RpoS-independent evolution reveals the importance of attenuated cAMPCRP regulation in high hydrostatic pressure resistance acquisition in E. coli

High hydrostatic pressure (HHP) processing is an attractive non-thermal alternative to food pasteurization. Nevertheless, the large inter- and intra-species variations in HHP resistance among foodborne pathogens and the ease by which they can acquire extreme resistance are an issue of increasing concern. Since RpoS activity has been considered as a central determinant in the HHP resistance of E. coli and its pathovars, this study probed for the potential of an E. coli MG1655 ΔrpoS mutant to acquire HHP resistance by directed evolution. Despite the higher initial HHP sensitivity of the ΔrpoS mutant compared to the wild-type strain, evolved lineages of the former readily managed to restore or even succeed wild-type levels of resistance. A number of these ΔrpoS derivatives were affected in cAMP/CRP regulation, and this could be causally related to their HHP resistance. Subsequent inspection revealed that some of previously isolated HHP-resistant mutants derived from the wild-type strain also incurred a causal decrease in cAMP/CRP regulation. cAMP/CRP attenuated HHP-resistant mutants also exhibited higher resistance to fosfomycin, a preferred treatment for STEC infections. As such, this study reveals attenuation of cAMP/CRP regulation as a relevant and RpoS-independent evolutionary route towards HHP resistance in E. coli that coincides with fosfomycin resistance

Carvacrol selective pressure allows the occurrence of genetic resistant variants of Listeria monocytogenes EGD-e

Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety

Hurdle Technology Approach to Control Listeria monocytogenes Using Rhamnolipid Biosurfactant

This study evaluates the combination of mild heat with a natural surfactant for the inactivation of L. monocytogenes Scott A in low-water-activity (aw) model systems. Glycerol or NaCl was used to reduce the aw to 0.92, and different concentrations of rhamnolipid (RL) biosurfactant were added before heat treatment (60 °C, 5 min). Using glycerol, RL treatment (50–250 µg/mL) reduced bacterial population by less than 0.2 log and heat treatment up to 1.5 log, while the combination of both hurdles reached around 5.0 log reduction. In the NaCl medium, RL treatment displayed higher inactivation than in the glycerol medium at the same aw level and a larger synergistic lethal effect when combined with heat, achieving ≥ 6.0 log reduction at 10–250 µg/mL RL concentrations. The growth inhibition activity of RL was enhanced by the presence of the monovalent salts NaCl and KCl, reducing MIC values from >2500 µg/mL (without salt) to 39 µg/mL (with 7.5% salt). The enhanced antimicrobial activity of RL promoted by the presence of salts was shown to be pH-dependent and more effective under neutral conditions. Overall, results demonstrate that RL can be exploited to design novel strategies based on hurdle approaches aiming to control L. monocytogenes

Comparative study on the impact of equally stressful environmental sporulation conditions on thermal inactivation kinetics of B. subtilis spores

Control of bacterial spores continues to be one of the main challenges for the food industry due to their wide dissemination and extremely high resistance to processing methods. Furthermore, the large variability in heat resistance in spores that contaminate foods makes it difficult to establish general processing conditions. Such heterogeneity not only derives from inherent differences among species and strains, but also from differences in sporulation environments that are generally ignored in spores encountered in foods. We evaluated heat inactivation kinetics and the thermodependency of resistance parameters in B. subtilis 168 spores sporulated at adverse temperatures, water activity (aw), and pH, applying an experimental approach that allowed us to quantitatively compare the impact of each condition. Reduction of incubation temperature from the optimal temperature dramatically reduced thermal resistance, and it was the most influential factor, especially at the highest treatment temperatures. These spores were also more sensitive to chemicals presumably acting in the inner membrane. Reducing sporulation aw increased heat resistance, although the magnitude of that effect depended on the solute and the treatment temperature. Thus, changes in sporulation environments varied 3D100°C values up to 10.4-fold and z values up to 1.7-fold, highlighting the relevance of taking such a source of variability into account when setting heat processing conditions. UV-C treatment and sodium hypochlorite efficiently inactivated all spore populations, including heat-resistant ones produced at low aw

Mejora de los test rápidos de detección de antibióticos en alimentos basados en el uso de esporos de Geobacillus spp. como indicadores biológicos

Los antibióticos se utilizan en producción animal para tratar y prevenir enfermedades infecciosas, pero la presencia de residuos en alimentos presenta diversos efectos adversos para el consumidor, como la aparición de reacciones alérgicas, y para las industrias de transformación, como la inhibición de los procesos de fermentación. Los límites máximos de residuos tolerables de sustancias farmacológicamente activas en alimentos de origen animal están regulados por la legislación, y para su control se necesitan métodos rápidos de cribado para la detección de los mismos. Entre ellos, destacan los métodos basados en la inhibición de la germinación y crecimiento de esporos de Geobacillus stearothermophilus por su rapidez y sensibilidad. Sin embargo, los test comerciales tienen una serie de limitaciones, como la alta concentración de esporos necesaria para tener tiempos de lectura rápidos, lo que a su vez encarece el coste de producción de los kits. En este trabajo, se investigaron estrategias para mejorar la producción de esporos de una cepa de G. stearothermophilus utilizada en un test comercial. Se consiguió aumentar el rendimiento de esporulación cambiando la composición del medio de cultivo, aunque los esporos producidos en este medio presentaban menor sensibilidad a ciertos antibióticos. Por otro lado, observamos que solo una pequeña proporción de los esporos son capaces de germinar durante el test. Además, el medio de detección del kit comercial no es apto para el crecimiento, e incluso favorece la muerte de las células germinadas mucho antes de que el indicador de pH (teoricamente el indicador de crecimiento/no crecimiento) cambie de color. Finalmente, se estudió si la adición de ciertos nutrientes, como glucosa o cisteína, permitía mejorar el porcentaje de esporos capaces de germinar.<br /