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The outbreaks of Listeria associated with food consumption are increasing worldwide concurrently with public concern about the need for natural growth inhibitors. In this context, propolis seems to be a promising bioactive product collected by honeybees, due to its antimicrobial activity against different food pathogens. This study aims to evaluate the efficacy of hydroalcoholic propolis extracts for controlling Listeria under several pH conditions. The physicochemical properties (wax, resins, ashes, impurities), the bioactive compounds (phenolic and flavonoid content), and the antimicrobial activity of 31 propolis samples collected from the half North of Spain were determined. Results showed similar trends in the physicochemical composition and bioactive properties, regardless of the harvesting area. Non-limiting pH conditions (7.04, 6.01, 5.01) in 11 Listeria strains (5 from collection and 6 wild strains from meat products) exhibited MICs (Minimum inhibition concentration) and MBCs (Minimum bactericidal concentration) ranging from 39.09 to 625 μg/mL. The antibacterial activity increased under acidic pH conditions, showing a synergistic effect at pH = 5.01 (p < 0.05). These findings suggest the potential of Spanish propolis as a natural antibacterial inhibitor to control Listeria growth in food products.

Fish is considered an essential food in the human diet due to its nutritional qualities and is widely consumed around the world. The source of fish destined for human use and consumption is through capture fisheries and aquaculture activities. Although fish is a food of nutritional quality, it is also a food susceptible to deterioration and microbiological contamination, putting the health of consumers at risk. The different viruses are considered hazards of biological origin in food that cause various outbreaks of diseases through the consumption of fish, and products derived from their contamination in distinct phases of the food chain, through contaminated water and food handlers. Therefore, this document aims to provide an overview of foodborne diseases and causative agents, especially viruses, through a bibliographic review. In the production and commercialization of foods such as fish and products, it is considered that actions to control and prevent viral diseases, sanitary regulation and microbiological analysis tests should be involved, all in favor of the promotion and safeguarding of public health through the availability and consumption of safe food and water. DOI: http://dx.doi.org/10.5281/zenodo.5865046

Hygiene failures in meat can be identified based on the evaluation of pathogenic microorganisms, which compromise the microbiological quality of food and can transmit food-borne diseases. The aim of the present study was to evaluate the hygienic quality of beef sold at supermarkets, butcher shops and public markets in the city of Campo Grande, state of Mato Grosso do Sul, Brazil, through the phenotypic and genotypic characterization of Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) as well as the investigation and quantification of Staphylococcus aureus. Seventy-one samples of beef from 17 commercial establishments were evaluated. Isolates were tested for antimicrobial susceptibility using the disk diffusion method recommended by the Clinical & Laboratory Standards Institute. Salmonella was found in 7.04% of the samples and 70.0% of the isolates were sensitive to the antimicrobials tested. A total of 25.35% of the samples were positive for Staphylococcus aureus, with counts ranging from 1.0 x 102 to 4.3 x 104 CFU/g; these isolates exhibited resistance to penicillin (87.5%), tetracycline (18.75%) and chloramphenicol (6.25%). None of the samples was positive for STEC. The detection of these pathogens in food poses a danger to public health, mainly due to the presence of antimicrobial-resistant isolates. These findings underscore the need for good hygiene and manufacturing practices at retail establishments. Resumo As falhas na qualidade higiênico-sanitária da carne podem ser identificadas a partir da avaliação de microrganismos patogênicos que comprometem a qualidade microbiológica do alimento e podem veicular doenças de origem alimentar. O presente estudo objetivou avaliar a qualidade higiênica-sanitária de carnes bovinas comercializadas em supermercados, açougues e mercados públicos da cidade de Campo Grande (Mato Grosso do Sul, Brasil) por meio da pesquisa e caracterização fenotípica e genotípica de Salmonella spp. e Escherichia coli produtora de toxina Shiga (STEC) e pesquisa e contagem de Staphylococcus aureus. Foram avaliadas 71 amostras de carne bovina de 17 estabelecimentos comerciais que foram submetidas a pesquisa de detecção de Salmonella spp., Escherichia coli produtora de toxina Shiga (STEC) e pesquisa e contagem de Staphylococcus aureus. Os isolados obtidos foram submetidos ao perfil de sensibilidade aos antimicrobianos pelo teste de difusão em disco, de acordo com o Clinical & Laboratory Standards Institute (CLSI). Constatou-se a presença de Salmonella em 7,04% das amostras avaliadas, sendo que 70,0% dos isolados foram sensíveis aos antimicrobianos testados. Em relação ao Staphylococcus aureus, 25,35% das amostras foram positivas com contagens variando entre 1,0 x 102 a 4,3 x 104 UFC/g, sendo que os isolados apresentaram resistência para penicilina (62,5%), tetraciclina (18,75%) e cloranfenicol (6,25%). Nenhuma amostra apresentou-se positiva para STEC. A detecção desses patógenos em alimentos representa um perigo a saúde pública, principalmente, devido a presença de isolados resistentes a antimicrobianos. Além disso, ressalta-se a necessidade do emprego das boas práticas de higiene e fabricação nos estabelecimentos varejistas.

