Analysis of the survival of Venezuelan equine encephalomyelitis virus and possible viral simulants in liquid suspensions.

AIMS: To compare the inactivation rate of Venezuelan equine encephalomyelitis (VEE) virus in liquids to that of Sindbis virus (SV, another alphavirus) and to a bacteriophage (MS2) generally used as a viral simulant in the development of countermeasures in biodefense. METHODS AND RESULTS: Viruses were inoculated into liquids and viral titres were determined at various times postinoculation. The viruses were stable in distilled-deionized (dd) water at 4 degrees C during the 21 days of the study. The inactivation rates of VEE and SV in dd water at 21 and 30 degrees C were very similar (between 0.12 and 0.14 log(10) per day), while MS2 was three-fold slower. In tap water (chlorine content between 4 and 5 ppm) at 21 degrees C, VEE and SV were inactivated at twice the rate measured in dd water. CONCLUSIONS: The inactivation rates of VEE and SV were similar to each other and faster than MS2 in all liquids tested. SIGNIFICANCE AND IMPACT OF THE STUDY: VEE is likely to remain viable for many days after release into water, snow, or even chlorinated tap water. SV can be used to estimate the persistence of VEE in liquids, but using MS2 as a simulant would overestimate of the stability of VEE.

Wet and dry density of Bacillus anthracis and other Bacillus species.

AIMS: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1.162 and 1.165 g ml(-1) and the other group included strains whose spores showed higher density values between 1.174 and 1.186 g ml(-1). Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R(2) = 0.95) between density and volume for the spores of all strains and species studied. CONCLUSIONS: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde.

AIMS: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. METHODS AND RESULTS: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality-controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. CONCLUSIONS: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time-duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. SIGNIFICANCE AND IMPACT OF THE STUDY: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde-based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.

Induction of oxidative single- and double-strand breaks in DNA by ferric citrate.

The relative risk of primary hepatocellular carcinoma in genetic hemochromatosis (GH) is estimated at over 200 times as that of control populations. Recently, ferric ion chelated to citrate (Fe-citrate) was identified as the major non-transferrin-bound iron in the serum of GH patients. We investigated whether low concentration of Fe-citrate plus reductant could damage supercoiled plasmid DNA under physiological pH and ionic strength. Incubation of Fe-citrate with either H2O2, L-ascorbate, or L-cysteine induced single- and double-strand breaks in supercoiled plasmid pZ189 in a concentration- and time-dependent fashion. DNA strand breaks produced by Fe-citrate plus H2O2 increased at reduced pH (< or = 6.9). Catalase and free radical scavengers inhibited the DNA breakage produced by Fe-citrate in combination with each reductant, suggesting that H2O2 and finally .OH are responsible DNA damaging species. The catalytic ability of Fe-citrate to induce DNA strand breaks, particularly double-strand breaks (DSBs), may contribute to the carcinogenic processes observed in GH.

Association between 8-hydroxy-2'-deoxyguanosine formation and DNA strand breaks mediated by copper and iron.

Oxidative DNA damage is involved in diverse biological phenomena and consists of several kinds of lesions, mainly, strand breaks, base modifications, and DNA-protein crosslinking. However, little is known about the existence of a chemical relationship among them or the ratio by which these different types of lesions are produced. In the present study we investigated whether a relationship exists between DNA strand breakage and base modification. We selected cupric [Cu(II)] and ferric [Fe(III)] ions for this study because these transition metals are active catalysts of DNA damage in vivo. Supercoiled plasmid DNA pZ189 was treated with Cu(II) or Fe(III) in the presence of different reducing agents. We measured in each sample both the number of DNA single-strand breaks (SSB) by quantitative electrophoresis and the amount of a modified DNA base, 8-hydroxy-2'-deoxyguanosine (8-OHdG) by HPLC with simultaneous electrochemical (EC) and spectrophotometric detection. The number of DNA SSBs produced was linearly related to the number of 8-OHdG present. The regression of the number of SSBs as a function of the number of 8-OHdG is expressed by the equation [SSBs] = b x [8-OHdG], where b = 1.7, 2.0, 2.7, 1.7, and 9.4, for Cu(II) in the presence of H2O2, L-cysteine and L-ascorbate, and for Fe(III) in the presence of H2O2 and L-ascorbate, respectively. The linear correlation we observed between the production of SSB and 8-OHdG mediated by Fe(III) and by Cu(II) suggests that these products may arise via a common chemical mechanism and could allow an easier and more precise estimation of DNA breakage.

