Effect of organic N-halamines on selected membrane functions in intact Staphylococcus aureus cells

Two N-halamine compounds, 3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone, were compared with free chlorine as to their effects on selected membrane functions of intact Staphylococcus aureus cells. Free chlorine was found to cause a loss of permeability control, as measured by the efflux of potassium from the cells and a dramatic increase in hydrogen ion permeability, and to affect cell respiration in a nonreversible fashion, as measured by oxygen uptake. The two N-halamines were found to have very little effect on permeability to either potassium or hydrogen ions but were both found to dramatically inhibit respiration in a reversible manner. It is proposed that the first step in the disinfection process by these N-halamines is an inhibition of respiratory enzymes that, if not reversed, ultimately leads to a loss of cell viability.     

Effect of organic N-halamines on selected membrane functions in intact Staphylococcus aureus cells.

Two N-halamine compounds, 3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone, were compared with free chlorine as to their effects on selected membrane functions of intact Staphylococcus aureus cells. Free chlorine was found to cause a loss of permeability control, as measured by the efflux of potassium from the cells and a dramatic increase in hydrogen ion permeability, and to affect cell respiration in a nonreversible fashion, as measured by oxygen uptake. The two N-halamines were found to have very little effect on permeability to either potassium or hydrogen ions but were both found to dramatically inhibit respiration in a reversible manner. It is proposed that the first step in the disinfection process by these N-halamines is an inhibition of respiratory enzymes that, if not reversed, ultimately leads to a loss of cell viability.     

Inactivation ofSalmonella enteritidis on shell eggs by novelN-halamine biocidal compounds

Summary Several newN-halamine compounds have been evaluated as potential replacements for free chlorine as disinfectants for the egg-processing industry. The compounds were tested againstSalmonella enteritidis on the surfaces of egg shells. Test procedure included spraying inoculated egg shells with solutions of several of theN-halamine compounds and free chlorine for comparison, suspending the most stableN-halamine compound in a thin coating of mineral oil on the egg shell and subsequent inoculation, and measuring the rates of diffusion of the compounds and free chlorine through the egg shells. Compounds DBC (1-bromo-3-chloro-2,2,5,5-tetramethylimidazolidin-4-one) and DC (1,3-dichloro-2,2,5,5,-tetramethylimidazolidin-4-one) were significantly more efficacious than free chlorine in inactivatingSalmonella in the spray experiments, while compound MC (1-chloro-2,2,5,5-tetramethylmidazolidin-4-one), in a mineral oil suspension, provided disinfection of the egg shells within 72 h of contact. None of the disinfectant compounds penetrated egg shells at a rate greater than 1 mg/l over a period of 6 h. Compound MC is recommended as a possible replacement for unstable, corrosive-free chlorine as a bactericide for the egg-processing industry.     

Catalysis by molecular iodine: a rapid synthesis of 1,8-dioxo-octahydroxanthenes and their evaluation as potential anticancer agents

Molecular iodine facilitated the reaction of 5,5-dimethyl-1,3-cyclohexanedione with aromatic aldehydes in iso-propanol affording a variety of 1,8-dioxo-octahydroxanthenes in high yields. Most of the compounds synthesized showed good anti-proliferative properties in vitro against three cancer cell lines and 9-(2-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione possessing a 2-hydroxy phenyl group at C-9 position was found to be promising. Further structure elaboration of the same compound and the crystal structure analysis and hydrogen bonding patterns of another compound that is, 9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione prepared by using this methodology is presented.     

Modifying aroylhydrazone prochelators for hydrolytic stability and improved cytoprotection against oxidative stress