Foodborne diseases continue to represent an important public health issue. The control of food spoilage and pathogenic microorganisms is achieved mainly by synthetic chemicals, unfortunately associated to several undesirable aspects. The growing requirement for new and safe alternative strategies has resulted in the research of agents from natural sources with antimicrobial properties, such as essential oils (EOs). This study’s purpose was to define the antibacterial profile of thyme (Thymus vulgaris) and cloves (Syzygium aromaticum) essential oils against both Gram-positive and Gram-negative important foodborne pathogenic bacteria. Gas chromatography mass spectrometry analysis was performed for EOs’ chemical composition. Qualitative in vitro antimicrobial assays (i.e., agar well diffusion method and disk-volatilization method) allowed for verification of the efficacy of EOs, used individually and in binary combination and both in liquid and vapor phase, against Staphylococcus aureus and Escherichia coli food isolates. Minimal inhibitory concentrations and minimal bactericidal concentration values have been used to quantitatively measure the antibacterial activity of EOs, while the fractional inhibitory concentration index has been considered as a predictor of in vitro antibacterial synergistic effects. The microbiological tests suggest that thyme and cloves EOs, rich in bioactive compounds, are able to inhibit the growth of tested foodborne bacteria, especially in vapor phase, also with synergistic effects. Results provide evidence to consider the tested essential oils as promising sources for development of new, broad-spectrum, green food preservatives.

Listeria monocytogenes is a food pathogen responsible for listeriosis, a relevant disease in public health worldwide. The genus Listeria spp., corresponds to cosmopolitan bacteria and capable of surviving different adverse conditions, which increases the risk for the food to be contaminated at any stage of the food chain. Fish and fish products are foods of high production level and, due to their chemical or nutritional composition, are highly susceptible to deterioration and contamination by pathogens in their productive chain relating to cases of listeriosis. Derived from the incidence and human mortality due to causative agents of listeriosis, along with their resistance to antimicrobials, they have acquired a greater emphasis on human health, animal health and food industry, resulting in the implementation of safety systems such as good hygiene practices, Hazard Analysis and Critical Control Points (HACCP) system, analytical methods and microbiological criteria, as some of the actions to contribute to the food safety and public health protection. The purpose of this review document is to provide, in a general way, aspects involved in foodborne illnesses, specifically listeriosis and its association with fish as a transmitting food, considering the prevention and control measures of this disease through food. It also includes aspects related to antimicrobial resistance by bacterial isolates obtained from fish, their implications and health risks.

The incidence of disease arising from food-borne pathogens is increasing continuously and has become a global public health problem. Rapid and accurate identification of food-borne pathogens is essential for adopting disease intervention strategies and controlling the spread of epidemics. Surface-enhanced Raman spectroscopy (SERS) has attracted increasing interest due to the attractive features including simplicity, rapid measurement, and high sensitivity. It can be used for rapid in situ sensing of single and multicomponent samples within the nanostructure-based confined space by providing molecular fingerprint information and has been demonstrated to be an effective detection strategy for pathogens. This article aims to review the application of SERS to the rapid sensing of food-borne pathogens in food matrices. The mechanisms and advantages of SERS, and detection strategies are briefly discussed. The latest progress on the use of SERS for rapid detection of food-borne bacteria and viruses is considered, including both the labeled and label-free detection strategies. In closing, according to the current situation regarding detection of food-borne pathogens, the review highlights the challenges faced by SERS and the prospects for new applications in food safety. Graphical abstractIn this review, the advances on the SERS detection of pathogens over the past decades have been reviewed, focusing on the improvements in sensitivity, reproducibility, specificity, and the performance of the SERS-based assay in complex analytical scenarios. Supplementary Information The online version contains supplementary material available at 10.1007/s00604-021-04864-4.

Buffalo milk is rich in nutrients and can serve as a substrate for the proliferation of microorganisms. Thus, the objective of the present study was to evaluate the growth kinetics of Salmonella Typhimurium and Listeria monocytogenes in buffalo milk under different processing and storage conditions. Samples of raw and pasteurized milk were inoculated with 1 CFU of each bacterium, separately and together, per 25 mL. After contamination, samples were stored at 8 °C or 37 °C, and bacterial counts were performed at 24, 48, and 168 h. In addition, the accompanying microbiota growth, pH, and the effect of these variables on the growth kinetics of microorganisms were monitored. The pathogens tested were able to proliferate under most conditions tested, reaching high titers throughout the experimental period. At 37 °C, there was a decrease in pH and an increase in the accompanying microbiota that interfered with the microbial growth curve. It was also observed that pasteurized milk subjected to 8 °C provided better conditions for the multiplication of bacteria. Therefore, it was concluded that care throughout the production chain, storage, and commercialization of milk must be adopted to guarantee the microbiological safety of this food. RESUMO: O leite bubalino é rico em nutrientes e pode servir de substrato para a proliferação de micro-organismos. Assim, o objetivo do presente estudo foi avaliar a cinética de crescimento de Salmonella Typhimurium e Listeria monocytogenes em leite bubalino, em diferentes condições de processamento e armazenamento. Para isso, utilizaram-se amostras de leite cru e pasteurizado, que foram inoculadas com 1 UFC/25 mL de cada bactéria separadamente e em conjunto. Após a contaminação, as amostras foram armazenadas nas temperaturas de 8 ºC e 37 ºC e realizadas contagens bacterianas em 24, 48 e 168h. Além disso, foi acompanhado o crescimento da microbiota acompanhante, o pH e o efeito dessas variáveis sobre a cinética de crescimento dos micro-organismos. Os patógenos testados conseguiram se proliferar na maioria das condições testadas, atingindo altos títulos durante todo o período experimental. Na temperatura de 37 ºC, houve uma diminuição do pH e um aumento da microbiota acompanhante, o que interferiu na curva de crescimento microbiana. Observou-se também que o leite pasteurizado e submetido a 8 ºC possibilitou melhores condições para a multiplicação das bactérias. Sendo assim, concluiu-se que cuidados durante toda a cadeia de produção, armazenamento e comercialização do leite devem ser adotados para garantir a segurança microbiológica desse alimento.

