Whole genome sequencing in the palm of your hand: how to implement a MinION Galaxy-based workflow in a food safety laboratory for rapid Salmonella spp. serotyping, virulence, and antimicrobial resistance gene identification.

Introduction: Whole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment. Methods: Herein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced. Results: After 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate. Discussion: The implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates.

Complete Genome Sequences of Six Salmonella enterica Strains (S. enterica subsp. arizonae, S. enterica subsp. diarizonae, and S. enterica subsp. salamae) Isolated from Poultry Houses.

Non-enterica subspecies of Salmonella enterica are associated with cold-blood animals. We report the complete genomes of six S. enterica strains (one S. enterica subsp. arizonae strain, four S. enterica subsp. salamae strains, and one S. enterica subsp. diarizonae strain) isolated from Spanish poultry houses. This will increase our knowledge of these non-enterica subspecies.

Determination of pharmaceuticals and heavy metals in groundwater for human and animal consumption and crop irrigation in Galicia.

Pharmaceuticals and heavy metals are contaminants present in groundwaters, which are the main source of drinking water in most parts of the world. In the northwest region of Spain, Galicia, groundwater harvesting is a common practice for drinking water supply, crop irrigation, cattle watering, as well as recreational use such as filling pools. In order to assess the quality of Galician groundwaters, the presence of 21 pharmaceuticals and 10 heavy metals was analysed by UPLC-MS/MS and ICP/MS methods, respectively, in a total of 118 groundwater samples from private wells. Seventeen of the 21 compounds studied were detected in 28% of the samples, with the highest presence of pharmaceuticals belonging to the antimicrobial group (52%), specifically the sulphonamides group in a range of concentration between 21 and 14.9 ng/L. In addition, 30% of the samples contained at least one heavy metal (Mn, As and Fe) above the legally permitted levels. Evaluation of the risk associated with the consumption of the analysed groundwater indicated no human risk for any of the detected pharmaceuticals but high cancer risk for children due to Cd, Cr and As concentrations was observe.

Salmonella spp. quorum sensing: an overview from environmental persistence to host cell invasion.

Salmonella spp. is one of the main foodborne pathogens around the world. It has a cyclic lifestyle that combines host colonization with survival outside the host, implying that Salmonella has to adapt to different conditions rapidly in order to survive. One of these environments outside the host is the food production chain. In this environment, this foodborne pathogen has to adapt to different stress conditions such as acidic environments, nutrient limitation, desiccation, or biocides. One of the mechanisms used by Salmonella to survive under such conditions is biofilm formation. Quorum sensing plays an important role in the production of biofilms composed of cells from the same microorganism or from different species. It is also important in terms of food spoilage and regulates the pathogenicity and invasiveness of Salmonella by regulating Salmonella pathogenicity islands and flagella. Therefore, in this review, we will discuss the genetic mechanism involved in Salmonella quorum sensing, paying special attention to small RNAs and their post-regulatory activity in quorum sensing. We will further discuss the importance of this cell-to-cell communication mechanism in the persistence and spoilage of Salmonella in the food chain environment and the importance in the communication with microorganisms from different species. Subsequently, we will focus on the role of quorum sensing to regulate the virulence and invasion of host cells by Salmonella and on the interaction between Salmonella and other microbial species. This review offers an overview of the importance of quorum sensing in the Salmonella lifestyle.

A Study on the Antimicrobial and Antibiofilm Peptide 1018-K6 as Potential Alternative to Antibiotics against Food-Pathogen Salmonella enterica.

Antimicrobial resistance has become one of the major global public health concerns, and it is indispensable to search for alternatives to conventional antibiotics. Recently, antimicrobial peptides have received great attention because of their broad-spectrum antimicrobial activity at relatively low concentrations, even against pathogens such as Salmonella enterica, which is responsible for most food-borne illnesses. This work aimed at evaluating the antimicrobial and antibiofilm activity of the innate defense peptide, named 1018-K6, against S. enterica. A total of 42 strains, belonging to three different subspecies and 32 serotypes, were included in this study. The antibiotic resistance profile of all the strains and the cytotoxic effects of 1018-K6 on mammalian fibroblast cells were also investigated. Results revealed that MIC (minimum inhibitory concentrations) and MBC (minimum bactericidal concentrations) values were in the ranges of 8-64 µg/mL and 16-128 µg/mL, respectively, although most strains (97%) showed MICs between 16 and 32 µg/mL. Moreover, sub-inhibitory concentrations of 1018-K6 strongly reduced the biofilm formation in several S. enterica strains, whatever the initial inoculum size. Our results demonstrated that 1018-K6 is able to control and manage S. enterica growth with a large potential for applications in the fields of active packaging and water disinfectants.