Cytotoxic and mutagenic effects of ferric nitrilotriacetate on L5178Y mouse lymphoma cells.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis that leads to a high incidence of renal adenocarcinoma in rodents. Others have shown that Fe-NTA induces modified DNA base products both in vitro and in vivo. However, Fe-NTA is negative in the Ames Salmonella test with or without S9 activation. The goal of this project was to determine if Fe-NTA is cytotoxic and mutagenic using the L5178Y (TK +/-) mouse lymphoma assay. Our experiments showed a relationship between the concentration of Fe-NTA (0 to 1 mM) and the decrease in relative survival. An exposure-dependent increase in the number of mutations was observed with increasing concentrations of Fe-NTA. At 14% relative survival, there was about a 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells. Ferric nitrate or nitrilotriacetic acid alone induced a relatively low 1.5- or 1.1-fold increase in mutation, respectively. Our results establish that Fe-NTA is mutagenic in the L5178Y mouse lymphoma assay system.

Cupric and ferric ions inactivate HIV.

Human immunodeficiency virus (HIV-1) was inactivated by either cupric or ferric ions when the virus was free in solution and also 3 hr after cell infection. Fifty percent inactivation of cell-free HIV was achieved with Cu(II) at a concentration between 0.16 and 1.6 mM, or by 1.8 to 18 mM Fe(III). Thus, the dose to inactivate 50% of infectious HIV (D50) by Cu(II) or Fe(III) is higher than that reported for glutaraldehyde (0.1 mM); between the D50 reported for sodium hypochlorite (1.3 mM) and sodium hydroxide (11.5 mM), and significantly lower than that required for HIV inactivation by ethanol (360 mM). Treatment of infected cells for 30 min at 20 degrees C with 6 mM Cu(II) or Fe(III) completely inhibited the formation of syncytia and the synthesis of virus-specific p24 antigen in HIV-infected cells, while still preserving cell viability. The virucidal properties of cupric and ferric ions could be exploited for the development of novel virucidal formulations efficient against HIV.

Microwave effects on plasmid DNA.

The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.

Comparative sporicidal effect of liquid chemical germicides on three medical devices contaminated with spores of Bacillus subtilis.

BACKGROUND: The relatively limited variety of surfaces and geometries challenged in current sporicidal testing reduces the predictive value of these analyses when extrapolated to the wide variety of medical devices. The unknown spore load being challenged and the qualitative nature (growth/no growth) of those tests further prevent precise comparison among liquid chemical disinfectants. Hence, the relative activity of different chemical substances has not been clearly established, hindering selection of the best agent for each clinical situation. METHODS: A micromethod was developed to assess sporicidal activity against Bacillus subtilis spores deposited on three different medical devices; carbon steel dental burs, silicone-rubber medical catheters, and titanium-alloy dental abutment screws. The spore load on each device and the recovery after three analytical steps were quantitatively assessed with spores radiolabeled with carbon 14 methionine. RESULTS: The killing of 2 to 7 x 10(6) spores loaded on three different devices and exposed to glutaraldehyde, formaldehyde, copper ascorbate, hydrogen peroxide, peracetic acid, sodium hypochlorite, or phenol for 30 minutes at 20 degrees C ranged from a 10(3)-fold decrease for 10% hydrogen peroxide to zero decrease for 5% phenol. Our results suggest that the nature of the surface being challenged may affect the sporicidal activity of some chemical agents. CONCLUSION: The quantitative data presented allow comparison of the sporicidal effect of different liquid chemical agents. These findings may help prevent an overestimation of sporicidal activity and possible transmission of pathogens from the surface of improperly decontaminated medical devices.

Comparative sensitivity of 13 species of pathogenic bacteria to seven chemical germicides.

BACKGROUND: The relative resistance of diverse human bacterial pathogens to commonly used germicidal agents has not been established. METHODS: We measured by titration the survival of thirteen different bacteria after exposure to glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate, sodium hypochlorite, or phenol. RESULTS: Our comparative experiments allowed classification of the organisms' survival into four groups: (a) Pseudomonas aeruginosa and Staphylococcus aureus showed the most resistance, (b) Clostridium perfringens, Salmonella typhimurium, Staphylococcus epidermidis, and Escherichia coli O157:H7 showed intermediate resistance, (c) Listeria monocytogenes, Shigella sonnei, and Vibrio parahaemolyticus survived some treatments with chemical agents only in the presence of protecting protein (serum albumin), and (d) Vibrio cholerae, Vibrio vulnificus, Bacillus cereus, and Yersinia enterocolitica did not survive any of the treatments applied. CONCLUSION: We found species that more frequently survived exposure to germicidal agents were also those most commonly reported in association with hospital infections. Our findings suggest that resistance to disinfectants may be more important than pathogenicity in determining the relative prominence of an organism as an agent responsible for nosocomial infections.