This work aimed to evaluate the chemical composition of the essential oils (EOs) of two cultivars of Allium sativum and their antibiofilm activity against the food pathogens Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. The crystal violet assay ascertained the susceptibility of the bacterial biofilms, while the MTT assay let to evaluations of the metabolic changes occurring in the bacterial cells within biofilms. Their chemical composition indicated some sulfuric compounds (i.e., allicin, diallyl disulfide, and allyl propyl disulfide), and decene as some of the main components of the EOs. The aerial parts and bulbs’ EOs from the two cultivars showed chemical differences, which seemed to affect the antibiofilm activity. The EOs from aerial parts of ‘Bianco del Veneto’ inhibited the biofilm formation of L. monocytogenes and E. coli (60.55% and 40.33%, respectively). In comparison, the ‘Staravec’ EO inhibited the cellular metabolism of E. coli (62.44%) and S. aureus (51.52%) sessile cells. These results indicate their possible use as preserving agents in the food industry and suggest their potential exploitation in the development of new formulations to avoid or limit nosocomial infections.

The increase in β-lactam-resistant Gram-negative bacteria is a severe recurrent problem in the food industry for both producers and consumers. The development of nanotechnology and nanomaterial applications has transformed many features in food science. The antibacterial activity of zinc oxide nanoparticles (ZnO NPs) and their mechanism of action on β-lactam-resistant Gram-negative food pathogens, such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Klebsiella pneumoniae, and Proteus mirabilis, are investigated in the present paper. The study results demonstrate that ZnO NPs possesses broad-spectrum action against these β-lactamase-producing strains. The minimal inhibitory and minimal bactericidal concentrations vary from 0.04 to 0.08 and 0.12 to 0.24 mg/mL, respectively. The ZnO NPs elevate the level of reactive oxygen species (ROS) and malondialdehyde in the bacterial cells as membrane lipid peroxidation. It has been confirmed from the transmission electron microscopy image of the treated bacterial cells that ZnO NPs diminish the permeable membrane, denature the intracellular proteins, cause DNA damage, and cause membrane leakage. Based on these findings, the action of ZnO NPs has been attributed to the fact that broad-spectrum antibacterial action against β-lactam-resistant Gram-negative food pathogens is mediated by Zn2+ ion-induced oxidative stress, actions via lipid peroxidation and membrane damage, subsequently resulting in depletion, leading to β-lactamase enzyme inhibition, intracellular protein inactivation, DNA damage, and eventually cell death. Based on the findings of the present study, ZnO NPs can be recommended as potent broad-spectrum antibacterial agents against β-lactam-resistant Gram-negative pathogenic strains.

This study aims at examining the contamination of coliform bacteria, Escherichia coli, Listeria monocytogenes, Vibrio vulnificus, and Vibrio cholerae, which carry extremely serious risks to the consumer health, in 700 seafood belonging to 4 different (raw sea fish, raw mussels, raw shrimp, and raw squid) categories. The total number of samples was determined as 700. When the obtained results were viewed in total, they were found to be 48.14%, 18.71%, 8.57%, and 3.42% for coliform bacteria, E. coli, L. monocytogenes, and V. vulnificus, respectively. V. cholerae, one of the factors studied, was not found. Conventional microbiological cultivation methods were used in the analysis stage as well as the real-time PCR method. This study aims at making a risk ranking modeling for consumer health based on product category and pathogens by interpreting the results of the analysis with statistical methods. According to the statistical analysis, significantly binary correlations were determined among some parameters that stimulate one another for reproducing. In the light of the obtained results of the study, it has been concluded that the studies of the most detailed examinations of the microbiological risks associated with seafood, forms of microbial pollution and microorganisms that cause deterioration in seafood and threaten consumer health and the path that their epidemiologies follow, are of primary importance to both protecting consumer health and obtaining safe and quality seafood. Istanbul University, Cerrahpasa Scientific Researches Project Unit

The use of peptides produced by lactic acid bacteria (LAB) as antimicrobial agents in food emerged from the increasing need of replacing chemicals with natural substances to ensure their safety and quality. A total of 30 LAB belonging to the genus Lactococcus sp. (10) and Enterococcus sp. (20) were isolated from native fruits of Ecuador subtropical rainforest. Among Lactococcus species, the isolates assigned Gt28, Gt29, and Ella8, identified as Lactococcus lactis subsp. lactis with 99% identity, showing highly inhibitory potential against four food pathogens were further characterized. The treatment of cell-free supernatant with proteolytic enzymes indicated the protein nature of released components, which displayed a broad antimicrobial activity against Gram-positive and -negative bacteria. Polymerase chain reaction analysis indicated the presence of lacticin 3147 gene in all isolates, lactococcin M gene in Gt28 and Gt29 but not in Ella8 and lactococcin A gene in Gt28 only. The antimicrobial activity was not linked to the presence of structural nisin gene as no amplification product was obtained. Treatment of Salmonella enterica ATCC 51741 and Escherichia coli ATCC 25922 at both vegetative and exponential phase of growth with the cell-free supernatant of Gt28 resulted in complete inactivation upon 3 h suggesting its bactericidal mode of action. An increment on inhibitory activity occurred when partial purified bacteriocin Gt28 was combined with ethylenediaminetetraacetic acid rather than bacteriocin only, indicating that the cells were sensitized in vitro by the chelator agent acting synergistically to induce the killing of pathogenic cells.

