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Tejuino is a popular and traditional beverage consumed in north and western of Mexico, due to its biological properties, it is considered a natural source of probiotics. Nevertheless, few studies have been performed on Tejuino microbiota. In this work, the probiotic potential of the tejuino isolated Lactiplantibacillus plantarum BI-59.1 strain was investigated. Its effectiveness was compared with a commercial Lactobacillus spp and identified by 16S rDNA sequence homology. Lactiplantibacillus plantarum BI-59.1 strain showed probiotic properties, i.e., production of antimicrobial compounds (lactic acid and presence of plantaricin A gene), inhibition of entero-pathogens by planktonic cells and metabolites (Salmonella enterica serovar Typhimurium inhibition to HT29-MTX adhesion), biofilm formation, bacterial adhesion (HT29-MTX, 3.96 CFU/cell), and tolerance to stimulated gastrointestinal conditions (tolerance to pH 3 and bile salts). The strain was gamma hemolytic, susceptible to most antibiotics and negative for gelatinase production; thus, the Lactiplantibacillus. plantarum BI-59.1 strain is suitable for its use as a probiotic for nutraceutical or pharmaceutical formulations.

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Salmonella Typhimurium (ST313) has caused an epidemic of invasive disease in sub-Saharan Africa and has been recently identified in Brazil. As the virulence of this ST is poorly understood, the present study aimed to (i) perform the RNA-seq in vitro of S. Typhimurium STm30 (ST313) grown in Luria-Bertani medium at 37°C; (ii) compare it with the RNA-seq of the S. Typhimurium SL1344 (ST19) and S. Typhimurium STm11 (ST19) strains under the same growing conditions; and (iii) examine the colonization capacity and expression of virulence genes and cytokines in murine colon. The STm30 (ST313) strain exhibited stronger virulence and was associated with a more inflammatory profile than the strains SL1344 (ST19) and STm11 (ST19), as demonstrated by transcriptome and in vivo assay. The expression levels of the hilA, sopD2, pipB, and ssaS virulence genes, other Salmonella pathogenicity islands SPI-1 and SPI-2 genes or effectors, and genes of the cytokines IL-1ß, IFN-γ, TNF-α, IL-6, IL-17, IL-22, and IL-12 were increased during ST313 infection in C57BL/6J mice. In conclusion, S. Typhimurium STm30 (ST313) isolated from human feces in Brazil express higher levels of pathogenesis-related genes at 37°C and has stronger colonization and invasion capacity in murine colon due to its high expression levels of virulence genes, when compared with the S. Typhimurium SL1344 (ST19) and STm11 (ST19) strains. STm30 (ST313) also induces stronger expression of pro-inflammatory cytokines in this organ, suggesting that it causes more extensive tissue damage.

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The lack of proper gastrointestinal models assessing the inter-strain virulence variability of foodborne pathogens and the effect of the vehicle (food matrix) affects the risk estimation. This research aimed to propose a dynamic and integrated in vitro/ex vivo gastrointestinal model to evaluate the probability and severity of infection of foodborne pathogens at different matrices. An everted gut sac was used to determine the adhesion and invasion of Salmonella enterica and tissue damage. S. Typhimurium ATCC 14028 was used as a representative bacterium, and two matrices (water and cheese) were used as vehicles. No differences (p > 0.05) in the probability of infection (Pinf) were found for intra-experimental repeatability. However, the Pinf of cheese-vehiculated S. Typhimurium was different compared to water- vehiculated S. Typhimurium, 7.2-fold higher. The histological analysis revealed Salmonella-induced tissue damage, compared with the control (p < 0.05). In silico proposed interactions between two major Salmonella outer membrane proteins (OmpA and Rck) and digested peptides from cheese casein showed high binding affinity and stability, suggesting a potential protective function from the food matrix. The results showed that the everted gut sac model is suitable to evaluate the inter-strain virulence variability, considering both physiological conditions and the effect of the food matrix.