Replication and physical parameters important for preparing purified Junin virus.

Junin virus (JV) is an Arenavirus and the causative agent of Argentine hemorrhagic fever (AHF), an often fatal human disease. The attenuated strain XJ-clone 3 (XJC13) of JV, after being tested in humans, has been considered a promising vaccine. We found that synthesis of JV XJC13 reaches a peak 2 days after infection and the kinetics of synthesis are little affected by the multiplicity of infection (MOI) in a range from 0.125 to 1.00. Virus synthesis is sensitive to actinomycin D, indicating that cellular biosynthesis is required for viral replication. Combined precipitation and ultracentrifugation of supernatant from virus-infected cells yielded large amounts of concentrated and purified virion that banded in sucrose as a single peak with average density 1.177 +/- 0.015 g/ml. Purified virions have an average diameter of 203 +/- 23 nm by electron microscopy and an average sedimentation coefficient of 454 +/- 27 S. The results from the present study should assist in the preparation of large amounts of attenuated Junin virus which are required for vaccination against and diagnosis of Argentine hemorrhagic fever.

Molecular characteristics of Junin virus.

Results suggest that protein, glycerophospolipid, galactoside, and sialyl glycoside residues are present in Junin virus (JV), are accessible to enzymatic digestion, and play an important role in infection. Four major protein bands with molecular masses (Mr) 64 +/- 2, 56 +/- 2, 52 +/- 3 (mean +/- standard deviation, n = 4) and approximately 12-18 kDa were consistently detected after denaturing gel electrophoresis analysis of purified attenuated JV. The 52 kDa protein showed a diffuse tail in the 52-56 kDa range and was considered to be the JV glycoprotein. By Western blotting, the 64 kDa protein bound a JV neutralizing antibody and was considered to be the viral nucleoprotein. Additional bands corresponding to larger proteins (approximately 200, 96, 86, and 78 80 kDa in size), as well as fainter and broader bands in the 23-44 kDa region were also present in purified JV preparations. The relative resistance of virus infectivity to RNase digestion demonstrates that the genome of JV is protected from enzymatic attack. Analysis of purified JV virions by electrophoresis resolved the viral small (S) RNA and large (L) RNA species, 3636 +/- 54 bases and 7667 +/- 154 bases long, respectively (average length +/- range, in four determinations). The (S) RNA of attenuated JV appeared slightly larger than that reported for a pathogenic strain, ruling out a large sequence deletion as a reason for attenuation.

Method for improving accuracy of virus titration: standardization of plaque assay for Junin virus.

Titrating infective virus is one of the most important and common techniques in virology. However, after many years of widespread use, the parameters governing the accuracy of titration values are still not well understood. It was found that under conditions currently used for virus titration, only a small percentage of virus in the inoculum is adsorbed onto the cells and thereby detected in the titration assay. The objective of our work was to establish the conditions for a plaque assay which could estimate more accurately the titer of Junin virus. Two different stain methods were compared and several parameters governing plaque formation were studied. The volume of the inoculum appeared as the most important factor affecting observed titer. A linear relationship between the volume of inoculum and the reciprocal apparent titer allowed us to estimate an absolute titer by extrapolation. The approach described here is likely to be applicable to the more accurate estimation of the titer of a wide range of virus.

Detection of Junin virus by the polymerase chain reaction.

Argentine hemorrhagic fever is an often fatal human disease caused by Junin virus, an RNA-containing virus and member of the Arenavirus family. This virus was detected in vitro by the polymerase chain reaction (PCR) procedure. A pair of Junin virus-specific PCR DNA oligonucleotide primers and an oligonucleotide probe were designed from a known portion of the viral RNA sequence. RNA was isolated from Junin virus-infected monkey kidney cells and used to produce complementary DNA (cDNA) by reverse transcription. A DNA segment, 151 +/- 24 bp long, was amplified from this cDNA and characterized by agarose gel electrophoresis and Southern blot hybridization with the Junin virus-specific DNA probe. Sensitivity experiments showed that Junin virus could be detected with nanogram quantities of RNA isolated from virus-infected cells. The rapid and sensitive assay described here may contribute towards the development of a procedure for the early diagnosis of Argentine hemorrhagic fever.

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine.