The objectives of this study were to evaluate the antimicrobial resistance and the biofilm-producing ability of Salmonella sp. strains isolated from frozen poultry carcasses. Antimicrobial susceptibility was tested by the disk-diffusion method. Biofilm-producing ability was determined in 96-well polystyrene microplates stained with crystal violet at 1%. Out of the 22 strains tested, all were multiresistant, that is, resistant to more than three antimicrobial classes, and 72.7% were able to form biofilms. The highest resistance rates obtained were against sulfonamides, tetracycline, and quinolones. On the other hand, 100% of the strains were sensitive to chloramphenicol. According to the rate of biofilm formation, 3 (13.6%) and 13 (59.1%) strains were classified as moderate and weak biofilm-producers, respectively, and 27.3% did not form biofilms. Biofilms increase the tolerance of microorganisms to stress, reducing their sensitivity to disinfectants and antimicrobials; favor equipment corrosion; and act as substrates for the adhesion of bacteria with lower biofilm-producing capacity. The results of the present study stress the importance of cleaning procedures in food processing plants and highlight the public health risks related to the emergence of multiresistant strains.

Foodborne safety has aroused tremendous research interest in recent years because of a global public health problem. The rapid and precise detection of foodborne pathogens can reduce significantly infection diseases and save lives by the early initiation of an effective treatment. This review highlights current advances in the development of biosensors for detection of Campylobacter spp. and Listeria monocytogenes that are the most common causes of zoonosis. The consumption of pathogen contaminated food is responsible for humans hospitalization and death. The attention focused on the recognition elements such as antibodies (Ab), DNA probes and aptamers able to recognize cells, amplicons, and specific genes from different samples like bacteria, food, environment and clinical samples. Moreover, the review focused on two main signal-transducing mechanisms, i.e., electrochemical, measuring an amperometric, potentiometric and impedimetric signal; and optical, measuring a light signal by OLED (Organic Light Emitting Diode), SPR (Surface Plasmon Resonance), and Optical fiber. We expect that high-performance of devices being developed through basic research will find extensive applications in environmental monitoring, biomedical diagnostics, and food safety.

Background Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient. Results In this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system. Conclusions Cassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required.

Juniperus communis and Satureja montana essential oils (EOs) were chemically characterized and their activity against Listeria monocytogenes was evaluated. Antilisterial effect was determined in vitro and in situ, in red wine-marinated beef, where the effect against common meat contaminants (aerobic heterotrophic mesophyll bacteria, Enterobacteriaceae and lactic acid bacteria) was also monitored. Microdilution assay on L. monocytogenes (strain ATCC19111 and three primoisolates) showed that minimal inhibitory concentrations (MICs) varied in the range 0.5–1% for both EOs. Effect of EOs mixtures was monitored in checkerboard assay revealing that all types of interactions including synergism were obtained only for isolates. In vitro time kill assay confirmed the mode of interactions: synergism for isolate from beef carcass (LMB), and its absence for ATCC19111 strain. The analysis of concentration dependent growth rates showed that estimated curve MIC values were notably lower than in microdilution assay (0.03–0.14%). In situ time kill assay against inoculated L. monocytogenes (ATCC19111 and LMB strains) and pre-existing meat spoiling contaminants was performed on marinated beef using EOs concentrations that were confirmed to be sensory acceptable. Basic red wine marinades and the ones containing each EO or their combination remarkably decreased the counts of all monitored groups comparing to saline control. The most pronounced effect was obtained with the marinade containing EOs mixture. For all monitored bacteria, the bactericidal effect during marination was followed by bacteriostatic effect during subsequent meat storage. Our results demonstrate for the first time the in situ efficacy of J. communis EO against meat contaminants and provide additional data on the preservation potential of S. montana EO.

Shiga toxigenic E. coli (STEC) are an important cause of foodborne disease globally with many outbreaks linked to the consumption of contaminated foods such as leafy greens. Existing methods for STEC detection and isolation are time-consuming. Rapid methods may assist in preventing contaminated products from reaching consumers. This proof-of-concept study aimed to determine if a metabolomics approach could be used to detect STEC contamination in spinach. Using untargeted metabolic profiling, the bacterial pellets and supernatants arising from bacterial and inoculated spinach enrichments were investigated for the presence of unique metabolites that enabled categorization of three E. coli risk groups. A total of 109 and 471 metabolite features were identified in bacterial and inoculated spinach enrichments, respectively. Supervised OPLS-DA analysis demonstrated clear discrimination between bacterial enrichments containing different risk groups. Further analysis of the spinach enrichments determined that pathogen risk groups 1 and 2 could be easily discriminated from the other groups, though some clustering of risk groups 1 and 2 was observed, likely representing their genomic similarity. Biomarker discovery identified metabolites that were significantly associated with risk groups and may be appropriate targets for potential biosensor development. This study has confirmed that metabolomics can be used to identify the presence of pathogenic E. coli likely to be implicated in human disease.

Antimicrobial peptides (AMPs) are excellent candidates to fight multi-resistant pathogens worldwide and are considered promising bio-preservatives to control microbial spoilage through food processing. To date, designing de novo AMPs with high therapeutic indexes, low-cost synthesis, high resistance, and bioavailability, remains a challenge. In this study, a novel decapeptide, named RiLK1, was rationally designed starting from the sequence of the previously characterized AMP 1018-K6, with the aim of developing short peptides, and promoting higher selectivity over mammalian cells, antibacterial activity, and structural resistance under different salt, pH, and temperature conditions. Interestingly, RiLK1 displayed a broad-spectrum of bactericidal activity against Gram-positive and Gram-negative bacteria, including multidrug resistant clinical isolates of Salmonella species, with Minimal Bactericidal Concentration (MBC) values in low micromolar range, and it was effective even against two fungal pathogens with no evidence of cytotoxicity on human keratinocytes and fibroblasts. Moreover, RiLK1-activated polypropylene films were revealed to efficiently prevent the growth of microbial spoilage, possibly improving the shelf life of fresh food products. These results suggested that de novo designed peptide RiLK1 could be the first candidate for the development of a promising class of decameric and multitask antimicrobial agents to overcome drug-resistance phenomena.

