Enteropathogenic Escherichia coli (EPEC) are important agents of acute diarrhea in children living in developing countries. A severe dysfunction of the intestinal epithelial barrier occurs during EPEC infection, leading to diarrhea and inflammation as consequences. EPEC main virulence factors include the adhesins intimin and bundle-forming pilus (BFP), as well as several effector proteins translocated to the enterocyte by the type three-secretion system. The initial interaction of EPEC with the host cell and the role of effector proteins in this process are well known. However, the role of the EPEC virulence factors in macrophage activation is not fully understood. Hence, we analyzed the ability of intimin and BfpA, to activate the innate response mediated by macrophages, where the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, and the anti-inflammatory cytokine IL-10 and chemokine MCP-1 were evaluated. Our results showed that recombinant intimin and BfpA activate macrophages in a dose-dependent manner, and the stimulated cells produced TNF-α, IL-12 and IL-6, IL-10 and MCP-1, but not IL-1β. No synergistic effect was observed in the production of proinflammatory cytokines by combining BfpA and intimin, although production of IL-10, an anti-inflammatory mediator, was potentiated at a higher dose. The effect observed was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. Thus, herein we showed that intimin and BfpA can activate the innate immune response, inducing the production of pro and anti-inflammatory cytokines, as well as chemokine’s, playing additional role as inflammatory molecules in the early steps of EPEC infection.

Cholera outbreaks are prevalent in countries with low Human Development Index (HDI) where people have limited access to safe drinking water, sanitation and hygiene (WASH). Intriguingly, state of Kerala which records the highest HDI in India is endemic to cholera. We discuss the epidemiology of a cholera outbreak reported among migrant workers of Kerala in 2017. Virulence genes of Vibrio cholerae, tox R and ctx A, were detected in the river and ground water samples collected from the outbreaks sites which indicates need of enhanced awareness on WASH practices among migrant workers. The pathogenic Vibrio cholerae isolated from four patients in two districts had a similar DNA band pattern when analysed using repetitive extragenic palindromic-PCR (BOX), which indicates their single clonal origin. The four isolates were serotyped as O1 Ogawa. These isolates were resistant to multiple antibiotics including the carbapenem like imipenem. Nevertheless, isolates of these pathogenic bacteria were susceptible to tetracycline identical to clinical isolates of Vibrio cholera reported earlier from this region. Current study highlights the importance of generating awareness on WASH protocols among migrant workers to prevent the outbreaks and considers community-based data as socioeconomic variable to predict the incidence of cholera.

Introduction. Bouvardia ternifolia is a Mexican endemic plant used in the past as a general curative source and contemporaneously as a remedy for dysentery, poison therapy, pain relief, and other afflictions. The hydroalcoholic extract shows acetylcholinesterase inhibition activity, as well as anti-inflammatory and antioxidant effects. “Bouvardin” is a cyclic hexapeptide found in B. ternifolia with in vitro cytotoxicity against B16 melanoma melanotic and P388 lymphocytic leukemia cell lines. These findings support the idea that this plant has potential as a source of therapeutic bioactive compounds. It is well known that many therapeutic secondary metabolites can be produced by plant-associated microorganisms and in the search of these, the characterization of species that live in conjunction with the plant becomes an important research activity. In this study, we report the characterization of the bacterial community associated with the Mexican medicinal plant B. ternifolia.

Material and methods. Five specimens and plant-soil samples were collected from different regions from the central part of Mexico and fractioned in flower, stem, leaf, and root. To establish the total and endophytic bacterial communities, each plant fraction was divided into two subfractions; the first one was processed directly and the second one was carefully tissue-surface sterilized before processing with chemical agents. Bacteria present in subfractions were collected by crushing down the plant tissue in a mortar followed by centrifugation steps. FavorPrep stool DNA isolation kit # FASTI001-1 and QIAGEN Dneasy PowerSoil kit were used to purify bacterial DNA which was employed to sequence a DNA library prepared from the V3 region of the 16S ribosomal gene by Ion Torrent PGM NGS system technology.