Probiotic Effects against Virus Infections: New Weapons for an Old War.

This review aimed to gather the available literature investigating the effects of probiotics against the most common viral infections using in vitro trials in cell lines and in vivo clinical trials in both experimental animals and humans. Probiotics were employed to prevent and reduce symptoms of infections caused by common viruses, especially respiratory tract viruses, but also for viral digestive infections (such as rotavirus, coronavirus, or norovirus) and other viral infections (such as viruses that cause hepatitis, human papillomavirus, human immunodeficiency virus, and herpes simplex virus). Different probiotics have been studied to see their possible effect against the abovementioned viruses, among which different Lactobacillus species, Bifidobacterium, Clostridium, Enterococcus, and Streptococcus can be highlighted. In many cases, mixtures of various probiotic strains were used. Although the results obtained did not show similar results, in most cases, probiotic supplementation improved both barrier and biochemical immune responses, decreased susceptibility to viral infections, and enhanced the effects of concomitant vaccines. Works collected in this review show a beneficial effect of probiotics in the prevention and treatment of different viral infections. We found interesting results related to the prevention of viral infections, reduction of the duration of diseases, and decrease of symptoms.

Antimicrobial Activity of Five Apitoxins from Apis mellifera on Two Common Foodborne Pathogens.

Antimicrobial resistance is one of today's major public health challenges. Infections caused by multidrug-resistant bacteria have been responsible for an increasing number of deaths in recent decades. These resistant bacteria are also a concern in the food chain, as bacteria can resist common biocides used in the food industry and reach consumers. As a consequence, the search for alternatives to common antimicrobials by the scientific community has intensified. Substances obtained from nature have shown great potential as new sources of antimicrobial activity. The aim of this study was to evaluate the antimicrobial activity of five bee venoms, also called apitoxins, against two common foodborne pathogens. A total of 50 strains of the Gram-negative pathogen Salmonella enterica and 8 strains of the Gram-positive pathogen Listeria monocytogenes were tested. The results show that the minimum inhibitory concentration (MIC) values were highly influenced by the bacterial genus. The MIC values ranged from 256 to 1024 µg/mL in S. enterica and from 16 to 32 µg/mL in L. monocytogenes. The results of this study demonstrate that apitoxin is a potential alternative agent against common foodborne pathogens, and it can be included in the development of new models to inhibit the growth of pathogenic bacteria in the food chain.

Animal-Origin Prebiotics Based on Chitin: An Alternative for the Future? A Critical Review.

The human gut microbiota has been revealed in recent years as a factor that plays a decisive role in the maintenance of human health, as well as in the development of many non-communicable diseases. This microbiota can be modulated by various dietary factors, among which complex carbohydrates have a great influence. Although most complex carbohydrates included in the human diet come from vegetables, there are also options to include complex carbohydrates from non-vegetable sources, such as chitin and its derivatives. Chitin, and its derivatives such as chitosan can be obtained from non-vegetable sources, the best being insects, crustacean exoskeletons and fungi. The present review offers a broad perspective of the current knowledge surrounding the impacts of chitin and its derived polysaccharides on the human gut microbiota and the profound need for more in-depth investigations into this topic. Overall, the effects of whole insects or meal on the gut microbiota have contradictory results, possibly due to their high protein content. Better results are obtained for the case of chitin derivatives, regarding both metabolic effects and effects on the gut microbiota composition.

Natural Compounds as Antimicrobial Agents.

During the first two decades of this century, conventional antimicrobial compounds have been found out to have more bacterial resistance. What has also been worrying is the rediscovery of the so-called "natural compounds", which in turn have a good name among the average citizen because of the former's plant or animal origin. However, they do not form a well-classified group of substances. This Special Issue consists of five reviews focusing on clinical bacteria applications in food and their specific effects upon virulent bacterial factors. You will also find a research on much needed, new antimicrobials sourced in extreme environments, and secondary metabolites of Burkholderia. This issue includes 12 original research papers which will provide you with an in-depth coverage of the protein extract activity, as well as the activity of other plant extracts, on fighting bacteria, fungi or diarrhea. Their use in broilers or laying eggs for production purposes has also been focused on in order to improve gut microbiota. Last but not least, we should not forget about honey and its effect; Allium sativum-fermented extracts, as well as other "natural" compounds, have been studied in their fight against biofilms. Furthermore, we have also examined the use of essential oils, which are currently used in edibles such as fresh sausages. The present work also deals with other applications such as natural compound derivatives as well as compound mixtures.

Potential Use of Marine Seaweeds as Prebiotics: A Review.

Human gut microbiota plays an important role in several metabolic processes and human diseases. Various dietary factors, including complex carbohydrates, such as polysaccharides, provide abundant nutrients and substrates for microbial metabolism in the gut, affecting the members and their functionality. Nowadays, the main sources of complex carbohydrates destined for human consumption are terrestrial plants. However, fresh water is an increasingly scarce commodity and world agricultural productivity is in a persistent decline, thus demanding the exploration of other sources of complex carbohydrates. As an interesting option, marine seaweeds show rapid growth and do not require arable land, fresh water or fertilizers. The present review offers an objective perspective of the current knowledge surrounding the impacts of seaweeds and their derived polysaccharides on the human microbiome and the profound need for more in-depth investigations into this topic. Animal experiments and in vitro colonic-simulating trials investigating the effects of seaweed ingestion on human gut microbiota are discussed.

Short Chain Fatty Acids Commonly Produced by Gut Microbiota Influence Salmonella enterica Motility, Biofilm Formation, and Gene Expression.

Short chain fatty acids (SCFAs) are commonly produced by healthy gut microbiota and they have a protective role against enteric pathogens. SCFAs also have direct antimicrobial activity against bacterial pathogens by diffusion across the bacterial membrane and reduction of intracellular pH. Due to this antimicrobial activity, SCFAs have promising applications in human health and food safety. In this study, the minimum inhibitory concentrations (MICs) of four SCFAs (acetic acid, butyric acid, propionic acid, and valeric acid) in Salmonella strains isolated from poultry were determined. The effect of subinhibitory concentrations of SCFAs in Salmonella biofilm formation, motility, and gene expression was also evaluated. Butyric acid, propionic acid, and valeric acid showed a MIC of 3750 µg/mL in all strains tested, while the MIC of acetic acid was between 1875 and 3750 µg/mL. Subinhibitory concentrations of SCFAs significantly (p < 0.05) reduced the motility of all Salmonella strains, especially in the presence of acetic acid. Biofilm formation was also significantly (p < 0.05) lower in the presence of SCFAs in some of the Salmonella strains. Salmonella strain. Salmonella Typhimurium T7 showed significant (p < 0.05) upregulation of important virulence genes, such as invA and hilA, especially in the presence of butyric acid. Therefore, SCFAs are promising substances for the inhibition of the growth of foodborne pathogens. However, it is important to avoid the use of subinhibitory concentrations that could increase the virulence of foodborne pathogen Salmonella.

Antimicrobial activity of apitoxin from Apis mellifera in Salmonella enterica strains isolated from poultry and its effects on motility, biofilm formation and gene expression.

Salmonella is a major global food-borne pathogen. One of the main concerns related to Salmonella and other food-borne pathogens is their capacity to acquire antimicrobial resistance and produce biofilms. Due to the increased resistance to common antimicrobials used to treat livestock animals and human infections, the discovery of new antimicrobial substances is one of the main challenges in microbiological research. An additional challenge is the development of new methods and substances to inhibit and destruct biofilms. We determined the antimicrobial and antibiofilm activities of apitoxin in 16 Salmonella strains isolated from poultry. In addition, the effect of apitoxin on Salmonella motility and the expression of biofilm- and virulence-related genes was evaluated. The minimum inhibitory concentrations (MIC) of apitoxin ranged from 1,024-256 µg/mL, with 512 µg/mL being the most common. Sub-inhibitory concentrations of apitoxin significantly reduced biofilm formation in 14 of the 16 Salmonella strains tested, with significant increases in motility. MIC concentrations of apitoxin destroyed the pre-formed biofilm by 27.66-68.22% (47.00% ±â€¯10.91). The expression of biofilm- and virulence-related genes and small RNAs was differentially regulated according to the strain and the presence of apitoxin. The transcription of the small RNAs dsrA and csrB, related to antimicrobial resistance, was upregulated in the presence of apitoxin. We suggest that apitoxin is a potential antimicrobial substance that could be used in combination with other substances to develop new drugs and sanitizers against food-borne pathogens.

