillin in the treatment of fulminant gas gangrene in mice caused by Clostridium perfringens. We hypothesize that antibiotic efficacy correlated with bactericidal or toxin-suppressing properties of these agents. To investigate the possibility that penicillin is only bacteriostatic against C. perfringens, we performed macrobroth dilution MIC and MBC determinations using C. perfringens ATCC 13124. Mean MICs were equal to MBCs for the following antibiotics (micrograms per milliliter): clindamycin, 0.07; tetracycline, 0.05; rifampin, 0.03; metronidazole, 0.69; and penicillin, 0.27. The MIC/MBCs of chloramphenicol were 1.50/3.10 (micrograms/ml). Because antibiotic efficacy did not correlate with bactericidal activity, we measured alpha-toxin activity and found complete suppression of alpha-toxin activity by tetracycline, metronidazole, rifampin, clindamycin, and chloramphenicol at concentrations equal to the MIC. In contrast, alpha-toxin activity persisted at concentrations of penicillin equal to and above the MIC. The dynamics of bacterial killing and kinetics of alpha-toxin production were next studied in log-phase cultures of C. perfringens with antibiotic concentrations 10 times the MIC. Clindamycin, metronidazole, and rifampin all caused rapid reductions in viability, turbidity, and alpha-toxin activity by 15 to 45 min. In contrast, penicillin demonstrated slower bacterial killing, increased turbidity (62.6% of control), and persistent alpha-toxin activity (80% of control values) for 2 h. Tetracycline and chloramphenicol were the least effective in reducing viability; however, the turbidity of cultures did not increase, and alpha-toxin activity was not detectable. Toxin suppression and rapid bacterial killing may in part explain the observed superior therapeutic efficacy of clindamycin, rifampin, and metronidazole compared with penicillin in the treatment of experimental gas gangrene.

Anti-Bacterial Agents↗

PubMed2002

Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect.

We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 micro g/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 micro g/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 micro M. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r = -0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.

Antifungal Agents↗

PubMed1999

Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro.

Mycobacterium tuberculosis has a relatively high resistance to killing by hydrogen peroxide and organic peroxides. Resistance may be mediated by mycobacterial catalase-peroxidase (KatG) and possibly by alkyl hydroperoxide reductase (AhpC). To determine the interrelationship between sensitivity to H2O2, catalase and peroxidase activities, and bacillary growth rates measured both intracellularly in human monocytes and in culture medium, we examined one laboratory strain, two clinical isolates, and three recombinant strains of M. tuberculosis with differing levels of KatG and AhpC. Five of the mycobacterial strains had intracellular doubling times of 27 to 32 h, while one KatG-deficient clinical isolate (ATCC 35825) doubled in approximately 76 h. Killing of mycobacteria by exogenously added H2O2 was more pronounced for intracellular bacilli than for those bacilli derived from disrupted monocytes. Strains with no detectable KatG expression or catalase activity were relatively sensitive to killing (43 to 67% killing) by exogenous H2O2. However, once even minimal catalase activity was present, mycobacterial catalase activity over a 10-fold range (0.56 to 6.2 U/mg) was associated with survival of 85% of the bacilli. Peroxidase activity levels correlated significantly with resistance of the mycobacterial strains to H2O2-mediated killing. An endogenous oxidative burst induction by 4beta-phorbol 12beta-myristate 13alpha-acetate treatment of infected monocytes reduced the viability of the KatG null strain (H37Rv Inhr) but not the KatG-overexpressing strain [H37Rv(pMH59)]. These results suggest that mycobacterial resistance to oxidative metabolites (including H2O2 and other peroxides) may be an important mechanism of bacillary survival within the host phagocyte.

Bacterial Proteins↗

PubMed1992

[Comparison of bidirectional collagenase perfusion with one way perfusion in the isolation of periportal or perivenous hepatocytes in rats].

