In vitro Susceptibility and Evaluation of Techniques for Understanding the Mode of Action of a Promising Non-antibiotic Citrus Fruit Extract Against Several Pathogens

Copyright © 2019 de Nova, Carvajal, Prieto and Rubio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.[EN] The screening for alternatives to antibiotics is an urgent need for the pharmaceutical industry. One of these alternatives seems to be the citrus fruit extracts, which are showing a significant antibacterial activity against Gram-negative and Gram-positive bacteria. One of these citrus extracts, named BIOCITRO®, is assessed in this study to elucidate its bacteriostatic and bactericidal effect and its mode of action on the important pathogens Campylobacter coli, C. jejuni, Escherichia coli, Salmonella enterica ssp. enterica, Clostridium difficile, C. perfringens, and Staphylococcus aureus. For most of the strains tested of these bacteria the product was bactericidal as well as bacteriostatic at the same concentration, and the minimum bactericidal concentrations ranged from 16 to 256 μg/mL. Regarding the mode of action, important changes in the permeability, structure, composition and morphology of the bacterial envelope were evidenced using flow cytometry, Fourier transform infrared spectroscopy and scanning electron microscopy. The main effect of the product was found over carbohydrates and polysaccharides, inducing the release of microvesicles by the cells in addition to other specific effects. During the study, the techniques used were evaluated to clarify their contribution to the knowledge of the mode of action of the product. The survival test elucidated whether the modifications displayed using other techniques affected the viability of the cells or on the contrary, the cells remained viable even with evident changes in their structure, composition or morphology. Flow cytometry showed that for some strains the proportion of cells detected with altered membrane permeability were higher than the number of non-viable cells, and therefore the damage did not affect the viability of some cells. On the contrary, some cells observed using scanning electron microscopy with no apparent damage, were demonstrated non-viable using the survival test, making this technique indispensable in studies of the mode of action of antimicrobials to make a correct interpretation of the data from other techniques.SIThe research was partly financed by PROBENA (2015/00119/001), the company commercializing BIOCITRO® in the EU, but without any influence over the experimental procedures neither results

Microbiological Safety and Shelf-Life of Low-Salt Meat Products—A Review

[EN] Salt is widely employed in different foods, especially in meat products, due to its very diverse and extended functionality. However, the high intake of sodium chloride in human diet has been under consideration for the last years, because it is related to serious health problems. The meat-processing industry and research institutions are evaluating different strategies to overcome the elevated salt concentrations in products without a quality reduction. Several properties could be directly or indirectly affected by a sodium chloride decrease. Among them, microbial stability could be shifted towards pathogen growth, posing a serious public health threat. Nonetheless, the majority of the literature available focuses attention on the sensorial and technological challenges that salt reduction implies. Thereafter, the need to discuss the consequences for shelf-life and microbial safety should be considered. Hence, this review aims to merge all the available knowledge regarding salt reduction in meat products, providing an assessment on how to obtain low salt products that are sensorily accepted by the consumer, technologically feasible from the perspective of the industry, and, in particular, safe with respect to microbial stability.S

Scientific Opinion on risk based control of biogenic amine formation in fermented foods.

A qualitative risk assessment of biogenic amines (BA) in fermented foods was conducted, using data from the scientific literature, as well as from European Union-related surveys, reports and consumption data. Histamine and tyramine are considered as the most toxic and food safety relevant, and fermented foods are of particular BA concern due to associated intensive microbial activity and potential for BA formation. Based on mean content in foods and consumer exposure data, fermented food categories were ranked in respect to histamine and tyramine, but presently available information was insufficient to conduct quantitative risk assessment of BA, individually and in combination(s). Regarding BA risk mitigation options, particularly relevant are hygienic measures to minimize the occurrence of BA-producing microorganisms in raw material, additional microbial controls and use of BA-nonproducing starter cultures. Based on limited published information, no adverse health effects were observed after exposure to following BA levels in food (per person per meal): a) 50 mg histamine for healthy individuals, but below detectable limits for those with histamine intolerance; b) 600 mg tyramine for healthy individuals not taking monoamino oxidase inhibitor (MAOI) drugs, but 50 mg for those taking third generation MAOI drugs or 6 mg for those taking classical MAOI drugs; and c) for putrescine and cadaverine, the information was insufficient in that respect. Presently, only high-performance liquid chromatography (HPLC)-based methods enable simultaneous and high sensitivity quantification of all BA in foods, hence are best suited for monitoring and control purposes. Monitoring of BA concentrations in fermented foods during the production process and along the food chain would be beneficial for controls and further knowledge. Further research on BA in fermented foods is needed; particularly on toxicity and associated concentrations, production process-based control measures, further process hygiene and/or food safety criteria development, and validation of analysis methods

A European questionnaire-based study on population awareness and risk perception of antimicrobial resistance