Transcriptomics: A powerful tool to evaluate the behavior of foodborne pathogens in the food production chain.

In recent years, the development of high-throughput transcriptomic tools such as RNA-seq and microarrays have revolutionized biological scientific research. Despite these techniques being widely used in biomedical research, their introduction into the field of food microbiology is still limited. However, transcriptomic research is of great importance to fully understand the resistance mechanisms and metabolic pathways involved in foodborne pathogen survival from farm to fork. Throughout this review, the main transcriptomic techniques available are described and discussed. Then, the main studies where transcriptomics were used to evaluate survival of foodborne pathogens in the different conditions of the food production chain are evaluated. Particularly, studies focused on biofilm formation, the food matrix and its environment, heat treatments, acid resistance, biocides, and food preservatives were specially reviewed. The data obtained from these studies are of great importance to develop new control strategies in any point of the food production chain.

Tracing recombinant bovine somatotropin ab(use) through transcriptomics: the potential of bovine somatic cells in a multi-dose longitudinal study.

In the European Union, the use of recombinant bovine somatotropin (rbST) in dairy cattle is forbidden. Monitoring rbST (ab)use by its direct detection in animal matrices still remains a challenging task. New monitoring methods based on indirect detection of the substance are necessary. A new transcriptomic system based on the use of high-throughput real-time PCR in combination with somatic cells was developed to control rbST administration in dairy animals. A total of nine cows, separated into control and rbST-treated groups, were included in the study. A subcutaneous injection containing 500 mg of rbST was administered to the treated group every 14 days, up to a total of 12 doses. Milk somatic cells (MSCs) were sampled from each animal at different time points throughout 8 months of study. It was possible to obtain the transcriptomic profile of 18 genes in MSCs of rbST-treated and control groups, and using univariate and multivariate statistical analysis control and treated animals were discriminated. The transcription of CCND1, IGF-1R, TNF and IL-1ß genes resulted strongly influenced by rbST treatment. The combination of MSCs, transcriptomic tools and statistical analysis has allowed the selection of four genes as potential biomarkers that could be used in a transcriptomic panel for monitoring rbST administration in cows.

Tracing Recombinant Bovine Somatotropin Ab(Use) Through Gene Expression in Blood, Hair Follicles, and Milk Somatic Cells: A Matrix Comparison.

The use of recombinant bovine somatotropin (rbST) in dairy cattle is forbidden in the European Union. Due to the very low circulating concentration of rbST in treated animals, its direct detection is still a challenge. Therefore, the use of indirect methods to detect the ab(use) of rbST in dairy cattle appears as a good alternative. In the past few years, gene expression demonstrated its utility in screening the use of illicit substances in both humans and animals. In this study, a comparison of three types of matrices (milk somatic cells, blood, and hair follicles) was carried out to evaluate their potential use for routine control of rbST using 15 gene-expression profiles. A total of six rbST-treated cows and three control cows were included in the study. A subcutaneous injection containing 500 mg of rbST was administered to the treated group. Samples of the three matrices were collected before rbST administration, and at three and nine days after treatment. The quality of RNA extracted was higher in the blood and hair-follicle samples than in the milk somatic cells. In the three matrices, there were significant differences in the expression of some genes, with milk somatic cells and blood presenting the the best matrices. On this note, the cyclin D1 (CCND1), interleukin 1 beta (IL-1ß), tumor necrosis factor (TNF), and insulin-like growth factor 1 receptor (IGF-1R) genes showed potential as biomarkers of rbST treatment. Therefore, blood, somatic cells, and follicle hair should be considered as promising sources of RNA, and can be used in gene-expression assays to routinely control the illicit use of rbST.

Simultaneous analysis of coccidiostats and sulphonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC.

Taking into consideration the maximum level (ML) for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulphonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulphonamides and coccidiostats in non-target feeds. The method was validated following Commission Decision 2002/657/EC, and recovery, repeatability and reproducibility were within the limits established in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the ML allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills and 10 interlaboratory feed samples.

