A Commonly Used Biocide 2-N-octyl-4-isothiazolin-3-oneInduces Blood-Brain Barrier Dysfunction via Cellular Thiol Modification and Mitochondrial Damage.

Isothiazolinone (IT) biocides are potent antibacterial substances commonly used as preservatives or disinfectants, and 2-n-Octyl-4-isothiazolin-3-one (OIT; octhilinone) is a common IT biocide that is present in leather products, glue, paints, and cleaning products. Although humans are exposed to OIT through personal and industrial use, the potentially deleterious effects of OIT on human health are still unknown. To investigate the effects of OIT on the vascular system, which is continuously exposed to xenobiotics through systemic circulation, we treated brain endothelial cells with OIT. OIT treatment significantly activated caspase-3-mediated apoptosis and reduced the bioenergetic function of mitochondria in a bEnd.3 cell-based in vitro blood-brain barrier (BBB) model. Interestingly, OIT significantly altered the thiol redox status, as evidenced by reduced glutathione levels and protein S-nitrosylation. The endothelial barrier function of bEnd.3 cells was significantly impaired by OIT treatment. OIT affected mitochondrial dynamics through mitophagy and altered mitochondrial morphology in bEnd.3 cells. N-acetyl cysteine significantly reversed the effects of OIT on the metabolic capacity and endothelial function of bEnd.3 cells. Taken together, we demonstrated that the alteration of the thiol redox status and mitochondrial damage contributed to OIT-induced BBB dysfunction, and we hope that our findings will improve our understanding of the potential hazardous health effects of IT biocides.

Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization.

INTRODUCTION: Several irrigants have been used for disinfection in regenerative endodontic procedures including chlorhexidine (CHX). In this context, the antibacterial properties of disinfectants are mainly in focus of research even though they may have an undesirable impact on the fate of stem cells. In this study, we hypothesized that CHX has both a direct effect when applied to stem cells of the apical papilla (SCAPs) and an indirect effect when SCAPs are exposed to dentin previously conditioned with CHX. METHODS: Cell toxicity was evaluated in vitro using the CellTox green fluorescence assay (Promega, Madison, WI) and CellTiter-Glo (Promega) after SCAPs were exposed directly to a dynamic concentration range of CHX; apical papilla explant cultures were stained with ApopTag (Merck Millipore, Billerica, MA) after culture with CHX. Furthermore, standardized slabs from human dentin were treated with CHX and consecutively rinsed in EDTA, L-α-lecithin (Sigma-Aldrich, St Louis, MO), or L-α-lecithin followed by EDTA. After that, SCAPs were cultured on the slabs for 5 days, and cellular viability was determined (indirect effect). Data were treated nonparametrically and analyzed using the Krukal-Wallis test (P ≤ .05). RESULTS: Direct exposure of SCAPs to CHX highly affected cell viability at concentrations above 10-3%, whereas lower concentrations had no adverse effect. During the initial 60 minutes, concentrations of 10-2% CHX or higher resulted in early pronounced toxicity with a maximum effect within 15 minutes after exposure. Likewise, CHX-conditioned dentin slabs were detrimental to SCAP survival; however, the deleterious effects were completely reversed by neutralization with L-α-lecithin. CONCLUSIONS: Chlorhexidine is toxic to SCAPs when applied directly or indirectly via conditioned dentin. If applied for a short time and neutralized by L-α-lecithin, it can be a gentle and cell-preserving disinfectant before endodontic regeneration.

Dichloroacetic acid-induced testicular toxicity in male rats and the protective effect of date fruit extract.