RESUMEN Con la finalidad de reducir el número de personas afectadas por enfermedades transmitidas por alimentos,se elaboraron películas biodegradables activas con propiedades antibacterianas a partir de ácido poliláctico, polietilenglicol, y aceite esencial de orégano (Origanum vulgare) al 1% (p/p) y 2 % (p/p). Las películas biodegradables activas fueron elaboradas mediante el método de evaporación del solvente. Se evaluó la acción antibacteriana de las películas biodegradables activas mediante un ensayo de inhibición a la adhesión microbiana contra Salmonella spp. y Staphylococcus aureus. Se pudo establecer que los porcentajes de aceite esencial aplicados tienen poco efecto inhibitorio sobre Staphylococcus aureus, mientras que para Salmonella spp. hay un efecto directo sobre la inhibición, cuando la concentración de aceite esencial es del 2% (p/p). Para una concentración del 1% (p/p), el efecto inhibidor observado fue pobre. Los resultados indican que las bacterias Gram-negativas Salmonella spp son más susceptibles al daño en la pared celular provocado por el aceite esencial de orégano, debido al menor espesor de la misma. En contraste, el mayor grosor de la pared celular que presentan las bacterias Gram-positivas Staphylococcus aureus exige, en principio, de un tiempo mayor para poder producir la lisis celular. Las películas biodegradables activas con aceite esencial de orégano pueden emplearse para la conservación de los alimentos como una tecnología de barrera en conjunto con otros mecanismos de control microbiano y así minimizar tratamientos térmicos severos o reducir la utilización de aditivos alimentarios, que en concentraciones inadecuadas pueden producir cierta toxicidad. Abstract In order to reduce the number of people affected by foodborne illnesses, active biodegradable films with antibacterial properties were made with polylactic acid, polyethylene glycol, and essential oil of oregano (Origanum vulgare) at 1% (w/w) and 2% (w/w). The active biodegradable films were made using solvent evaporation method. The active biodegradable films antibacterial action was evaluated by microbial adhesion inhibition test against Salmonella spp. and Staphylococcus aureus. It was established that the percentages of essential oil used have little inhibitory effect on Staphylococcus aureus, while for Salmonella spp. there is a direct effect on inhibition, when the essential oil concentration is 2% (w/w). For a concentration of 1% (w/w), the observed inhibitory effect was poor. The results indicate that Gram-negative bacterias Salmonella spp are more susceptible to cellular wall damage caused by oregano essential oil, due to its lower thickness. In contrast, the greater thickness of the cell wall that Staphylococcus aureus, Gram-positive bacteria has, requires, in principle, a longer time to produce cell lysis. Biodegradable active films with oregano essential oil can be used for food preservation as a barrier technology in conjunction with other microbial control mechanisms, thus minimizing severe heat treatments or reducing the use of food additives that, in inadequate concentrations can produce some toxicity.&nbsp

Food security is currently a global socio-political pressure point that is exacerbated by the ever-increasing world population [...]

The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed.

Very low cell count detection of Escherichia coli O157:H7 in foods is critical, since an infective dose for this pathogen may be only 10 cells, and fewer still for vulnerable populations. A flow cytometer is able to detect and count individual cells of a target bacterium, in this case E. coli O157:H7. The challenge is to find the single cell in a complex matrix like raw spinach. To find that cell requires growing it as quickly as possible to a number sufficiently in excess of matrix background that identification is certain. The experimental design for this work was that of a U.S. Food and Drug Administration (FDA) In-House Level 3 validation executed in the technology’s originating laboratory. Using non-selective enrichment broth, 6.5 h incubation at 42°C, centrifugation for target cell concentration, and a highly selective E. coli O157 fluorescent antibody tag, the cytometry method proved more sensitive than a reference regulatory method (p = 0.01) for detecting a single target cell, one E. coli O157:H7 cell, in 25 g of spinach. It counted that cell’s daughters with at least 38× signal-to-noise ratio, analyzing 25 samples in total-time-to-results of 9 h.

Combination of oregano and rosemary essential oils (EOs) was proposed to obtain a better antimicrobial and antioxidant effect on fresh‐cut broccoli compared to the use of individual oils. In in vitro assays, EOs combinations (oregano + rosemary) were more effective to inhibit the growth of Escherichia coli O157:H7, Salmonella Choleraesuis, Listeria monocytogenes, and Staphylococcus aureus compared to individual EOs doses. Oregano EO showed a higher antioxidant power compared to rosemary tested by DPPH and TEAC methods. Moreover, oregano + rosemary EOs (concentrations of 0.24 + 0.91 mg/ml) added within pectin coatings reduced coliform counts by 1 log unit and yeasts and molds by 1.8 log of coated broccoli; meanwhile, individual application of EOs (oregano at 0.48 and rosemary at 1.82 mg/ml) was not as effective. It was demonstrated that a mixture of EOs offered antimicrobial protection to fresh‐cut broccoli without negatively affecting its sensorial acceptability, even when no significant increment in its antioxidant capacity was observed.Practical applicationsCombination of essentials oils with different composition reduces the amount andcosts of agents needed to inhibit bacterial growth and survival. The use of essential oils as oregano (Lippia graveolens) and rosemary (Rosmarinus officinalis) combined a diverse mixture of antibacterial compounds that could be a solution to avoid bacterial resistance developed for the treatment with single compounds. Additionally, the negative sensorial impact of higher single oils doses could be reduced when combined, creating a new range of flavors and odors. Fil: Alvarez, María Victoria. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Departamento de Ingeniería Química. Grupo de Investigación en Ingeniería en Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina Fil: Ortega Ramírez, Luis A.. Centro de Investigación E/alimentos y Desarrollo A.c; México Fil: Silva Espinoza, Brenda A.. Centro de Investigación E/alimentos y Desarrollo A.c; México Fil: González Aguilar, Gustavo Adolfo. Centro de Investigación E/alimentos y Desarrollo A.c; México Fil: Ayala Zavala, Jesús Fernando. Centro de Investigación E/alimentos y Desarrollo A.c; México

