Influence of environmental factors on bacterial biofilm formation in the food industry: a review

Formation and development of bacterial biofilms in the food industry could be a cause of food contamination, compromising food safety and shelf-life. Among the factors modulating biofilm formation, this review will focus in conditions normally encountered by bacteria in food environments, especially in biofilm initiation and development. The effect of environmental factors (substratum, temperature, oxygen concentration, hydrodynamic effects, food matrix composition, and microbial interactions) on biofilm formation is multifaceted and, in many circumstances, their influence could be compensatory. A better knowledge of these factors would allow for a better control of biofilm formation, either by avoiding and/or eradicating biofilms or by defining adequate Hazard Analysis and Critical Control Point systems in the food industry

Emerging mutant populations of Listeria monocytogenes EGD-e under selective pressure of Thymbra capitata essential oil question its use in food preservation

Due to their excellent antimicrobial properties, essential oils (EO) have been proposed as potential preservatives for certain kinds of foods, such as dairy products. However, the occurrence of pathogenic populations that are resistant to EOs could pose a health risk. This report seeks to assess the emergence of resistant populations in Listeria monocytogenes EGD-e growth at 37 °C under selective pressure of Thymbra capitata EO (TCO), to characterise their resistance in laboratory media, and to identify their genotypic changes, as well as to evaluate the resistance in skimmed milk. TCO cyclic treatment allowed the isolation of two L. monocytogenes EGD-e resistant strains against the EO: LmSTCO by sublethal doses (75 µL/L TCO) and LmLTCO by lethal doses (300 µL/L TCO) after 20 and 30 cycles, respectively. Both strains displayed an increase of the minimum inhibitory and bactericidal concentration against TCO and a higher survival rate after lethal treatments than the wild-type strain (LmWT). Growth kinetics revealed a better adaptation of LmSTCO in presence of TCO, while LmLTCO grew more slowly compared to LmWT, even in the absence of the antimicrobial. Moreover, a slight increase in cross-resistance to antibiotics was observed: LmSTCO to β-lactams and LmLTCO to a series of broad-spectrum antibiotics. The genomic study revealed one sole nucleotide change in LmSTCO located in plsC gene codifying an enzyme involved in the production of phosphatidic acid, a precursor in cell membrane synthesis. Five genetic variations were found in LmLTCO: among them, the deletion of an ATP-synthesis system involved in slowing bacterial growth. Inhibition and inactivation assays in skimmed milk confirmed the increased resistance of both strains, thereby indicating a safety risk in case these strains emerge in the food chain. These results strongly suggest that the occurrence of such resistances should be taken into account in order to ensure the efficacy of natural antimicrobials in the design of food preservation strategies

Salmonella enterica serovar Typhimurium genetic variants isolated after lethal treatment with Thymbra capitata essential oil (TCO) showed increased resistance to TCO in milk

The high prevalence of Salmonella enterica in milk poses a risk of considerable concern in the preservation of certain dairy products, mainly those elaborated from raw milk. Essential oils (EOs) have been proposed as a promising food preservative for such products due to their strong antimicrobial properties. Additionally, these natural antimicrobials have been shown to be effective against multi-drug resistant strains. They can thus also be utilized to prevent the dissemination of antimicrobial resistances (AMR). However, recent evidence of the development of bacterial resistance under EO treatments may call their use into question. This study sought to assess the emergence of antimicrobial resistant genetic variants of S. enterica serovar Typhimurium from survivors after cyclic exposure to lethal doses (>5 log10 cycles of inactivation) of Thymbra capitata EO (TCO), in order to evaluate the impact that it could have on milk preservation, to ascertain whether cross-resistance to antibiotics occurs, and to identify the genomic changes responsible for their phenotype. Isolated strains by TCO (SeTCO) showed a two-fold increase in minimum inhibitory and bactericide concentrations (MIC and MBC) of TCO compared to Salmonella enterica serovar Typhimurium wild-type strain (SeWT) in laboratory growth medium, as well as a greater adaptation and growth rate in the presence of the EOs and a higher survival to TCO treatments in buffers of pH 4.0 and 7.0. The increased resistance of SeTCO was confirmed in skimmed milk: 300 μL/L TCO reduced only 1 log10 cycle of SeTCO population, whereas it inactivated more than 5 log10 cycles in SeWT. Moreover, SeTCO showed an increased cross-resistance against aminoglycosides, quinolones and tetracyclines. Whole genome sequencing revealed 5 mutations in SeTCO: 2 in genes involved in O-antigens synthesis (rfbV and rfbX), 2 in genes related to adaptation to the growing medium (trkA and glpK), and 1 in a redox-sensitive transcriptional regulator (soxR). The phenotypic characterization of a constructed SeWT strain with mutant soxRSeTCO demonstrated that the mutation of soxR was the main cause of the increased resistance and tolerance observed in SeTCO against TCO and antibiotics. The emergence of resistant strains against EOs might jeopardize their use as food preservatives. Further studies will thus be required to determine under which conditions such resistant strains might occur, and to assess the food risk they may pose, as well as to ascertain their impact on the spread of AMR

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

Phenotypic and genotypic comparison of antimicrobial-resistant variants of Escherichia coli and Salmonella Typhimurium isolated from evolution assays with antibiotics or commercial products based on essential oils

On account of the widespread development and propagation of antimicrobial-resistant (AMR) bacteria, essential oils (EOs) have emerged as potential alternatives to antibiotics. However, as already observed for antibiotics, recent studies have raised concerns regarding the potential emergence of resistant variants (RVs) to EOs. In this study, we assessed the emergence of RVs in Escherichia coli and Salmonella enterica Typhimurium after evolution assays under extended exposure to subinhibitory doses of two commercial EOs (AEN and COLIFIT) as well as to two antibiotics (amoxicillin and colistin). Phenotypic characterization of RVs from evolution assays with commercial EOs yielded no relevant increases in the minimum inhibitory concentration (MIC) of E. coli and did not even modify MIC values in S. Typhimurium. Conversely, RVs of E. coli and S. Typhimurium isolated from evolution assays with antibiotics showed increased resistance. Genotypic analysis demonstrated that resistance to commercial EOs was associated with enhanced protection against oxidative stress and redirection of cell energy toward efflux activity, while resistance to antibiotics was primarily linked to modifications in the cell binding sites of antibiotics. These findings suggest that AEN and COLIFIT could serve as safe alternatives to antibiotics in combating the emergence and dissemination of antimicrobial resistance within the agrifood system

An integrative model for media literacy and digital competence in Primary Education

Tras un amplio estudio acercadela formación sobre y con los medios en el marco regulatorio español vigente, se estima oportuno incorporar un modelo integrador, TPACK, para facilitar el desarrollo de actividades para la mejora de la alfabetización mediática con los medios en la Educación Primaria. Para llevar a cabo este trabajo, se optó por un método bajo un enfoque exploratorio y concluyente con metodología cuasi-experimental, realizándose cuestionarios pretest y postest, tras la implementación de los campos de conocimiento que se incluyen en las matrices trabajadas y que dan lugar al desarrollo de actividades de competencia digital, en función del contexto o ecosistema educativo digital, de entre los reconocidos por la UNESCO. Los resultados sobre la valoración didáctica y pedagógica de los profesores, acerca de la confección de los materiales didácticos digitales resultantes, concluyó en la conveniencia y validez de dichas matrices tanto a nivel disciplinar, pedagógico, como en el aprovechamiento tecnológico.After an extensive study on media training in the current Spanish regulatory framework, it is considered appropriate to incorporate an integrative model, TPACK, to facilitate the development of activities for the improvement of media literacy in Primary Education. To carry out this research, a quasi-experimental methodology was chosen and an exploratory and conclusive approach adopted Pre-test and post-test questionnaires were administered after the implementation of the fields of knowledge included in the matrices. These help develop activities to improve students’ digital competence in the different digital education ecosystems recognized by UNESCO. The results regarding teachers’ assessment of the digital teaching materials created point to the validity and suitability of the matrices used, both at the pedagogical and the technological level