BACKGROUND: The present study was designed to investigate the protective effect of aqueous date extract (ADE) against the dichloroacetic acid (DCA)-induced testicular injury in rats. METHODS: Forty-eight male Wistar rats were randomly divided into six groups of eight: group I served as the control; group II was given ADE (4 ml/kg) by gavage; groups III and IV received DCA at 0.5 and 2 g/L drinking water, respectively; and groups V and VI received DCA at 0.5 and 2 g/L drinking water, respectively, before ADE administration. The experiment was performed for two months. RESULTS: Results showed that the absolute weights of testes and epididymis were decreased following the DCA administration. The testosterone, FSH and LH levels were also decreased. Severe histopathological changes in testes were observed including degeneration of seminiferous tubules and depletion of germ cells. These changes were associated with alterations of oxidative stress markers. Levels of lipid peroxidation and SOD and CAT activities were increased, while activity of GPx and GSH levels were decreased. Pretreatment with ADE has effectively alleviated the oxidative stress induced by DCA thereby restoring these parameters to normal values. CONCLUSIONS: These results suggest that ADE has a protective effect over DCA-induced oxidative damage in rat testes.

Resistance of Clostridioides difficile spores from the NAP CR1 strain (Clostridiales: Peptostreptococcaceae) to sodium dichloroisocyanurate / Resistencia de esporas de Clostridioides difficile de la cepa NAPCR1 (Clostridiales: Peptostreptococcaceae) al dicloroisocianurato de sodio

Abstract Introduction: Clostridioides difficile is a significant cause of diarrhea in hospitals and the community. This bacterial pathogen is transmitted through the ingestion of endospores, which are challenging to eliminate due to intrinsic resistance to a variety of chemical disinfection agents. The well-characterized laboratory strain CD630 displays low virulence, has not caused outbreaks, and is highly susceptible to disinfectants. Nonetheless, a closely related strain termed NAPCR1 caused outbreaks in Costa Rica and later became endemic in many hospitals from this country. This strain causes disease through unusual mechanisms and is genotypically distinct from CD630. Consequently, its epidemic potential could be influenced by as yet unknown spore phenotypes, such as increased resistance to disinfectants. Objective: To determine whether the NAPCR1 strain is more resistant to a conventional and highly effective C. difficile sporicidal agent than strain CD630 and to identify potential explanatory mechanisms at the genomic level. Methods: We used an in vitro dilution-neutralization method to calculate the sporicidal activity of sodium dichloroisocyanurate (DCC) against purified spores from three subtypes of NAPCR1 isolates (LIBA-2945, LIBA-5761, and LIBA-6276), CD630, and a representative of the highly virulent and epidemic NAP1 strain (LIBA-5758). This phenotypic characterization was complemented with a genomics-steered search of polymorphisms in 15 spore- or sporulation-related genes. Results: Whereas DCC at a final concentration of 0.1 % (w/v) eradicated CD630 endospores with high efficacy (log10 reduction factor (LFR) ≥ 5), it only partially inactivated NAPCR1 (average LFR range: = 1.77-3.37) and NAP1 endospores (average LRF = 3.58). As hypothesized, the three NAPCR1 subtypes tested were more resistant to DCC than strain CD630 (ANOVA, P < 0.05), with LIBA-5761 showing the highest level of DCC resistance overall (ANOVA, P < 0.05). All three NAPCR1 isolates showed large deletions in bclA1. Besides, isolates LIBA-5761 and LIBA-6276 had deletions in bclA2. Conclusions: Our in vitro tests revealed a differential resistance of spores from the C. difficile NAPCR1 strain to DCC. They highlight the importance of continuously evaluating the efficacy of deployed disinfection agents against circulating strains and hint to a potential role of structural proteins from the exosporium in resistance to disinfectants in C. difficile.

Resumen Introducción: Clostridioides difficile es una causa importante de diarrea a nivel hospitalario y comunitario. Esta bacteria se transmite por medio de la ingestión de endosporas, las cuales son difíciles de erradicar por su resistencia intrínseca a diferentes agentes químicos de desinfección. La cepa de referencia CD630 está bien caracterizada, es poco virulenta, no ha causado brotes, y es altamente susceptible a los desinfectantes. Además, pertenece al mismo clado MLST y es filogenéticamente muy cercana a la cepa NAPCR1. Sin embargo, solo la última ha causado brotes en Costa Rica y se ha convertido en una cepa endémica en varios hospitales locales. La cepa NAPCR1 causa enfermedad por mecanismos poco usuales y es genotípicamente diferente a la cepa CD630. Por lo tanto, su potencial epidémico podría estar influenciado por cambios fenotípicos en sus esporas, como una resistencia incrementada a los desinfectantes. Objetivo: Determinar si la cepa NAPCR1 presenta mayor resistencia que CD630 a un desinfectante de alta eficacia utilizado a nivel hospitalario y dilucidar posibles mecanismos a nivel genómico. Métodos: Se utilizó el método de dilución-neutralización para evaluar la actividad esporicida in vitro del dicloroisocianurato de sodio (DCC) contra esporas de 3 subtipos de la cepa NAPCR1 (LIBA-2945, LIBA-5761, y LIBA-6276), CD630 y un aislamiento representativo de la cepa epidémica e hipervirulenta NAP1 (LIBA-5758). Esta caracterización fenotípica fue complementada con una búsqueda genómica de polimorfismos en 15 genes relacionados con la estructura de la endospora o el proceso de esporulación. Resultados: El DCC a una concentración final de 0.1 % (p/v) erradicó las endosporas de la cepa CD630 con gran eficacia (factor de reducción logarítmica; FRL ≥ 5) y eliminó parcialmente las de las cepas NAPCR1 (FRL promedio = 1.77-3.64) y NAP1 (FRL promedio = 3.58). El perfil de susceptibilidad del aislamiento NAPCR1 LIBA-5761 fue único, ya que mostró un mayor nivel de resistencia hacia el DCC que los otros aislamientos NAPCR1 y la cepa NAP1 examinada (ANOVA, P < 0.05). Los tres aislamientos NAPCR1 mostraron deleciones en bclA1 y los aislamientos LIBA-5761 y LIBA-6276 tenían deleciones adicionales en bclA2. Conclusiones: Nuestros experimentos in vitro confirman la resistencia incrementada a los desinfectantes de la cepa NAPCR1 y una susceptibilidad diferencial en sus tres subtipos. Adicionalmente, señalan la importancia de evaluar continuamente la eficacia de los desinfectantes contra cepas circulantes y asignan un posible papel en la resistencia a los desinfectantes gracias a las proteínas del exosporio de C. difficile.

Efficacy of some neutralizers in suspension tests determining the activity of disinfectants.

The ability of six mixtures to neutralize glutaraldehyde, o-phthalaldehyde and peracetic acid was tested using four reference strains: Pseudomonas aeruginosa CIP A22, Escherichia coli CIP 54127, Staphylococcus aureus CIP 53154, and Enterococcus faecium CIP 5855. Glutaraldehyde was the hardest to neutralize, and peracetic acid the easiest. The most effective mixture was Tween 80 with sodium bisulphate, sodium thioglycolate, lecithin and cysteine, and the least effective was Tween 80, lecithin and histidine. The efficacy of the neutralizers may indicate a propensity loss of activity from interfering substances when disinfectants are used in practice.

Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat-killed microbial whole cells.

The antibacterial activity of chlorhexidine digluconate and iodine potassium iodide on Enterococcus faecalis A197A was tested in the presence of dentin, dentin matrix, dentin pretreated by EDTA and citric acid, collagen, and heat-killed cells of Enterococcus faecalis and Candida albicans. Medications were preincubated for 1 h with each of the potential inhibitors and tested for their antibacterial activity against E. faecalis, strain A197A. Surviving bacteria were sampled after 1 and 24 h of incubation. Dentin matrix and heat-killed microbial cells were the most effective inhibitors of chlorhexidine, whereas dentin pretreated by citric acid or EDTA showed only slight inhibition. Dentin and skin collagen showed some inhibition at 1 h but not after 24 h. Iodine potassium iodide was effectively inhibited by dentin, dentin matrix, and heat-killed microbial cells. Skin collagen and dentin pretreated by EDTA or by citric acid showed little or no inhibitory effect on iodine potassium iodide. Different components of dentin are responsible for the divergent patterns of inhibition of the antibacterial activity of chlorhexidine digluconate and iodine potassium iodide. Chemical treatment of dentin before applying the medication into the root canal may alter the antibacterial effect of the medication.

Evaluation of glycine as an inactivator of glutaraldehyde.

Glycine was evaluated as an inactivator of the sporicidal activity of glutaraldehyde. Spores from glucose depleted cultures of Bacillus stearothermophilus grown in a chemically defined medium were used. When glycine is used as an inactivator of glutaraldehyde, it lowers the pH value of the solution. Glycine 1% failed to inactivate 0.5% or higher concentrations of alkaline glutaraldehyde in sporicidal studies. If viable counts cannot be performed within the first hour after inactivation, the concentration of glycine should be at least 2% to inactivate effectively 2% alkaline glutaraldehyde.

Inhibition of bleach-induced luminol chemiluminescence.

The luminol chemiluminescence presumptive test for blood is based on the mild peroxidase activity of hemoglobin in basic peroxide solution. However, this test is subject to interference by strong oxidants, certain transition metal ions, and true peroxidases. This paper reports methods for reducing the interference caused by hypochlorite-containing bleaches. Amines such as 1,2-diaminoethane react rapidly with hypochlorite without interfering significantly with the hemoglobin-catalyzed oxidation. Thus, addition of 0.1 mol/L 1,2-diaminoethane to a standard luminol-peroxide spray lead to almost complete inhibition of hypochlorite-induced chemiluminescence while satisfactory chemiluminescence was still observed from bloodstains. If time allows, an alternative method for reducing interference from hypochlorite bleach is to wait several days until the bloodstains have dried thoroughly, by which time the hypochlorite will have decomposed.

[Characteristics of gram-negative microflora on environmental objects in puerperal wards and their resistance to antibacterial preparations and disinfectants]. / Kharakteristika gramotritsatel'noi mikroflory ob''ektov okruzhaiushchei sredy poslerodovykh otdelenii i ee rezistentnost' k antibakterial'nym preparatam i dezinfektantam.

The species composition of gram-negative opportunistic bacteria isolated from different objects at three puerperal wards of a maternity clinic was studied. Escherichia coli and Acinetobacter were found to have a fairly wide circulation. The objects most contaminated by these bacteria were determined. The study showed that up to 33.3% of the isolated hospital strains of gram-negative bacteria were characterized by multiple resistance to antibiotics used in medical practice and to sulfathiazole. The strains showed the highest sensitivity to gentamicin and kanamycin. Most of the hospital strains were sensitive to chloramine and nirtan, but 4-13% of Klebsiella and Pseudomonas aeruginosa strains showed enhanced resistance to 0.1% chloramine solution.

[The sensitivity to antibiotics and disinfecting substances of opportunistic microorganisms isolated from the oral cavity of patients with orthodontic pathology]. / Chuvstvitel'nost' k antibiotikam i dezinfitsiruiushchim veshchestvam uslovno-patogennykh mikroorganizmov, vydelennykh iz polosti rta bol'nykh so ortopedicheskoi patologiei.

Antibiotic and disinfectant sensitivities of opportunistic bacteria, isolated from the oral cavity of normal subjects and orthodontic patients, were under study. The findings evidence that oral microflora of patients with orthodontic conditions is more rich in microorganisms that can induce pyoinflammatory diseases than that in health. Among the isolated opportunistic bacteria strains were found that were resistant to both: antibiotics and disinfectants. Antibiotic sensitivities of the microorganisms isolated from normal subjects and patients were found virtually the same, whereas the strains isolated from the patients have shown a higher sensitivity to disinfectants. The authors emphasize that effective prevention of pyoseptic complications in the maxillofacial area involves not only bacteriologic monitoring and studies of the microorganism sensitivity to antibiotics, but analysis of the bacterial sensitivity to disinfectants as well.

Inappropriate use of D-values for determining biocidal activity of various antimicrobials.

The objective of this study was to investigate the application of established D-value calculations to survival curves for various bacteria using the following antimicrobials: acidified sodium chlorite, triclosan, octanoic acid, and sodium hydroxide. D-values can be calculated in 3 ways, a linear regression, an endpoint calculation, or an average of multiple endpoint calculations. The assumption made in calculating a D-value is that the rate of kill follows 1st-order kinetics under specified treatment conditions. Each antimicrobial solution was challenged with approximately 108 CFU/mL of Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica subsp. enterica, and Escherichia coli independently and in triplicate. Test systems were sampled at each of the 10 time points over a period of 7 min, neutralized, pour plated then incubated at 35 °C for 48 h (AOAC official method 960.09). Survival curves using the log-transformed data were calculated using regression analysis. Correlations coefficients for all linear regression analyses ranged between 0.291 and 0.982, with 6 of the 16 different treatment systems having an R2 value below 0.7. Methods used for calculating D-values should lead to the same result if the survival curve in a given condition is linear. The calculated D-values were different using endpoint analysis (Stumbo method), linear regression, and average of multiple endpoints. This study demonstrates the nonlinearity of inactivation curves of antimicrobials. D-value estimations cannot be reliably used to illustrate biocidal activity in antimicrobial test systems.

Desinfectantes en aerosol como alternativa de control ambiental de los hongos anemófilos en viviendas / Disinfectant spray as an alternative environmental control of airborne fungi in houses

El objetivo de este trabajo fue evaluar el efecto de productos desinfectantes comerciales en aerosol sobre la carga de hongos ambientales en habitaciones de viviendas, teniendo en cuenta que las personas, principalmente los niños, pasan la mayor parte del día en las mismas. Además, se estudió, a través de encuestas, las preferencias del público a la hora de adquirir los desinfectantes comerciales ensayados. El número de UFC/placa fue variable dentro de la misma habitación para las distintas mediciones realizadas antes de la aplicación de los productos desinfectantes. Los porcentajes de inhibición del crecimiento fúngico fueron variables dependiendo del producto desinfectante ensayado y de la habitación muestreada. Dichos porcentajes no fueron diferentes significativamente entre los desinfectantes. Los valores de temperatura estuvieron dentro de un rango de 11 a 19,5 ºC y el porcentaje de humedad relativa varió entre un 64 a 85 por ciento. El 89 por ciento de los encuestados conocía la existencia de desinfectantes ambientales en aerosol. El 78 por ciento conocía el Lysoform®, seguido por Glade® antitabaco y Ayudin®. Dentro de los productos más utilizados, el 54 por ciento de los encuestados indicó el producto Lysoform®. La mayoría de los consumidores eligieron a los desinfectantes por lo que conocen y los medios de comunicación influyeron en el 29 por ciento de los consumidores al momento de elegir el desinfectante. Estos productos ensayados son recomendables en una habitación para disminuir la carga de hongos ambientales y así reducir el efecto perjudicial de estos hongos sobre las personas que la utilizan.

The aim of this study was to evaluate the effect of commercial disinfectant spray on the concentration of airborne fungi in bedrooms, considering that people, especially children, spend most of the day in them. Also, public preferences through surveys were studied when acquiring commercial disinfectants tested. The number of CFU/Petri dish was variable within the same bedroom for the different measurements performed before application of the disinfectant. Percent inhibition of fungal growth were variable, depending on the disinfecting product tested and sampled bedroom. These percentages were not significantly different between the disinfectants. The temperature values were within a range of 11 to 19.5° C and relative humidity percentage ranged from 64 to 85 percent. Eighty nine percent of respondents knew about environmental disinfectant spray. Seventy-eight percent knew about Lysoform®, followed by antismoking Glade® and Ayudin®. Among the most widely used products, 54 percent of respondents said the Lysoform® product was the most used. Most consumers chose known disinfectants and, when choosing a disinfectant, the media influenced 29 percent of consumers. The used products are recommended to be used in a bedroom to decrease the number of airborne fungi and thus reduce the harmful effect of these fungi on the people that use this room.

Neutralization of the activity of eight disinfectants by organic matter.

The effect of organic matter on the activity of eight disinfectants was evaluated. Three types of interfering substrates (whole milk powder, dried beef blood and fish meal) were tested according to the method of Whitmore and Miner adapted to the AOAC use-dilution method. Glutaraldehyde and to a certain extent, chlorhexidine acetate and the amphoteric surfactant kept their disinfecting activity after contact with high concentrations of organic matter. The quaternary ammonium compound as well as the quaternary ammonium-glutaraldehyde complex were more readily neutralized whereas anionic acid, iodophor and sodium hypochlorite did not tolerate the presence of organic matter. The neutralizing activity of powders was correlated to their solubility and composition.

[Inactivation of disinfectants by proteins and calcium ions as a function of their chemical constitution and bacterial species]. / Inactivation par les protéines et les ions calcium des désinfectants en fonction de leur nature chimique et des espèces bactériennes.

Activity of nine disinfectants was evaluated with interfering substances (albumin-yeast mixture and hard water), by two methods (NFT. 72 170, NFT. 72 171 and without interfering substance NFT. 72 150, NFT 72 151 respectively). Interference indexes were defined as the ratio of bactericidal concentration without interfering substances to bactericidal concentration with interfering substances. Five different ratings of interference effects were arbitrarily defined (no effect, moderate, average, strong, very strong). Proteins and hard water significantly inactivate aldehydes and quaternary ammonium salts : interference index is usually 25 but may reach 100-500. Inactivation of phenolic products by these two interfering substances is either lacking or inconsiderable. Conversely to Gram negative bacteria, activity of disinfectants on Gram positive bacteria is not significantly affected by proteins or hard water. P. aeruginosa can survive despite high concentrations of disinfectant in the presence of proteins and calcium ions. This finding may be a reason why this microorganism is difficult to eliminate from hospital wards.

[Bactericidal activity of various disinfectants with or without interfering protein substances]. / Activité bactéricide de quelques désinfectants en présence ou non de substances interférentes protéiques.

Determination of bactericidal activity of 15 disinfectants by AFNOR standard NF T 72-151 (in distilled water) and AFNOR standard NF T 72-171 (in the presence of protein) provides practical guidelines for optimal use of the different available products. Inhibition by dirt is weak for products composed chiefly of phenol derivatives, while it is very strong for quaternary ammonium compounds as well as chlorine which therefore require prior cleansing of surfaces to be disinfected. Two strains (out of the five indicated by AFNOR standards), i.e. P. aeruginosa and M. smegmatis, are especially resistant when residual organic material is present. According to specific conditions of use, such as whether surfaces are clean or dirty, or whether a hospital or food factory is involved, appropriate AFNOR standards must be chosen to study the activity of disinfectants in various settings and on different strains.

Efficacy of inactivators against 14 disinfectant substances.

More than 24 inactivators were tested for their suitability against 14 disinfectant substances by a quantitative suspension test. Even simple inactivators were found to be efficace: 0.1% cysteine and 0.5% sodium thiosulfate for mercuric chloride and the iodophor; 0.1% sodium sulfite for these two substances and for the QAC in the lower concentration; 1.0% polysorbate 80 for hexylresorcinol, o-phenylphenol and the QAC in the lower concentration; 0.5% sodium thioglycolate for mercuric chloride, the iodophor and chloramine-T. The inactivator active against most disinfectants was the combination LPHT or 0.3% lecithin / 3.0% polysorbate 80 /0.1% histidine / 0.5% sodium thiosulfate. Also active were: LPWT (0.5% lecithin / 1.0% polysorbate 80 /1.0% Lubrol W / 1.0% sodium thiosulfate), LPT (2.0% lecithin / 2.0% polysorbate 80 / 0.5% sodium thiosulfate) and PS (3.0% polysorbate 80 / 3.0% saponin).

The use of inactivators in the evaluation of disinfectants.

Since the earliest days of disinfectant testing bacteriostatic effect has misled many workers in this field. This problem has long been appreciated by some and a considerable battery of neutralizers has been employed, often with great success. Other anti-bacteriostatic measures, used without control, fail to revive damaged though viable organisms.This paper sets out to describe some of the problems that are encountered in this confused field and the means whereby they may be overcome.