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Introduction. The Salmonella Typhimurium monophasic variant (1,4,[5],12:i:-) is currently the most commonly detected variant in Salmonella surveillance programs worldwide. In Colombia, the Salmonella enterica monophasic variant is the fourth most common clinical isolate recovered through the laboratory surveillance of the Grupo de Microbiología from the Instituto Nacional de Salud; however, it is unknown whether these isolates are closely related to the monophasic Typhimurium variant, which circulates globally, and their genetic and phenotypic characteristics have not been reported. Objective. To characterize monophasic Salmonella enterica isolates identified in Colombia from 2015 to 2018 by the Instituto Nacional de Salud. Materials and methods. Two hundred eighty-six clinical isolates of the monophasic Salmonella enterica variant were analyzed by PCR or whole-genome sequencing to confirm whether they corresponded to the Salmonella Typhimurium monophasic variant while the genetic structure of the operon encoding the second flagellar phase was determined in 54 isolates. Motility, growth, and expression of the outer membrane proteins were evaluated in 23 isolates. Results. During the study period in Colombia, 61% (n=174) of Salmonella monophasic isolates belonged to Salmonella Typhimurium serovar monophasic (1,4,[5],12:i-). Of these, 64.8% (n=35/54) were related to the European/Spanish clone and 13% (n=7/54) to the U.S. clone. Two isolates recovered from urine samples showed differences in motility, growth, and the absence of the OmpD porin in M9 minimal medium. Conclusions. Most of the monophasic Salmonella Typhimurium variants that have circulated in Colombia since 2015 lacked the second phase of operon fljAB, which is related to the European/Spanish clone. The results evidenced phenotypic changes in urine samples suggesting bacterial adaptation in the case of these invasive samples.

Introducción. La variante monofásica (1,4,[5],12:i:-) de Salmonella Typhimurium ocupa los primeros lugares en los programas de vigilancia de Salmonella a nivel mundial. En Colombia, Salmonella enterica variante monofásica alcanza el cuarto lugar en cuanto a los aislamientos clínicos recuperados por medio de la vigilancia por laboratorio del Grupo de Microbiología del Instituto Nacional de Salud, pero se desconoce si dichos aislamientos están relacionados con la variante monofásica de Typhimurium que circula a nivel global, y con sus características genéticas y fenotípicas. Objetivo. Caracterizar los aislamientos de Salmonella monofásica recuperados en Colombia entre el 2015 y el 2018 por el Grupo de Microbiología del Instituto Nacional de Salud. Materiales y métodos. Se analizaron 286 aislamientos clínicos de Salmonella enterica variante monofásica mediante PCR o secuenciación del genoma completo (Whole Genome Sequencing, WGS) para confirmar si correspondían a Salmonella Typhimurium variante monofásica, en tanto que, en 54 aislamientos, se determinó la estructura genética del operón que codifica la segunda fase flagelar y, en 23, se evaluó la motilidad, el crecimiento y la expresión de las proteínas de membrana externa. Resultados. El 61 % (n=174) de los aislamientos de Salmonella monofásica correspondió a Salmonella Typhimurium serovar monofásico. El 64,8 % (n=35/54) se relacionó con el clon europeo-español y, el 13 % (n=7/54), con el estadounidense. En dos aislamientos de orina se encontró una diferencia significativa en la motilidad y el crecimiento, así como ausencia de la porina OmpD en medio mínimo M9. Conclusiones. En el periodo de estudio, circuló en Colombia la variante monofásica de Salmonella Typhimurium relacionada con el clon europeo-español, y se registró ausencia total del operón fljAB. Los resultados evidenciaron cambios fenotípicos en los aislamientos provenientes de muestras de orina que sugieren adaptación en procesos invasivos.

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Citrus by-products are inexpensive sources of polyphenols, important bioactive compounds with wide pharmaceutical and food applications. This study aimed to investigate the effect of enzymatic treatment of citrus by-products on the polyphenolic profile of extracts and assess the influence of extracts on the growth and adhesion of probiotics and foodborne pathogenic bacteria and on the inflammatory response of epithelial cells. Enzyme-assisted extraction altered the polyphenolic profile (as assessed by HPLC-DAD), increasing the content of aglycone flavanones (naringenin and hesperetin). Enzymatic extracts and aglycone flavanones exhibited higher antibacterial and prebiotic activities than non-enzymatic extracts and glycoside flavanones. However, a higher content of aglycones was not associated with higher anti-adhesion activity. Citrus extracts significantly (P ≤ 0.05) decreased the inflammatory response of Caco-2 cells to Salmonella Typhimurium adhesion. These results support the sustainable reuse of citrus agroindustrial wastes and indicate the potential of citrus extracts in preventing infection by foodborne pathogenic bacteria and inducing proliferation of probiotics in foods and the gut environment.

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Salmonella can enter on the viable but non-culturable state (VBNC), characterized by the loss of ability to grow in routine culture media hindering detection by conventional methods and underestimation of the pathogen. Despite advances in research done so far, studies comparing conditions that lead Salmonella into the VBNC state are scarce. The main objective of this study was to evaluate different stresses to induce Salmonella to the VNBC state. Osmotic (1.2 M NaCl), acid (peracetic acid, 5.66 mg/mL) and oxidative (hydrogen peroxide, 1.20 mg/mL) stress were used at 4 °C to induce Salmonella enterica serovars Enteritidis and Typhimurium to the VBNC state. The culturability loss was monitored in the brain heart infusion (BHI) broth and agar, and the viability was determined by fluorescence microscopy, using the Live/Dead® kit, and by flow cytometry. Besides, the morphological characterization by atomic force microscopy (AFM) was performed. Storage in 1.2 M NaCl at 4 °C induced the VBNC state in Salmonella cells for periods longer than 121 days, and the percentage of viable cells has reached above 80.9%. More aggressive stress conditions promoted by peracetic acid and hydrogen peroxide induced the VBNC state in periods of, at most 0.14 day, and resulted in percentages of 8.5% to 45.5% viable cells, respectively. The counts of viable cells in the flow cytometer corroborate the results obtained by microscopic counts. The VBNC cells obtained in 1.2 M NaCl at 4 °C showed morphological changes, reducing the size and changing the morphology from bacillary to coccoid. No morphological change was observed on the cells stressed by acid or oxidant compounds.

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Non-typhoidal Salmonella is considered a major public health concern. The growing relevance of pigs as reservoir of Salmonella spp. has prompted several countries to set up surveillance and control programs to fight Salmonella infection in swine and reduce public health risk. In the last decade, pork production in Córdoba increased significantly to become one of the most important pig production provinces in Argentina. The aim of this study was to estimate Salmonella spp. prevalence and associated risk factors in large scale-farms in this province. Mesenteric lymph nodes (MLN) of 580 pigs from 20 finishing large-scale farms were collected between 2014 and 2015 to estimate Salmonella infection. A prevalence of 41.5% (95%CI: 37.6-45.6%) was observed. Two major risk factors were significantly associated with Salmonella infection, both related to the pre-slaughter period (distance from the farm to the slaughterhouse and lairage time), highlighting the need to pay special attention to pre-slaughter practices in the province. Shortening transport times and complying with national regulations for lairage time at slaughter may help to reduce the prevalence of infection. Sixteen different serovars were identified, being S. Anatum and S. Typhimurium the most prevalent ones. Moreover, two isolate of the monophasic variant of Salmonella Typhimurium (I 4,5,12:i:-) resistant to enrofloxacin and which also displayed multidrug resistance was isolated for first time from pigs in Córdoba. The moderate to high levels of antimicrobial resistance detected for antibiotics commonly used in the pig sector suggested the need for implementing a plan to limit their use in the province.