The antimicrobial activity of quaternary ammonium salts of pyridinium oxime against four food pathogenic bacteria, the Gram positive Bacillus subtilis and Listeria monocytogenes, as well as the Gram negative Escherichia coli and Yersinia enterocolitica, were evaluated. Changes of proteome in these bacteria grown under stress conditions were identified. By the application of a new method for sample preparation, followed by both in-gel and in-solution digestion and LC–MS/MS, both characterization and comparison of proteomes of these food pathogens were achieved. It was shown in all investigated bacteria that some of the proteins of key importance for protein turnover and bacterial metabolism are down regulated. Some stress proteins involved in protein folding and degradation were up regulated. Most of both up- and down-regulated proteins belong to the group of proteins with high abundance. Flagellin is the only protein of lower abundance that was found to be down regulated in two strains, B. subtilis and E. coli. The presented results give the better view into the proteome of food pathogens, and pave the way for further investigation of their virulence, pathogenicity and detection of biomarkers for tracing the ways and sources of food contamination.

It is known that raw milk can contain microorganisms that are harmful to health. In light of the increasing number of machines where raw milk is sold via raw milk vending systems, the Federal Institute for Risk Assessment (BfR) has assessed the risk for food infections with the Campylobacter bacterium resulting from the milk dispensed from these raw milk vending machines. The assessment also addresses the question of whether the more frequent occurrence of outbreaks of illness due to Campylobacter infections (Campylobacter enteritis or Campylobacteriosis) could be associated with the increase in the number of points of sale for raw milk and in particular raw milk vending machines. As there are no validated empirical data, the preliminary assessment of the BfR is based on the working hypothesis that changes in consumer behaviour entailing the increased consumption of raw milk from the so-called "milk vending machines" is leading to more frequent outbreaks of Campylobacter. One of the main sources of contamination of the raw milk is faecal contamination during the milking process. Unlike most known foodborne pathogens, these bacteria cannot multiply in the raw milk. However, just a few Campylobacter cells are enough to cause infection. The BfR therefore advises consumers to always comply with the instruction displayed at the point of sale stating "raw milk – boil before consumption". The BfR also advises against preparing and consuming non-heated ("cold") cocoa drinks or other milkshakes made from raw milk both directly on-site and at home (also see the BfR FAQ on the consumption of raw milk http://www.bfr.bund.de/cm/349/questions-and-answers-on-the-consumption-of-raw-milk.pdf ). The full version of this BfR Information is available in German on http://www.bfr.bund.de/cm/343/rohmilch-abkochen-schuetzt-vor-infektionen-mitcampylobacter.pdf

This study was conducted to evaluate the capability of the Vitek immunodiagnostic assay system easy Salmonella (VIDAS ESLM) method and a specific real-time PCR system (LightCycler, LCPCR) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella from a total of 105 naturally contaminated samples comprised of poultry meat and poultry meat products. The detection limit of ISO and LCPCR was 9 cfu/mL for both poultry meat and poultry meat products, whereas that of VIDAS ESLM with both sample types was determined to be 90 cfu/mL. Twelve (33.33%), 11 (30.55%), and 18 (50.00%) out of 36 poultry meat samples were positive for Salmonella by ISO, VIDAS ESLM, and LCPCR, respectively. Salmonella detection rates from poultry meat products were 5.80% for ISO and 8.69% for LCPCR, whereas none of these products tested positive by VIDAS ESLM. In poultry meat samples, VIDAS ESLM and LCPCR detection results were in substantial agreement with ISO, with the relative accuracy, sensitivity, and specificity rates of 97.2, 91.7, and 100%, respectively, for VIDAS ESLM and 83.3, 100, and 75%, respectively, for LCPCR. This is the first report on the evaluation of both VIDAS ESLM and LCPCR to complement ISO for the rapid detection of Salmonella in poultry meat and meat products. We determined that both VIDAS ESLM and LCPCR have the potential to complement the ISO standard culture method in the rapid screening of Salmonella from naturally contaminated poultry meats. For the poultry meat products, VIDAS ESLM and LCPCR can be used for rapid primary screening, and they should be complemented absolutely by ISO. Although LCPCR can preferentially be used for initial screening poultry meat products, the results should definitely be confirmed by ISO. Also, the VIDAS ESLM did not seem to be a suitable method for detecting Salmonella in poultry meat products.

In order to meet the increasing demand for food quality and safety, the control of pathogenic microorganisms from “farm to fork” is a continuous challenge. This challenge has become more important due to changes in animal production, product processing and distribution, new food habits, higher numbers of consumers at risk for infection and increased awareness. This review is focused on the use of protective and probiotic cultures as “natural” intervention measures to control and prevent the transmission of pathogens along the food chain and on the most used technologies to produce these cultures at the large scale.

A great improvement in food safety and quality controls worldwide has been achieved through the development of biosensing platforms. Foodborne pathogens continue to cause serious outbreaks, due to the ingestion of contaminated food. The development of new, sensitive, portable, high-throughput, and automated platforms is a primary objective to allow detection of pathogens and their toxins in foods. Listeria monocytogenes is one common foodborne pathogen. Major outbreaks of listeriosis have been caused by a variety of foods, including milk, soft cheeses, meat, fermented sausages, poultry, seafood and vegetable products. Due to its high sensitivity and easy setup, electrochemical impedance spectroscopy (EIS) has been extensively applied for biosensor fabrication and in particular in the field of microbiology as a mean to detect and quantify foodborne bacteria. Here we describe a miniaturized, portable EIS platform consisting of a microfluidic device with EIS sensors for the detection of L. monocytogenes in milk samples, connected to a portable impedance analyzer for on-field application in clinical and food diagnostics, but also for biosecurity purposes. To achieve this goal microelectrodes were functionalized with antibodies specific for L. monocytogenes. The binding and detection of L. monocytogenes was achieved in the range 2.2 &times, 103 cfu/mL to 1 &times, 102 with a Limit of Detection (LoD) of 5.5 cfu/mL.

Hundreds of foodborne infection cases occur around the world, and up to one third of the population in industrialized nations suffers from foodborne illness each year. The advent of genetic-based technologies made feasible developing sensitive and specific screening tests for the detection of microbial pathogens. Microarray-based technologies represent an advance in nucleic acid testing methods whose main features include miniaturization, ability to parallelize sample processing, and ease of automation. Many applications, based on both commercial and custom arrays, have already been reported. The constant attempt to obtain reliable, sensitive, and robust methods has also driven the development of different molecular methods, relying on hybridization or on enzymatic techniques (such as minisequencing/ extension or ligation). At the same time, probe design strategies and data analysis pipelines have been refined as well, in order to make result evaluations faster and less prone to errors. The high number of genetic information already available allowed reaching a resolution below the species level, being able to discriminate among serovars or strains, thanks to the careful choice of variable regions. Currently, the sensitivity of the assays can be as low as 1 CFU/g for some bacteria after enrichment. In this review, we present an overview of the most important advances in microarray technologies in the foodborne pathogen detection field.

This study aims to determine the serogroup profiles of randomly collected 46 chicken meat and 15 turkey meat samples, following the U. S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual Chapter 5: Salmonella and International Organization for Standardization (ISO) Method 6579 culture methods. The total number of poultry meat samples with more than one serogroup isolated by the FDA and ISO culture methods were 10 (37.0%) and 21 (77.8%) of 27, respectively. Presence of multiple serogroups per sample was more frequently observed in chicken meat samples than in turkey meat samples. The profile of Salmonella serogroup isolates of chicken meat samples in descending order were serogroups D and E4 (15.8%), B and C2 (8.8%), C1 (5.3%), G (3.5%), and E1 and F (1.7%). The serogroup distribution of turkey meat sample isolates were serogroups B (27.2%), E4 (18.2%), and C2 (9.1%). On the basis of our findings that a selective plate in Salmonella culture method can harbor more than one serogroup, and that the FDA and ISO methods could detect different serogroups from chicken and turkey meats, we suggest screening multiple suspect colonies from each plate, if possible, and considering the collective and comparative use of the FDA and ISO culture methods and/or including several selective and differential media to ensure the detection of Salmonella and the possible detection of multiple serogroups from samples.

Several recent reports in the literature show that some microemulsions and nanoemulsions have interesting antimicrobial properties. However, those studies lacked appropriate controls showing the action of the isolated components at similar concentrations. In this study we demonstrate that the bactericidal effects of two previously studied emulsions are due predominantly to one of their components. In fact, the bioactivity of those compounds, namely tributyl phosphate and n-pentanol, is diminished when incorporated in the dispersed oil phases. Therefore, we suggest that future work regarding the antimicrobial action of emulsions include controls similar to the ones carried out in this research.

In 1994, in response to the outcomes of a joint Advisory Committee of Microbiological Safety of Food (ACMSF) and Steering Group on the Microbiological Safety of Food (SGMSF) meeting, a Working Group was set up to investigate the science and epidemiology of Foodborne Viral Infections. The Working Group assessed the risk from viruses that were believed to be the primary cause of foodborne illness. This report provides an update to this information and provides a new focus on the viruses which are currently the major route of foodborne illness. Since the publication of the 1998 report, with the exception of two minor risk assessments on hepatitis E and avian influenza, no formal review on viruses had been performed by the ACMSF. It was decided that as significant developments had been made not only in the detection of foodborne viruses, but also in the amount of information obtained from the Infectious Intestinal Disease (IID) Study in England (published in 2000), which indicated a significant disease burden from enteric viruses in the community, it was important that an Ad-Hoc Group was convened to revisit these issues and to provide an update to the 1998 risk assessment.

Detection of Salmonella by bacteriologic methods is known to be time consuming. Therefore, we have developed a real-time probe-specific polymerase chain reaction (PCR) to rapidly detect Salmonella invA gene-based PCR products from chicken feces and carcasses by a fluorescence resonance energy transfer assay. The sensitivity and the specificity of this system were determined as 3 colony-forming units ml(-1) and 100%, respectively. Overnight tetrathionate broth enrichment cultures of chicken feces and carcass samples were used in template preparation for PCR. Also, a standard bacteriology was performed (National Poultry Improvement Plan-U.S. Department of Agriculture, Bacteriological Analytical Manual-Food and Drug Administration Center for Food Safety and Applied Nutrition) for confirmation. Seventy-two cloacal swab, 147 intestine, and 50 carcass (neck) samples were examined. Thirteen (8.8%) and 25 (17%) of the intestinal samples were found to harbor Salmonella by bacteriology and PCR, respectively. Forty-five of 50 (90%) carcass samples were Salmonella positive by both methods. Salmonella was not detected from cloacal swab samples. Results indicate that this assay has the potential for use in routine monitoring and detection of Salmonella in infected flocks and carcasses.