BSIH ((E)-N′-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) is a prodrug version of the metal chelator SIH ((E)-N′-(2-hydroxybenzylidene)isonicotinohydrazide) in which a boronate group prevents metal chelation until reaction with hydrogen peroxide releases SIH, which is then available for sequestering iron(III) and inhibiting iron-catalyzed oxidative damage. While BSIH has shown promise for conditionally targeting iron sequestration in cells under oxidative stress, the yield of SIH is limited by the fact that BSIH exists in cell culture media as an equilibrium mixture with its hydrolysis products isoniazid and 2-formylphenyl boronic acid. In the current study, several BSIH analogs were evaluated for their hydrolytic stability, reaction outcomes with H₂O₂, and prochelator-to-chelator conversion efficiency. Notably, the para-methoxy derivative (p-OMe)BSIH ((E)-N′-(5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) and the meta-, para-double substituted (MD)BSIH ((E)-N′-((6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxol-5-yl)methylene)isonicotinohydrazide) showed 1.3- and 1.9-fold improved hydrolytic stability compared to BSIH, respectively, leading to a 22 and 50% increase in chelator released. Moreover, both prochelators were found to protect retinal pigment epithelial cells stressed with either H₂O₂ or paraquat insult.     

Iron complexes of dioxoborolanylbutadienes

The first [2,3-dialkyl-1,4-bis(1,3-dioxo-4,4,5,5-tetramethyl-2-borolanyl]-1,3-butadiene]Fe(CO)₃ complexes (2, alkyl=n-butyl; 3, alkyl=cyclopentyl; 4, ‘alkyl’=phenyl; 5, alkyl=3-chloropropyl) have been prepared from the reaction of bis(cis-cyclooctadiene)Fe(CO)₃ (1) with the borolanylbutadienes. The X-ray crystal structures of 2 and 3 are reported. The geometries of the coordinated ligands are compared with those of the free ligands.     

Bactericidal properties of a new water disinfectant.

The N-chloramine compound 3-chloro-4,4-dimethyl-2-oxazolidinone (agent I) has been compared with calcium hypochlorite as to its efficacy as a bactericide for the treatment of water. The study included concentration, contact time, pH, temperature, and water quality as controlled variables. The species of bacteria tested were Staphylococcus aureus, Pseudomonas aeruginosa, and Shigella boydii. In general, for highly pure, demand-free water, calcium hypochlorite was the more rapid disinfectant at a given total chlorine concentration, although for water containing a controlled amount of organic load, agent I was the better disinfectant. The differences in efficacy of each of the two disinfectants can be attributed primarily to their different stabilities in water at various controlled conditions.     

Bactericidal properties of a new water disinfectant

The N-chloramine compound 3-chloro-4,4-dimethyl-2-oxazolidinone (agent I) has been compared with calcium hypochlorite as to its efficacy as a bactericide for the treatment of water. The study included concentration, contact time, pH, temperature, and water quality as controlled variables. The species of bacteria tested were Staphylococcus aureus, Pseudomonas aeruginosa, and Shigella boydii. In general, for highly pure, demand-free water, calcium hypochlorite was the more rapid disinfectant at a given total chlorine concentration, although for water containing a controlled amount of organic load, agent I was the better disinfectant. The differences in efficacy of each of the two disinfectants can be attributed primarily to their different stabilities in water at various controlled conditions.     

Chemical Disinfection of Holding-Tank Sewage

A number of chemical disinfectants were evaluated for their bactericidal and virucidal effectiveness in holding-tank sewage. It was found that the disinfection efficiencies of formaldehyde, benzalkonium chloride, cetylpyridinium chloride, and methylene blue were markedly improved if the pH of the sewage was raised from 8.0 to 10.5. When formaldehyde, benzalkonium chloride, and methylene blue were tested with either 2-week holding times with no sewage additions or 10-day holding times with daily sewage additions, disinfection effectiveness was maintained as long as the sewage pH was kept at 10.5 and the disinfectant concentration was kept at 100 mg/liter or more. Calcium hypochlorite, zinc sulfate, and phenol were found to be relatively ineffective disinfectants for holding-tank sewage.     

Insecticidal compound isolated from Syzygium lineare Wall. (Myrtaceae) against Spodoptera litura (Lepidoptera: Noctuidae)

A new crystal compound 2,5-diacetoxy-2-benzyl-4,4,6,6-tetramethyl-1,3-cyclohexanedione was isolated from the leaves of Syzygium lineare. The insecticidal activity of the compound was assessed against fourth instar larvae of Spodoptera litura. Its activity was better than the positive control azadirachtin. The compound was responsible for growth inhibition on S. litura. It induced larval, pupal and adult deformities even at low concentration. The compound may be useful as a botanical pesticide.     

Evaluation of Disinfection Techniques for, and Their Effects on, Rectal Thermocouple Catheters

The antibacterial activities of an iodophor (Wescodyne G), a quaternary ammonium compound (Roccal), and an iodine tincture as agents for the cold disinfection of rectal catheters contaminated in vitro were determined. Following thorough cleaning with an alcoholic solution of soft soap, each of the three disinfectants tested showed satisfactory results (100% kill) in 5 min against the enteric test bacteria (Escherichia coli and Salmonella typhosa) as well as a test species of the genus Pseudomonas, among the bacteria most resistant to surface-active agents.

An aqueous solution of Wescodyne G containing 75 ppm available iodine was used both as a wiping solution and for subsequent disinfection of rectal catheters contaminated in vivo. Total bacterial destruction was found to follow a 60-min soak preceded by the wiping procedure.

Rectal catheters subjected to prolonged immersion in each of the test disinfectants were found to be essentially unaffected, retaining their initial calibrations within a permissible tolerance. Neither Roccal nor Wescodyne G solutions were found to measurably attack bare thermocouples. Alcoholic iodine 0.5% did, however, exert a deteriorating effect on bare thermocouples in a short time, as measured by change in resistance characteristics.

The results of this study have led to the recommendation that Wescodyne G containing 75 ppm available iodine be used in standing operating procedures for the initial cleaning and subsequent disinfection of rectal thermocouple catheters.

     

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.     

Development and Testing of a Microbiological Assay To Detect Residual Effects of Disinfectant on Hard Surfaces

We describe a glucuronidase bioassay for detecting residual bactericidal activity from the use of disinfectants on hard surfaces; in this assay we used formaldehyde, ethanol, isopropanol, chlorine, and a commercial preparation containing 2-bromo-2-nitro-1,3-propanediol. Chlorine and the commercial preparation showed bactericidal activity (53.5% and 98.2%, respectively) for a week after disinfection.     

Investigating the suitability of the Calgary Biofilm Device for assessing the antimicrobial efficacy of new agents

This study investigated the suitability of the Calgary Biofilm Device (CBD), originally designed as a test surrogate for indwelling medical devices, for assessing the efficacy of antimicrobials developed for food and food contact surface disinfection applications. The conditions for the development of uniform biofilms from pure and mixed bacterial cultures of wild type Escherichia coli and Listeria innocua were optimized. We were able to recover approximately 2 x 10(6) colony forming units (CFU) from the biofilms formed on the individual pegs of the device in 24 h. Further, the parameters for the consistent release of the cells from the biofilms were optimized; test showed that the number of cells released was uniform and reproducible. The consistency and reproducibility of the biofilms formed on the pegs was evaluated using scanning electron microscopy and by plate count method. The efficacies of disinfectants on cells residing in biofilms versus planktonic cells were compared. For both species, higher concentrations of disinfectants were needed to eliminate attached cells as compared with planktonic cells. This study establishes the value of the CBD for generating consistent biofilms from either pure or mixed cultures. These biofilms can be used to assess efficacies of disinfectants against cells that have colonized the surfaces of foods and food-processing equipment. Such a system could serve as a standard surrogate for evaluating new disinfectants designed to reduce or eliminate biofilms from food-contact surfaces.     

Synthesis of monosaccharides by oxidation of furfural derivatives

dl-Ribose, dl-arabinose, and dl-xylose have been synthesized from 2-furaldehyde. Condensation of 2-furaldehyde with pinacol followed by oxidation of the product with bromine water or lead tetra-acetate and reduction with sodium borohydride gave dl-2-(1,4-dihydroxy-cis-but-2-enyl)-4,4,5,5-tetramethyl-1,3-dioxolane (4a). Hydroxylation of the dibenzoate of 4a followed by debenzoylation and hydrolysis gave dl-ribose and dl-arabinose. Epoxidation of 4a with monoperoxysuccinic acid followed by hydrolysis gave dl-xylose.     