Results. We find ~1,000 OTUs shared among total plant, endophytic, and soil samples, ~2,000 OTUs exclusively for soil bacteria, ~1,500 OTU’s for total plant bacteria, and ~250 OTUs for endophytic bacteria. Proteobacteria (46-79%) was the most representative phylum followed by Actinobacteria and Acidobacteria. The phylum Cyanobacteria (8.5%) was more representative in endophytes samples than in total plant samples (1%). Bacterial richness and diversity by plant fractions and soil, from highest to lowest was observed in the following order: Soil > Root > Leaf > Stem > Flower. Linear discriminant effect size analysis showed the order Streptophyta, the families Oxalobacteraceae, Ruminococcaceae, Lachnospiraceae, and the genera Propionilbacterium, Paracoccus, Lactobacillus as the most representative taxa in endophyte bacterial samples.

Conclusions. This study provides evidence for the composition and diversity of the bacterial communities present in soil and tissues of the Mexican medicinal plant B. ternifolia which can be considered for subsequent analysis to profiling therapeutic and bioactive compounds that give this species its medicinal properties.

Acknowledgments. This work was financed by Cinvestav, FONCICYT 2 267416, CONACYT-BMBF-267416, and CONACyT-163235 INFR-2011-01.

Prickly pear Opuntia ficus-indica (L.) Mill.,1768) is a succulent plant belonging to the Cactaceae family, native to Central America but now diffused both in the the Mediterranean area (mainly Sicily, Calabria, Puglia, Sardinia and Malta) and in the temperate areas of America, Africa, Asia and Oceania. The oil obtained from its seeds, rich in polyunsatured fatty acids, has also known antimicrobial and antioxidant properties. Herein we evaluated not only the capacity of this oil to inhibit the formation of biofilm by different pathogens but also to block the metabolic changes taking place in the microbial cells included in the biofilm. The oil was capable to inhibit at 38.75% the biofilm of Escherichia coli just at a concentration of 1 microliter/ml, and, when tested against Pseudomonas aeruginosa, the inhibition reached the 74%. The oil was also effective against Pseudomonas aeruginosa (71.84% of inhibition) and the phytopathogen Pectobacterium carotovorum (63.06%). The metabolic activity of the microbial cells present within the biofilm was also strongly inhibited and, when the oil was tested against P. carotovorum, microbial cell metabolism was completely inhibited. The action of the prickly pear seeds oil was effective also in blocking at 64.97 % the metabolism of Listeria monocytogenes cells, which therefore had conversely demonstrated to be more resistant compared to the other bacteria (31% of biofilm inhibition). Data from the present study indicates a wide field of application of this oil, with interesting potentialities for food and health purposes.

Introduction. Anaerobic digestion (AD) is considered one of the most beneficial technologies for the treatment of organic wastes, from which energy in form of biogas and digestate as a soil amendment can be obtained. However, AD often suffers from process instability due to high levels of inorganic toxic substances such as ammonium, phosphates, sulfates or metal ions, variations in operating parameters such as temperature and pH, as well as a lack of proper balance between microbial groups. One of the alternatives to combat these imbalances is bioaugmentation, which is defined as the addition of specific microorganisms (either pure cultures or microbial consortia) into the system to accomplish a certain function. In this work, hydrogenotrophic methanogens were used to improve the methane yield of a thermophilic AD process of the organic fraction of municipal solid wastes (OFMSW) in Mexico City. Methods. The OFMSW (substrate) and leachate (inoculum) were collected from a composting plant in Mexico City. The consortium containing the hydrogenotrophic methanogens was isolated from the leachate. A biochemical methane potential (BMP) test was performed to know if the consortium influenced the methane yield. A 1:1 volatile solids (VS) ratio substrate/inoculum was operated in 125 mL serum bottles with a working volume of 60 ml. The consortium was added to different volume ratios. The process lasted 32 days at 55° C and 60 rpm. The identification of the microbial communities of all treatment groups and consortium were done by high throughput DNA sequencing of bacterial and archaeal 16S rDNA libraries. Results. The highest yield >600 ml CH₄/g VS_added was achieved when 25% of the consortium was added followed by 50%; however, between these two treatments, there was not a statistically significant difference. The rest of the treatments were under 600 ml CH₄/g VS_added. Nevertheless, it should be noted that the treatment at 25% reached its methane peak after 10 days, meanwhile, the treatment at 50% reached it on approximately 8 days, suggesting in this last treatment, the generation of methane was more accelerated. The methane yield increased by approximately 7% and 5% compared to the control (0%), while using 25% and 50% of the consortium, respectively. Regarding the bacterial communities, it was found that the consortium was mainly composed of members of the phylum Synergistetes. Members of this phylum are known for their acetate oxidizing properties. In all the treatments the phyla Firmicutes, Thermotogae, and Synergistetes were dominant and as the concentration of the consortium in the treatments increased, the presence of the family Thermotogaceae also increased. Conclusions. In this research, we detected that the bioaugmentation using hydrogenotrophic methanogens improved the methane yield of the AD process by more than 5%. The bacterial communities present in the consortium have capacities to degrade cellulolytic compounds and act as acetate oxidants, which play a role in syntrophy with hydrogenotrophic methanogens, however, it is still necessary to know the dynamics of the methanogens. Acknowledgments. The project was supported by CIIEMAD IPN SIP 20172277, 20180942, 20195607, and CONACyT-163235 INFR-2011-01. AKGB was granted a CONACyT-700337 doctorate scholarship.