Pregnant women are considered a special risk population group for food-borne illnesses, due to their specific susceptibility to certain infectious diseases and the severity of the complications that may appear. In Spain, official incidence of food-borne disease outbreaks in this population group is low. However, due to their special vulnerability and the strict sanitary control associated to pregnancy, they constitute a group in which prevention measures based on risk communication can be very effective. Listeriosis is one of the more relevant food-borne diseases during pregnancy since, although rare, it affects seriously the foetus, causing abortion or premature birth, stillbirth, meningitis or sepsis. Toxoplasma gondii can also cause foetal serious infection, resulting in miscarriages, perinatal mortality or congenital lesions in the brain, eyes and other organs. Other pathogens transmitted by food which, although infrequently, may seriously affect pregnant women or foetus are Brucella spp., Hepatitis E virus, verotoxigenic E. coli, Salmonella spp. and Campylobacter spp. Health education, together with prevention measures taken during production, processing and distribution, is the main factor to reduce microbiological hazards derived from food consumption. Therefore, in order to establish the basis for performing management and risk communication activities, the Scientific Committee has studied the main food-borne pathogens of special risk during pregnancy, analysing the factors affecting their survival and growth, as well as the foods most frequently involved in their transmission. This report includes a list of foods to avoid during pregnancy in order to minimize the risk of infection mainly by Listeria monocytogenes and Toxoplasma , but also by the rest of studied pathogens. The need to include instructions about the hygienic handling of food at home in any risk communication campaign is highlighte, ES; en; pfefsa@msssi.es

This study presents Salmonella Enteritidis incidence in chicken layer flocks in Turkey determined by real-time PCR (rPCR) and by International Organization for Standardization (ISO) method 6579:2002/Amd 1:2007. A total of 259 samples, composed of 1,036 individual samples each pooled into 4, including 175 cloacal swab, 14 intestine, 35 gizzard swab, and 35 cecal swab samples, belonging to 6 major companies, were collected from 50 layer flocks and tested by rPCR and ISO culture methods. Overall incidence of Salmonella in layer flocks by rPCR and culture was 61.0 and 55.6%, respectively, where 70.1% of these Salmonella isolates were determined as Salmonella Enteritidis. Incidences of Salmonella Enteritidis in culture-positive samples were 65.3% in cloacal swabs, 50.0% in intestines, 73.9% in gizzard swabs, and 87.5% in cecal swabs. The rPCR results were in 100% agreement (100% sensitivity and specificity) with culture results when cecal swabs were selected as the sample type. The relative accuracy of rPCR was 92.4, 91.4, and 84% for intestine, gizzard, and cloacal swab samples, respectively. As a result, by using rPCR and ISO culture, we determined that the Salmonella Enteritidis incidence in layer flocks in Turkey was high and that the use of cecal swab and intestine samples in Salmonella detection would yield reliable results. To reduce this high Salmonella Enteritidis incidence in layer flocks, Salmonella Enteritidis-specific vaccination should be implemented properly in conjunction with a well-designed biosecurity plan, including verifiable corrective actions.

Aims: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. Methods and Results: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 × 108 CFU ml−1 of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 × 101 and 6 × 104 CFU ml−1, respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml−1 with TTB and SCB, and 6 × 101 CFU ml−1 with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. Conclusions: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. Significance and Impact of the Study: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.

The demonstration of a labeless immunosensor for the detection of pathogenic bacteria using screen printed gold electrodes (SPGEs) and a potassium hexacyanoferrate(II) redox probe is reported. Gold electrodes were produced using screen printing and the gold surfaces were modified by a thiol based self assembled monolayer (SAM) to facilitate antibody immobilisation. SAMs based on the use of thioctic acid (TA), mercaptopropionic acid (MPA) and mercaptoundecanoic acid (MUA) were evaluated. Following antibody immobilisation via the optimum SAM, the redox behaviour and diffusion co-efficient (D) of the potassium hexacyanoferrate(II) probe was monitored in the absence and presence of analyte. In the presence of analyte, a change in the apparent diffusion co-efficient of the redox probe was observed, attributable to impedance of the diffusion of redox electrons to the electrode surface due to the formation of the antibody-bacteria immunocomplex. No change in the diffusion co-efficient was observed when a non-specific antibody (mouse IgG) was immobilised and antigen added. The system has been demonstrated with Listeria monocytogenes and Bacillus cereus.

3rd Seminar on Risk Management by Hygienic Design and Efficient Sanitation Programs; Tallinn; Estonia; 4 May 2009 through 6 May 2009, Risk assessment (RA) which is a holistic approach involves hazard identification, exposure assessment, dose-response assessment, and risk characterization (Oscar, 2004). Microbiological risk assessments (MRAs) have been subdivided into four steps, which comprise, hazard identification, hazard characterization, exposure assessment and risk characterization. RAs are well developed for chemical hazards, and much effort has been put into the application of this type analysis to microbial food safety risks, especially for human foodborne diseases. The methods used for MRA, as well as the underlying concepts for evaluating risk derived from foodborne organisms and the effects of control measures on reducing risk are still new to many countries including developing countries. It is important to demonstrate examples of practical data generation and modelling even if they are limited in data size.

The behaviour of Listeria monocytogenes and Escherichia coli O157:H7 was studied during the manufacture and ripening of two traditional Italian Alps cheeses. Each cheese type was manufactured in a pilot plan from raw cow milk (without the addition of starter cultures) artificially inoculated with L. monocytogenes and E. coli O157:H7 to a final concentration of about 4 log CFU/mL. The pathogens were enumerated throughout the cheese making and ripening processes to study their behaviour. When the milk was inoculated with 4 Log CFU/mL, the pathogens counts increased in the first time during the manufacturing process and then remained constant, until the end of ripening, or decreased significantly. Results indicate that the environment and nature of food borne pathogens affected the concentration of the bacteria during the manufacturing and ripening process. Thus, the presence of low cells numbers of L. monocytogenes and E. coli O157:H7 in milk destined for the production of raw milk cheeses characterized by a cooking of the curd less than 48°C can constitute a hazard for the consumer.