The interaction of chelators and reducing agents is of particular importance in understanding iron-associated pathology since catalytic iron undergoes cyclic reduction and oxidation in vivo. Therefore, we treated plasmid DNA with free or chelated Fe(III) in the presence of biological reductants, and simultaneously measured the number of single strand breaks (SSBs) and oxidative base modification (8-hydroxy-2'-deoxyguanosine; 8-OHdG) by quantitative gel electrophoresis and HPLC with electrochemical detection, respectively. Production of SSBs and 8-OHdG was linearly correlated suggesting that these two different lesions share a common chemical mechanism. The levels of both lesions were enhanced when Fe(III) was chelated to citrate or nitrilotriacetic acid. Reducing agents showed different potency in inducing DNA damage catalyzed by chelated iron (L-ascorbate > L-cysteine > H2O2). Chelation increased SSB formation by approximately 8-fold and 8-OHdG production by approximately 4-fold. The ratio of SSB/8-OHdG catalyzed by chelated iron, which is twice as high as by unchelated iron, indicates that chelation affects iron-catalyzed oxidative DNA damage in a specific way favoring strand breakage over base modification. Since iron is mostly chelated in biological systems, the production of genomic and mitochondrial DNA damage, particularly strand breaks, in diseases involving iron overload is likely to be higher than previously predicted from studies using unchelated iron.

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.

Cytotoxicity of liquid disinfectants.

This study was prompted by toxic responses to disinfecting agents reported in patients after surgical procedures and in sensitized health care personnel. We evaluated the cytotoxicity of seven substances used in the formulation of common liquid chemical disinfectants and sterilants. We found that a standard method based on direct microscopic examination of cell cultures was insensitive and may result in an underestimation of the risk that disinfectants pose to health care personnel or patients who are exposed to these substances. Using independent quantitative tests measuring the integrity of the cellular membrane, metabolic activity, or cell growth, we found that there is a several-hundredfold difference in the relative toxicity of various disinfecting substances. The concentration toxic in 50% of the cell population (TC(50)) that was found for each disinfectant was similar in a variety of cell lines from human, monkey, or mouse origin. Statistical analysis of TC(50)s suggests that liquid disinfecting agents could be classified in three main groups according to their relative toxicity, with: (1) mild (TC(50) > 1 mM, including phenol, hydrogen peroxide, and formaldehyde); (2) moderate (1mM > TC(50) > 0.1 mM, sodium hypochlorite); and (3) severe (TC(50) < 0.1 mM, glutaraldehyde, cupric ascorbate, and peracetic acid) toxicity. These data suggest a vast difference in the potential risk of various disinfectants and sterilants. The data presented in this study should help to define the relative toxic risk of different disinfecting substances to patients and health care personnel and assist in the selection of safer microbicidal formulations.

Resistance of Pseudomonas aeruginosa to liquid disinfectants on contaminated surfaces before formation of biofilms.

A comparison was made of the effectiveness of popular disinfectants (Cavicide, Cidexplus, Clorox, Exspor, Lysol, Renalin, and Wavicide) under conditions prescribed for disinfection in the respective product labels on Pseudomonas aeruginosa either in suspension or deposited onto surfaces of metallic or polymeric plastic devices. The testing also included 7 nonformulated germicidal agents (glutaraldehyde, formaldehyde, peracetic acid, hydrogen peroxide, sodium hypochlorite, phenol, and cupric ascorbate) commonly used in disinfection and decontamination. Results showed that P. aeruginosa is on average 300-fold more resistant when present on contaminated surfaces than in suspension. This increase in resistance agrees with results reported in studies of biofilms, but unexpectedly, it precedes biofilm formation. The surface to which bacteria are attached can influence the effectiveness of disinfectants. Viable bacteria attached to devices may require dislodging through more than a one-step method for detection. The data, obtained with a sensitive and quantitative test, suggest that disinfectants are less effective on contaminated surfaces than generally acknowledged.

Effects of salt and serum on the sporicidal activity of liquid disinfectants.

This study compares the effects of various concentrations of salt or serum in the killing of Bacillus subtilis spores by either glutaraldehyde, sodium hypochlorite, cupric ascorbate, hydrogen peroxide, peracetic acid, formaldehyde, or phenol. Salt affected only glutaraldehyde, its sporicidal activity increasing with an increase in concentration of sodium bicarbonate or sodium chloride. The sporicidal activity of glutaraldehyde was minimal when the concentrations of aldehyde groups and lysine residues from protein were similar. We present an equation describing the effect of serum on spore survival as a function of glutaraldehyde concentration that fits the data with a regression coefficient of 0.9. Cupric ascorbate and peracetic acid were inhibited by serum, but this effect was linked to a rise in pH. Sodium hypochlorite was the agent most sensitive to protein, with its sporicidal activity nearly disappearing in the presence of 2% serum or an equivalent amount of purified protein.