Introduction. The ready-to-eat food microbiota are the microorganisms present in the dishes that are currently consumed during the meals. These microorganisms include those that may have a health benefit, potentially pathogenic microorganisms, among others that have not yet been given a function. Foods that are suitable for human consumption are not free of microorganisms, however, within the food industry only yeasts have been given a beneficial function ready-to-eat food, while other microorganisms such as filamentous fungi and bacteria have been studied for their negative effect for food. Methods. Using High-throughput DNA sequencing of 16S rDNA libraries, we determined the bacterial diversity in different samples of high demanded freshly prepared unspoiled ready-to-eat Mexican dishes. Results. We found a great bacterial diversity. The most abundant bacterial phyla were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, TM7, Thermi, among others. The phyla included bacteria with remarkable abundance per type of dish. For instance, we found in the soups, the genera Microbacterium, and Anoxybacillus; in the main dishes the family Lachnospiraceae, the genus Dorea; in the dairy products, the genera Thermus, and Blautia; in the side plates, members of the family Aeromonadaceae, and the genus Kaistobacter; the beverages contained members of the family Enterobacteriaceae and the genus Lactobacillus, and finally the desserts, contained bacteria of the family Ruminococaceae, and the genus Victoria. We found a large abundance of chloroplast sequences, which were eliminated by filtering. The alfa diversity analyses showed that the main dishes had the largest diversity. The beta-diversity analyses clustered the bacterial communities of soups, side plates, desserts, and beverages, and some main dishes. Conclusions. Based on our results we conclude that unspoiled ready-to-eat Mexican dishes contain a rich diversity in the bacterial community, which may contribute to the organoleptic properties of the dishes without representing a sanitary risk for the consumers. Acknowledgments. The project was supported by Cinvestav and CONACyT 163235 INFR-2011-01.

Rhizobacteria play an important role in maintaining soil balance, among these bacteria there are those which have shown their ability to promote the growth of plants known as Plant Growth Promoting Rhizobacteria (PGPR). In our work, we are interested in characterizing 110 bacterial strains isolated in the field in the region of ben badis (Constantine Algeria) from 5 varieties of bean. Phenotypic and biochemical characterization showed that most of the isolates are cream colored, slightly raised, flat and opaque, gram-, catalase + and oxidase-, Bacillus form. PCA analysis allowed us to select 40 isolates with a high degree of variability to continue our work. The results obtained have directed us towards different taxonomic groups (rhizobium, Pseudomonas, bacillus etc.). The evaluation of the PGPR potential of bacteria (phytostimulation, biofertilization and biocontrol), showed that 100 % of bacteria are able to produce auxin at different concentrations with the highest concentration (177.77µg / ml) for the isolate 6, that more than 50% of isolates are capable of producing nitrogen, ammonia and phytate mineralization. These PGPR traits are a direct effect on plant growth of five varieties of the bean and can be used to select the best performing bacteria for inoculation tests.