Influence of food consumption patterns and Galician lifestyle on human gut microbiota

The proportion of different microbial populations in the human gut is an important factor that in recent years has been linked to obesity and numerous metabolic diseases. Because there are many factors that can affect the composition of human gut microbiota, it is of interest to have information about what is the composition of the gut microbiota in different populations in order to better understand the possibilities for improving nutritional management. A group of 31 volunteers were selected according to established inclusion and exclusion criteria and were asked about their diet history, lifestyle patterns, and adherence to the Southern European Atlantic Diet. Fecal samples were taken and subsequently analyzed by real-time PCR. The results indicated different dietary patterns for subjects who consumed a higher amount of fruits, vegetables, legumes, and fish and a lower amount of bakery foods and precooked foods and snacks compared to Spanish consumption data. Most participants showed intermediate or high adherence to Southern European Atlantic Diet, and an analysis of gut microbiota showed high numbers of total bacteria and Actinobacteria, as well as high amounts of bacteria belonging to the genera Lactobacillus spp. and Bifidobacterium spp. A subsequent statistical comparison also revealed differences in gut microbiota depending on the subject's body weight, age, or degree of adherence to the Southern European Atlantic Diet

In vitro evaluation of the prebiotic effect of red and white grape polyphenolic extracts

Nowadays, the strong relationship between diet and health is well known. Although the primary role of diet is to provide nutrients to fulfill metabolic requirements, the use of foods to improve health and the state of well-being is an idea increasingly accepted by society in the last three decades. During the last years, an important number of scientific advances have been achieved in this field and, although in some situations, it is difficult to establish a distinction between "harmful" and "good" bacteria, experts agree in classifying the genera Bifidobacterium and Lactobacillus as beneficial bacteria. Thus, several strategies can be used to stimulate the proliferation of these beneficial intestinal bacteria, being one of them the consumption of prebiotics. The development of new prebiotics, with added functionality, is one of the most serious challenges shared not only by the scientific community but also by the food industry. The objective of this work was to evaluate the potential prebiotic effect of red and white grape residues, both obtained during the winemaking process. For such purpose, an in vitro study with pure cultures of Lactobacillus and Bifidobacterium was first conducted. Secondly, a study with mixed cultures using human fecal inocula was carried out in a simulator of the distal part of the colon. The obtained results showed an increase in the Lactobacillus and Bifidobacterium population, indicating that these ingredients are serious candidates to be considered as prebiotics

A comprehensive review of non-enterica subspecies of Salmonella enterica.

Salmonella is a major foodborne pathogen with a complex nomenclature. This genus is composed of two species, S. enterica and S. bongori. S. enterica is divided into six subspecies. S. enterica subspecies enterica is composed of more than 1500 serotypes with some of great importance, such as S. Typhimurium and S. Enteritidis. S. enterica subsp. enterica is responsible of more than 99% of human salmonellosis and therefore it is widely studied. However, the non-enterica subspecies of S. enterica have been little studied. These subspecies are considered to be related to cold-blooded animals and their pathogenicity is very limited. Phenotype and genotype information generated from different studies of non-enterica subspecies reveal poor ability to invade host cells and the absence or modification of important virulence factors. Also, the great majority of human infections due to non-enterica subspecies are related to a previous depressed immune system. Therefore, we propose to treat these subspecies only as opportunistic pathogens. For establish this premise, the present review evaluated, among other things, the genomic characteristics, prevalence, antimicrobial resistance and reported human cases of the non-enterica subspecies.

Influence of food consumption patterns and Galician lifestyle on human gut microbiota.

The proportion of different microbial populations in the human gut is an important factor that in recent years has been linked to obesity and numerous metabolic diseases. Because there are many factors that can affect the composition of human gut microbiota, it is of interest to have information about what is the composition of the gut microbiota in different populations in order to better understand the possibilities for improving nutritional management. A group of 31 volunteers were selected according to established inclusion and exclusion criteria and were asked about their diet history, lifestyle patterns, and adherence to the Southern European Atlantic Diet. Fecal samples were taken and subsequently analyzed by real-time PCR. The results indicated different dietary patterns for subjects who consumed a higher amount of fruits, vegetables, legumes, and fish and a lower amount of bakery foods and precooked foods and snacks compared to Spanish consumption data. Most participants showed intermediate or high adherence to Southern European Atlantic Diet, and an analysis of gut microbiota showed high numbers of total bacteria and Actinobacteria, as well as high amounts of bacteria belonging to the genera Lactobacillus spp. and Bifidobacterium spp. A subsequent statistical comparison also revealed differences in gut microbiota depending on the subject's body weight, age, or degree of adherence to the Southern European Atlantic Diet.