Versión post-printTo tackle antimicrobial resistance (AMR) is of outmost importance for the general population to understand the severity and the relevance of different routes of transmission. Respondents of different age groups, educational and occupational backgrounds, area of living, diet and household composition participated in an online survey with questions concerning socio-demographics, personal use of antibiotics, awareness, general knowledge, sources of information, behavior and attitude toward antibiotics, and risk perception on antibiotics and AMR. Descriptive and logistic regression analyses were carried out. A total of 1252 respondents, mainly from EU, participated in the survey. About 57.7% declared they consumed antibiotics in the last year and some misguided behaviors were identified, especially for those not having a food- or health-related background, who more frequently failed in giving the right answer to uncontroversial true/false questions (ANOVA, P < 0.05). The youngest respondents were less confident on the information received from traditional media (OR = 0.425), the national government (OR = 0.462), and consumer organizations (OR = 0.497), while they frequently obtained information from social networks and online media, which could therefore be exploited as a channel for educational campaigns targeting this population group. New measures, strategies and policy agenda at a European level aimed at improving awareness on AMR among targeted community groups must be taken into consideration.S

Plasma-activated water: A cutting-edge technology driving innovation in the food industry

.Innovation regarding food production and processing is required to meet the emerging challenges of ensuring worldwide food security and meeting consumer demands for high-quality, safe and nutritious food products. This review provides insights into the current state-of-the-art of the emerging applications of Plasma Activated Water (PAW) in the food industry. PAW antimicrobial properties, inactivation mechanisms and the critical factors determining the lethal effect, as well as the bases for other technological applications are discussed. Overall, this review article describes the degree of success achieved by PAW technology in different applications and illustrates its feasibility and applicability in the food-processing industryS

High pressure processing at the early stages of ripening enhances the safety and quality of dry fermented sausages elaborated with or without starter culture

[EN] To study the quality of chorizo de León dry fermented sausages (DFS), high pressure processing (HPP) applied at the early stages of ripening and the use of a functional starter culture were evaluated as additional safety measures. Furthermore, the ability to control the populations of artificially inoculated Listeria monocytogenes and Salmonella Typhimurium was investigated and the evolution of microbial communities was assessed by amplicon 16S rRNA metataxonomics. The use of HPP and the starter culture, independently or combined, induced a reduction of Listeria monocytogenes of 1.5, 4.3 and > 4.8 log CFU/g respectively, as compared to control. Salmonella Typhimurium counts were under the detection limit (<1 log) in all treated end-product samples. Both additional measures reduced the activity of undesirable microbiota, such as Serratia and Brochothrix, during the production of DFS. Moreover, the starter culture highly influenced the taxonomic profile of samples. No adverse sensory effects were observed, and panelists showed preference for HPP treated DFS. In conclusion, this new approach of applying HPP at the early stages of ripening of DFS in combination with the use of a defined starter culture improved the safety and quality of the meat productS

Biofilm formation ability and tolerance to food-associated stresses among ESBL-producing Escherichia coli strains from foods of animal origin and human patients

[EN] The dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli continues to be an important health concern, with the food production chain as a suggested reservoir. A collection of 31 strains, including ESBL-producing E. coli strains isolated from foods of animal origin and from human patients and reference collection strains, was evaluated regarding their biofilm formation ability, tolerance to food-associated stresses (heat, acid, non-thermal atmospheric plasma (NTAP) and UV-C light) and RpoS status. The most relevant phenotypic differences among strains were observed for biofilm formation and heat resistance, being related to the source of isolation (clinical vs food vs reference) or to the sequence type (ST131 vs other STs), while only minor differences were related to the occurrence of the ESBL genes (blaTEM, blaCTX-M, blaSHV). The biofilm formation ability on stainless steel was significantly higher for the field isolates, both clinical- and food-related, than for the reference strains. Food isolates were more thermotolerant (58 oC-1 and 2 min) than clinical isolates. Minor differences among strain categories were observed for their tolerance to the other inactivation technologies. Some polymorphisms in the rpoS gene sequences were detected, but loss-of-function mutations were not found, with the clustering of strains mainly based on their sequence typeSIThis work was founded by “Consejería de Educación” (Junta de Castilla y León) and “Fondo Europeo de Desarrollo Regional” (FEDER; European Commission) [LE113P17]. P. Fernández-Gómez is grateful to Junta de Castilla y León and the European Social Fund (ESF) for awarding her a pre-doctoral grant [BOCYL-D-15122017-4]. Cristina Albalá Díez and Sandra Rodríguez de la Torre were involved in the performance of some of the laboratory experiments. The stainless steel plates used for biofilm formation determinations were provided by the Department of Mechanical Engineering of the University of La Rioj

Assessment of a plasmid conjugation procedure to monitor horizontal transfer of an extended-spectrum β-lactamase resistance gene under food chain scenarios