To selectively isolate hepatocytes from the periportal (PP) and perivenous (PV) regions of rat liver acinus, we compared two different perfusion methods with collagenase: the bidirectional perfusion method (2-P) and the one way perfusion method (1-P). We determined the optimal conditions for each method on the basis of the zonal selectivity of isolated hepatocytes with a hematoxylin-eosin stained liver specimen. By both methods, hepatocytes were selectively isolated from the PP and PV regions. Comparing cell yield and cell viability after the two perfusion methods, 1-P was found to be better than 2-P. Density gradient centrifugation with Percoll was found to be an effective procedure for removing the damaged hepatocytes. We concluded that 1-P could isolate viable PP and PV hepatocytes with a normal glucagon-cyclic AMP response and ultrafine structure in high yield from rat liver.

Animals↗

PubMed1992

OmpC and OmpF porins influence viability and culturability of Escherichia coli cells incubated in seawater.

The contribution of the major outer membrane porins OmpF and OmpC to the maintenance of viability and culturability of Escherichia coli cells in seawater was analyzed using isogenic mutant strains lacking one or both porins. Cells that possessed OmpF and OmpC survived better than those lacking one or both of them. However, the results differed, depending on whether the cells were adapted to high osmolarity or not before transfer to seawater. When cells were grown at low osmolarity, survival was largely influenced by porins, the OmpF+ strains surviving better than those lacking this porin. Addition of an OmpF plasmid to OmpF- OmpC- cells also improved their viability. When grown at high osmolarity, the role of porins was less critical since both the viability and culturability of the cells increased. However, cells that expressed only OmpC showed the most dramatic loss of viability. Cells lacking both OmpF and OmpC exhibited a higher loss of viability and culturability in seawater. Regarding the influence of porins on survival, these results show that the conditions that prevail during the growth of cells before their transfer to seawater are highly influential: cells that express the porin corresponding to the growth conditions they are in at the time of transfer survive better.

Colony Count, Microbial↗

PubMed2005

Genes and molecules involved in Aspergillus fumigatus virulence.

Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systemic diseases (invasive aspergillosis) with high mortality rates. Pathogenicity depends on immune status of patients and fungal strain. There is no unique essential virulence factor for development of this fungus in the patient and its virulence appears to be under polygenetic control. The group of molecules and genes associated with the virulence of this fungus includes many cell wall components, such as beta-(1-3)-glucan, galactomannan, galactomannanproteins (Afmp1 and Afmp2), and the chitin synthetases (Chs; chsE and chsG), as well as others. Some genes and molecules have been implicated in evasion from the immune response, such as the rodlets layer (rodA/hyp1 gene) and the conidial melanin-DHN (pksP/alb1 gene). The detoxifying systems for Reactive Oxygen Species (ROS) by catalases (Cat1p and Cat2p) and superoxide dismutases (MnSOD and Cu, ZnSOD), had also been pointed out as essential for virulence. In addition, this fungus produces toxins (14 kDa diffusible substance from conidia, fumigaclavin C, aurasperon C, gliotoxin, helvolic acid, fumagilin, Asp-hemolysin, and ribotoxin Asp fI/mitogilin F/restrictocin), allergens (Asp f1 to Asp f23), and enzymatic proteins as alkaline serin proteases (Alp and Alp2), metalloproteases (Mep), aspartic proteases (Pep and Pep2), dipeptidyl-peptidases (DppIV and DppV), phospholipase C and phospholipase B (Plb1 and Plb2). These toxic substances and enzymes seems to be additive and/or synergistic, decreasing the survival rates of the infected animals due to their direct action on cells or supporting microbial invasion during infection. Adaptation ability to different trophic situations is an essential attribute of most pathogens. To maintain its virulence attributes A. fumigatus requires iron obtaining by hydroxamate type siderophores (ornitin monooxigenase/SidA), phosphorous obtaining (fos1, fos2, and fos3), signal transductional falls that regulate morphogenesis and/or usage of nutrients as nitrogen (rasA, rasB, rhbA), mitogen activated kinases (sakA codified MAP-kinase), AMPc-Pka signal transductional route, as well as others. In addition, they seem to be essential in this field the amino acid biosynthesis (cpcA and homoaconitase/lysF), the activation and expression of some genes at 37 degrees C (Hsp1/Asp f12, cgrA), some molecules and genes that maintain cellular viability (smcA, Prp8, anexins), etc. Conversely, knowledge about relationship between pathogen and immune response of the host has been improved, opening new research possibilities. The involvement of non-professional cells (endothelial, and tracheal and alveolar epithelial cells) and professional cells (natural killer or NK, and dendritic cells) in infection has been also observed. Pathogen Associated Molecular Patterns (PAMP) and Patterns Recognizing Receptors (PRR; as Toll like receptors TLR-2 and TLR-4) could influence inflammatory response and dominant cytokine profile, and consequently Th response to infec tion. Superficial components of fungus and host cell surface receptors driving these phenomena are still unknown, although some molecules already associated with its virulence could also be involved. Sequencing of A. fumigatus genome and study of gene expression during their infective process by using DNA microarray and biochips, promises to improve the knowledge of virulence of this fungus.