Patterns of trees’ endogenous rhythmic growth (ERG) and paralleled C allocation shift between root and shoot systems have been studied, but there is still a need to understand their impact in shaping soil microbiomes. Moreover, impact of plants on soil microbial communities can be modulated or overweighed by time-induced plant and/or seasonal changes. Thus, we intended to analyze the structure of soil microbiomes as response to simultaneous alternated host tree root and shoot flushes and time-induced changes within one vegetation period at two sites in Central Germany. In this study, we utilized oak phytometers (Quercus robur L., clone DF159) as host trees, and made use of their ERG, whereby consecutive root and shoot flushes make a complete growth cycle. We studied two complete growth cycles during the same vegetation period, performed a non-destructive soil sampling and applied high-throughput amplicon sequencing of the bacterial 16S gene and the fungal ITS2 region. As C allocation shifts between the tree root and shoot, released root exudates and consequently the nutrient availability alternate for soil microorganisms. We therefore anticipated different microbial communities in the host tree root zone along the growth cycles until senescence. In our results, the bacterial community exhibited a directional change over time along the vegetation period. In contrast, the fungal community appeared sample specific. Our findings enlarge the current understanding of the temporal microbial assembly in the host tree root zone.

An approach of new and sustainable uses for by-products generated in the wine production industry, one of the agro-food sectors of importance, has been studied. Wine lees, a sediment obtained in different processes of decantation of wine, have been used to produce biomass of microalgae enriched in carotenoids as high added value biomolecules. Experiments to incorporate chemical components of wine lees into microalgae biomass to understand the effect of these residues on the growth and biosynthesis of carotenoids into commercial microalgae Chlorella sorokiniana have been done. Algae culture system has been optimized and preparation of culture media have been obtained by extracting in water the soluble nutrients contained in the lees at different concentrations between 5% and 50% p/v. Optimal growth was obtained using extraction of wine residues at 5% and 10% w/v. At 10 % oxidative stress, measured as carotenoids production (specially lutein) and antioxidant activity (DPPH method), was more intense than the obtained using residues at 5%. Our results show that growth in culture media prepared with wine lees extracts at 10% stimulated the antioxidant activity and the production of carotenoids in C. sorokiniana cells. Preliminary information, not only to produce sustainable growth media for biomass of microalgae enriched in high value molecules, but also to reuse nutrients contained in wine industry by-products what is of particular interest in the context of a circular economy is provided.
Funding: This research was funded by PROGRAMA OPERATIVO FEDER ANDALUCIA (Spain) 2014-2020. Proyectos de I+D+I., grant number UHU-1265804.

The increasing trend of antibiotic resistance by bacteria accentuates the necessity to exploit alternative potential therapeutic agents capable to act as effective and natural antibacterial agents. Therefore, use of honey as antibacterial agent back to ancient times. Aim of this study was to demonstrate the susceptibility of biofilm formation by different pathogens to some types of monofloral honey, Hedera helix (ivy), Arbutus unedo (strawberry tree), Lavandula angustifolia (lavender), sulla (Hedysarum coronarium), and Ailanthus altissima (tree of heaven). In addition, we assessed the capacity of the honey to block the metabolic changes taking place in the microbial cells included in the biofilm. The inhibitory action was variable. Listeria monocytogenes was the most sensitive bacteria, so that the formation of biofilm was inhibited up to 72.20 % (when we tested 11.42 microliters/ml of sulla honey) and never lower than 27.32% (by assaying 5.71 microliters/ml of lavender honey). Pseudomonas aeruginosa was less sensitive; however, some types of honey, such as tree of heaven and sulla, caused a biofilm inhibition up to 40.41% and 35.85%, respectively. The types of monofloral honey were able to act on the P. aeruginosa metabolism, with percentage of inhibition not inferior than 46.07% (with 11.42 microliters of ivy honey), reaching even 75.24% (with 11.42 microliters of tree of heaven honey). St. aureus, which showed greater resistance to the biofilm-inhibitory action of the types of honey, was instead more sensitive at the metabolic level, with percentages of inhibition reaching 61.63% (in the presence of the tree of heaven honey).