[EN] Plasmids are relevant reservoirs of antimicrobial resistance genes (ARGs) which confer adaptive advantages to their host and can be horizontally transferred. The aims of this study were to develop a conjugation procedure to monitor the horizontal transfer of a 193 kb plasmid containing the extended-spectrum β-lactamase production gene blaCTX-M-14 between two Escherichia coli strains under a range of food chain-related scenarios, including temperature (20–37 °C), pH (5.0–9.0) or the presence of some biocidal agents (benzalkonium chloride, sodium hypochlorite or peracetic acid). The average conjugation rate in LB broth after 18 h at 37 °C was 2.09e-04 and similar rates were observed in a food matrix (cow's milk). The conjugation was reduced at temperatures below 37 °C, at alkaline pH (especially at pH 9.0) or in the presence of benzalkonium chloride. Peracetic acid and sodium hypochlorite slightly increased conjugation rates, which reached 5.59e-04 and 6.77e-03, respectively. The conjugation procedure described can be used to identify risk scenarios leading to an enhanced ARGs transmission via plasmid conjugation, as well as to identify novel intervention strategies impairing plasmid conjugation and tackling antimicrobial resistance.SIThis work was supported by the Ministry of Science, Innovation and Universities of the Spanish Government (AGL2016-78085-P and PID2020-118813GB-I00). AAM is grateful to the Ministry of Science, Innovation and Universities of the Spanish Government for awarding him a pre-doctoral grant (grant number FPI BES-2017-079753

Tuberculosis Epidemiology and Badger (Meles meles) Spatial Ecology in a Hot-Spot Area in Atlantic Spain

[EN] We provide a temporal overview (from 2012 to 2018) of the outcomes of tuberculosis (TB) in the cattle and badger populations in a hot-spot in Asturias (Atlantic Spain). We also study the badger’s spatial ecology from an epidemiological perspective in order to describe hazardous behavior in relation to TB transmission between cattle and badgers. Culture and single intradermal tuberculin test (SITT) were available for cattle as part of the National Program for the Eradication of TB. A field survey was also carried out in order to determine the paddocks and buildings used by each farm, and the information obtained was stored by using geographic information systems. Moreover, eighty-three badgers were submitted for necropsy and subsequent bacteriological studies. Ten badgers were also tracked, using global positioning system (GPS) collars. The prevalence of TB in cattle herds in the hot-spot increased from 2.2% in 2012 to 20% in 2016; it then declined to 0.0% in 2018. In contrast, the TB prevalence in badgers increased notably (from 5.55% in 2012–2015 to 10.64% in 2016–2018). Both cattle and badgers shared the same strain of Mycobacterium bovis. The collared badgers preferred paddocks used by TB-positive herds in spring and summer (when they were more active). The males occupied larger home ranges than the females (Khr95: males 149.78 ± 25.84 ha and females 73.37 ± 22.91 ha; Kcr50: males 29.83 ± 5.69 ha and females 13.59 ± 5.00 ha), and the home ranges were smaller in autumn and winter than in summer. The averages of the index of daily and maximum distances traveled by badgers were 1.88 ± (SD) 1.20 km and 1.99 ± 0.71 km, respectively. One of them presented a dispersive behavior with a maximum range of 18.3 km. The most preferred habitat was apple orchards in all seasons, with the exception of winter, in which they preferred pastures. Land uses and landscape structure, which have been linked with certain livestock-management practices, provide a scenario of great potential for badger–cattle interactions, thus enhancing the importance of the badgers’ ecology, which could potentially transmit TB back to cattle in the futureSIThis work was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) projects (FEDER co-funded): RTA2011-00010-00-00, RTA2014-00002-C02-01; by the Agencia Estatal de Investigación (AEI) reference project RTI2018-096010-B-C21 (FEDER co-funded); and by PCTI 2018–2020 (GRUPIN: IDI2018-000237) and FEDER.We received funds from RTI2018-096010-B-C21 (FEDER co-funded) to cover publication cost

Evaluation of the performance of a three-strains lactic acid bacteria cocktail for the control of Listeria monocytogenes on marinated lean pork

[EN] The aim of this study was to evaluate the addition of a cocktail of three lactic acid bacteria (LAB) (Lactococcus lactis, Lacticaseibacillus paracasei and Lactiplantibacillus plantarum) as a protective culture into marinated pork. The pork was artificially inoculated with two Listeria monocytogenes strains and stored for 12 days. Two packagings were used: vacuum or modified atmosphere packaging (MAP). LAB, L. monocytogenes, psychrotrophic bacteria, pH, aw, color, the metataxonomic profile and the sensorial quality of the product were evaluated. The growth of L. monocytogenes under vacuum and MAP was reduced with the use of the LAB up to 0.8 and 0.7 log10 CFU/g, respectively. LAB counts gradually increased, which was accompanied by a slight decrease in pH. In LAB samples, psychrotrophic bacteria showed a reduction at day 12 as compared to non-inoculated samples. Some minor differences were also observed among samples for color and sensory parameters. Regardless of the type of packaging, the microbiota of the marinated pork was dominated initially by Photobacterium and subsequently during storage by a diversity of LAB. The application of LAB could help to obtain a safe product, although further evaluation would be required to optimize the application of the LAB cocktail in real-scale commercial scenarios.SIPublicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL