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Clostridium botulinum is a foodborne pathogen responsible for severe neuroparalytic disease associated with the ingestion of pre-formed toxin in food, with processed meats and canned foods being the most affected. Control of this pathogen in meat products is carried out using the preservative sodium nitrite (NaNO2), which in food, under certain conditions, such as thermal processing and storage, can form carcinogenic compounds. Therefore, the objective was to use nanoemulsified essential oils (EOs) as natural antimicrobial agents, with the aim of reducing the dose of NaNO2 applied in mortadella. The antimicrobial activity of nanoemulsions prepared with mixtures of EOs of garlic, clove, pink pepper, and black pepper was evaluated on endospores and vegetative cells of C. botulinum and Clostridium sporogenes (surrogate model) inoculated in mortadella prepared with 50 parts per million NaNO2. The effects on the technological (pH, water activity, and color) and sensory characteristics of the product were also evaluated. The combinations of EOs and their nanoemulsions showed sporicidal effects on the endospores of both tested microorganisms, with no counts observed from the 10th day of analysis. Furthermore, bacteriostatic effects on the studied microorganisms were observed. Regarding the technological and sensorial characteristics of the product, the addition of the combined EOs had a negative impact on the color of the mortadella and on the flavor/aroma. Despite the strong commercial appeal of adding natural preservatives to foods, the effects on flavor and color must be considered. Given the importance of controlling C. botulinum in this type of product, as well as the reduction in the amount of NaNO2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.

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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.

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Abstract The aim of this study was to evaluate the antimicrobial effect of the essential oils of cinnamon, cardamom, clove, oregano, and thyme and their synergism on vegetative cells and endospores of Clostridium perfringens type A inoculated in meat sausage (mortadella), as well as the influence of blends on the color, and lipid oxidation through the determination of thiobarbituric acid reactive substances (TBARS index). The anticlostridial action of the oil blends was established. The two added oil blends (Treat. 1: oregano, clove, and thyme; Treat. 2: oregano, clove, and cinnamon) in combination with reduced nitrite content (75 ppm) promoted a lower growth of C. perfringens in mortadella stored at 15 °C for 21 days in comparison to treatments containing only 75 ppm of nitrite. The essential oil blends showed antioxidant action and did not alter food color, thus possessing potential application as a preservative for the meat products industry.

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BACKGROUND: Clostridioides difficile is transmitted through endospores. Most disinfection procedures for these structures deploy high concentrations of chlorine-derived compounds such as sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC). However, these substances are linked to undesirable public health and environmental issues. AIM: To compare the efficacy of NaCl-derived electrochemically activated solution (ECAS, 0.18% w/v NaOCl, pH=9.6-10.3), commercial bleach (5000 ppm, 2.83% w/v NaOCl, pH=5.6) and NaDCC (1000 ppm, pH=6.8) to inactivate C. difficile endospores on surfaces using a standard quantitative test (EPA MO-21-03). FINDINGS: Ten representative reference and field strains from multi-locus sequence typing Clades 1-5 were assayed (N=10). Irrespective of the phylogenetic background of the strains, ECAS showed comparable or better log reduction values [mean=3.22, 95% confidence interval (CI) 0.40-5.56] than bleach (mean=2.74, 95% CI 0.12-5.50) and NaDCC (mean=2.02, 95% CI 0.10-5.12). Cyclic voltammetry measurements revealed similar electrochemical behaviours and open-circuit potentials for ECAS and NaOCl. Congruently, similar morphologies for spores treated with these two compounds were observed by transmission electron microscopy. A factorial design demonstrated that exposure time, but not activation time, influenced the efficacy of ECAS. CONCLUSIONS: ECAS and NaOC were found to have functional equivalence and may have a common mechanism of action.

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El objetivo de este trabajo fue emplear endosporas de Bacillus subtilis con actividad probiótica sobre indicadores fermentativos en órganos digestivos e inmunológicos de pollos de engorde. Se estudiaron tres cepas de B. subtilis obtenidos en trabajos previos (C-31, C-34 y E-44). Las tres cepas resistieron condiciones de pH ácido y sobrevivieron a la presencia de sales biliares. La cepa E-44 tuvo mayor capacidad de crecimiento y producción de endosporas, escogiéndose para la realización de los estudios posteriores. Se realizó un experimento con pollos de engorde y tres tratamientos con endosporas: control sin endosporas; baja dosis con 1,0x10(6); dosis media con 1,0x10(7) y dosis alta con 1,0x10(8) UFC.mL-1.kg de alimento. En el ciego de los pollos, la cepa E-44 a la dosis alta produjo mayor acidez y mayores contenidos de ácidos láctico, butírico, propiónico, acético y ácidos grasos de cadena corta totales. El peso relativo de la bolsa de Fabricio y el bazo, así como la inhibición de la hemaglutinación, fueron mayores en los animales que recibieron endosporas con la dosis media a los 42 días. Se concluye que se produjo una mejoría en la respuesta del sistema inmune de los pollos con el empleo del cultivo de endosporas de B. subtilis E-44 evaluado.

The aim of this study was the use of endospores of Bacillus subtilis with probiotic activity on fermentative indicators in digestive and immune organs of broilers. Three B. subtilis strains isolated from previous studies (C-31, C-34 and E-44) were used. The three strains resisted acid pH conditions and survived the presence of bile salts. E-44 strain had greater capacity for growth and endospore production was chosen to carry out further studies. An experiment with broilers and three treatments were performed with endospores at different concentrations administered with food. Control: without endospores; low dose: 1.0x10(6); medium and high dose 1.0x10(7) and 1.0x10(8) CFU/mL per Kg of food respectively. Results showed that strain E-44 at high dose, in the ceca of the chickens produced higher acidity and higher content of lactic, butyric, propionic, acetic and total short chain fatty acids. The relative weight of the bursa of Fabricius and spleen, as well as hemagglutination inhibition, after 42 days of treatment, were greater in animals receiving endospores at the medium dose. We concluded that there was an improvement in the immune response of chickens with the use of endospores of B. subtilis E-44 strain at the given dose.