Studies on Disinfection of Clinical Thermometers: II. Oral Thermometers from a Tuberculosis Sanatorium

A study was made of the chemical disinfection of oral thermometers used by patients with active pulmonary tuberculosis. Only 10% of these thermometers were found to be contaminated with acid-fast bacilli. Because of this small number, the use of a phenol coefficient type test with Mycobacterium tuberculosis was suggested as an alternative method for evaluating tuberculocidal activity. These data, in conjunction with data from Part I of these studies dealing with disinfection of oral thermometers from a general hospital, were the criteria used to judge the efficacy of the disinfectants. It was concluded that 70% ethyl alcohol, 2% phenolic no. 1, and 3% phenolic no. 3 were reliable disinfectants for thermometers. Fifty per cent ethyl alcohol, 2% phenolic no. 2, 2% iodophor (300 ppm available iodine), and 0.1% benzalkonium chloride, aqueous or tincture, were unreliable.     

Residue aspects of disinfectants used in the food industry

Disinfectants are frequently used in the food industry against harmful (micro-)organisms on equipment (surfaces, pipelines etc.) which can come into contact with food. In The Netherlands, such uses of disinfectants are only allowed when registered according to the provisions of our national Pesticide Law. In most cases, (registered) uses require an operating procedure consisting of 3 consecutive steps: cleaning, disinfection and rinsing the equipment with clean water, thus avoiding contamination of food as much as possible. However, in some cases traces of the used compound(s) in food are inevitable. Therefore, Maximum Residue Limits (MRLs) for the disinfectants involved are established in food in the Netherlands. As remaining residues must be safe when ingested by the public, intake must be well below the Acceptable Daily Intake (ADI) for the respective chemicals. Also, sensitive and easy-to-operate methods of residue analysis, capable of detecting low levels of the compounds in food, must be available for enforcement purposes. In the Netherlands, experience with the existing legislation was gained over the past 25 years, resulting in the establishment of some 15–20 MRLs for residues of disinfectants in food.Harmonisation of the use of disinfectants (and other biocides) in the EU will be achieved in a draft ‘Biocide Directive’ in which the residue aspects of disinfectants are also covered, in a more-or-less comparable way as has been layed down in the Dutch legislation.Recent developments include the use of disinfectants in food as such, to reduce micro-organisms and to prolong the shelf-life of food products. Although in some surrounding countries this technique may already have become common practice. The Netherlands is reluctant to register those uses, and restricts it to cases where there is an inevitable technical need for disinfection, to minimize the exposure of the consumer to residues of (potentially) dangerous chemicals.     

Exposure of biofilms to slow flow fields: the convective contribution to growth and disinfection

A previously introduced degenerate diffusion-reaction model of biofilm growth and disinfection is extended to account for convective transport of oxygen and disinfectants in an aqueous environment. To achieve this in a computationally efficient manner we employ a thin-film approximation to the (Navier)-Stokes equations that can be solved analytically. In numerical experiments, we investigate how the convective transport of nutrients and disinfectants due to bulk flow hydrodynamics affects the balance between growth and disinfection processes. It is found that the development of biofilms can be significantly affected by the flow field even at extremely low Reynolds numbers. While it is natural to expect that increased bulk flow velocities imply increased mass transfer of both, nutrients and disinfectants, and hence an acceleration of both, growth and decay of biomass, it is found, furthermore, that in many instances the actual flow conditions, determine the success or failure of disinfection, i.e. persistence or extinction of a biofilm community.     

Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log₁₀ units) with LP-UV (20 mJ/cm²) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log₁₀ unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.     

Carbonic anhydrase inhibitors: Crystallographic and solution binding studies for the interaction of a boron-containing aromatic sulfamide with mammalian isoforms I–XV

We investigated the inhibition of carbonic anhydrase (CA, EC 4.2.1.1) isoforms I–XV with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylsulfamide and other simple or sugar sulfamides, a class of less investigated CA inhibitors (CAIs). The crystal structure of the adduct of hCA II with the boron-substituted sulfamide shows the organic scaffold of this compound bound in the hydrophilic half of the active site where it makes a large number of van der Waals contacts with Ile91, Gln92, Val121, Phe131, Leu198, and Thr200. The data here reported provide further insights into sulfamide binding mechanism confirming that this zinc-binding group could be usefully exploited for obtaining new potent and selective CAIs.