Animals↗

PubMed2006

Microbial exopolymers link predator and prey in a model yeast biofilm system.

Protistan grazing on biofilms is potentially an important conduit enabling energy flow between microbial trophic levels. Contrary to the widely held assumption that protistan feeding primarily involves ingestion of biofilm cells, with negative consequences for the biofilm, this study demonstrated preferential grazing on the noncellular biofilm matrix by a ciliate, with selective ingestion of yeast and bacterial cells of planktonic origin over attached and biofilm-derived planktonic cells. Introducing a ciliate to two biofilm-forming Cryptococcus species, as well as two bacterial species in a model biofilm system, fluorescent probes were applied to determine ingestion of cellular and noncellular biofilm fractions. Fluoromicroscopy, as well as photometric quantification, confirmed that protistan grazing enhanced yeast biofilm metabolism, and an increase in biofilm biomass and viability. We propose that the extracellular polymeric matrix of biofilms may act as an interface regulating interaction between predator and prey, while serving as source of nutrients and energy for protists.

Animals↗

PubMed1999

Interference of modern antibacterials with bacillus Calmette-Guerin viability.

PURPOSE: We determine the effects of modern antibacterial chemotherapeutics on bacillus Calmette-Guerin (BCG) viability, particularly those of cycloserine, which has been recommended for treating BCG induced sepsis. MATERIALS AND METHODS: The minimal inhibitory concentrations of 31 antibacterial drugs against Connaught BCG strain were determined in vitro by the radiometric BACTEC 460TB method. Minimal inhibitory concentrations were compared with the drug concentrations achievable in blood and urine to estimate systemic or intravesical susceptibility. Susceptibility testing of cycloserine was performed with Connaught, Tice and RIVM BCG strains, using the modified proportion method on Lowenstein-Jensen agar. RESULTS: Connaught BCG was susceptible to quinolones in systemic infections but resistant to beta-lactams, macrolides and some aminoglycosides. It was resistant to pyrazinamide but showed good susceptibility toward the other antituberculosis drugs tested. All 3 BCG strains analyzed were resistant to cycloserine. Most antibacterials may interfere with BCG in the bladder because of high urinary recovery. CONCLUSIONS: Antibacterial drug interference with BCG viability should be avoided during intravesical instillation therapy. In cases of severe complications quinolones rather than cycloserine may be given in addition to standard triple antituberculosis drug therapy or if one of these drugs is not tolerated. Our data may contribute toward enhancing the therapeutic safety and efficacy of intravesical BCG immunotherapy by the appropriate use of antibacterial drugs.

Antibiotics, Antitubercular↗

PubMed2005

Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound.

AIMS: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS: According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS: The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.

Cell Membrane↗

PubMed2000

Comparative killing rates of fluoroquinolones and cell wall-active agents.

Killing rates of fluoroquinolones, beta-lactams, and vancomycin were compared against Enterobacteriaceae, Staphylococcus aureus, pneumococci, streptococci, and Enterococcus faecalis. The times required for fluoroquinolones to decrease viability by 3 log(10) were 1.5 h for Enterobacteriaceae, 4 to 6 h for staphylococci, and >/=6 h for streptococci and enterococci. Thus, the rate of killing by fluoroquinolones is organism group dependent; overall, they killed more rapidly than beta-lactams and vancomycin.

Anti-Bacterial Agents↗