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The virulence genes of Salmonella are modulated during infection by several regulatory systems, and the RcsCDB system is one of the most important of these. The S. Typhimurium EG14873 (rcsC11) strain harbours the rcsC11 point mutation, displaying a constitutive activation of this system, which is characterized by mucoid colonies and attenuated virulence phenotypes. In this work, the stability of the rcsC11 mutation was analysed under stress conditions. Under acid and anaerobic stresses, we observed the appearance of small and non-mucoid colonies of the rcsC11 strain. The sequencing of the rcsC gene from these colonies showed that the mutation is conserved. Moreover, we found that small colonies were also generated when the wild-type strain grew in acid and anaerobic conditions. It is worth noting that the transition from normal to atypical colonies of both strains only took place after several days of incubation and was not observed during eukaryotic cell infection. Therefore, the appearance of these atypical colonies is a characteristic feature of S. Typhimurium strains under stressful situations and does not involve a reversion of the rcsC11 allele and nor does it imply any risk to mammalian cells. Therefore, we propose that the S. Typhimurium rcsC11 strain is a good candidate for the development of attenuated vaccines.

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Salmonella enterica serotypes have been reported as the agent of various outbreaks occurred after the consumption of low water activity (aw) foods. When the pathogen encounters harsh conditions, several regulatory networks are activated through dynamic differential gene expression that lead to cell survival for prolonged periods. In this work, the transcriptome of S. enterica serovar Typhimurium using RNA-Seq, after cells' inoculation in four distinct types of low aw foods (milk chocolate, powdered milk, black pepper, and dried pet food), following storage at 25 °C per 24 and 72 h was studied. The findings of this study suggest that gene regulation is influenced by the food composition mainly in the first 24 h post-inoculum, proceeded by the induction of similar genes shared among all samples. It was possible to evaluate the differences on each type of food matrix regarding the bacteria adaptation, as well as the similarities provoked by low aw. The results reveal genes that may play key roles in response to desiccation in Salmonella, as well as the pathways in which they are involved.

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The Salmonella enterica serovar Typhimurium RcsCDB system regulates the synthesis of colanic acid and the flagellum as well as the expression of virulence genes. We previously demonstrated that the rcsC11 mutant, which constitutively activates the RcsB regulator, attenuates Salmonella virulence in an animal model. This attenuated phenotype was also produced by deletion of the slyA gene. In this work, we investigated if this antagonistic behavior is produced by modulating the expression of both regulator-encoding genes. We demonstrated that SlyA overproduction negatively regulates rcsB transcription. A bioinformatics analysis enabled us to identify putative SlyA binding sites on both promoters, P rcsDB and P rcsB , which control rcsB transcriptional levels. We also determined that SlyA is able to recognize and bind to these predicted sites to modulate the activity of both rcsB promoters. According to these results, SlyA represses rcsB transcription by direct binding to specific sites located on the rcsB promoters, thus accounting for the attenuated/virulence antagonistic behaviors. Moreover, we showed that the opposite effect between both regulators also physiologically affects the Salmonella motility phenotype. In this sense, we observed that under SlyA overproduction, P rcsB is repressed, and consequently, bacterial motility is increased. On the basis of these results, we suggest that during infection, the different RcsB levels produced act as a switch between the virulent and attenuated forms of Salmonella Thereby, we propose that higher concentrations of RcsB tilt the balance toward the attenuated form, while absence or low concentrations resulting from SlyA overproduction tilt the balance toward the virulent form.IMPORTANCE The antagonistic behavior of RcsB and SlyA on virulence gene expression led us to hypothesize that there is interplay between both regulators in a regulatory network and these could be considered coordinators of this process. Here, we report that the SlyA virulence factor influences motility behavior by controlling rcsB transcription from the P rcsB promoter. We also demonstrate that SlyA negatively affects the expression of the rcsB gene by direct binding to P rcsDB and P rcsB promoters. We suggest that different levels of RcsB act as a switch between the virulent and attenuated forms of Salmonella, where high concentrations of the regulator tend to tilt the balance toward the attenuated form and low concentrations or its absence tilt it toward the virulent form.

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