Insecticide Efficacy of Green Synthesis Silver Nanoparticles on Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of Liberibacter asiaticus Jagoueix et al. and Liberibacter americanus Teixeira et al., causal agents of the critical yellow dragon disease or Huanglongbing (HLB), which affects citrus production worldwide. Recently, green synthetic nanoparticles have emerged as a potential alternative to control of agricultural insect pests. The insecticide effect of silver nanoparticles (AgNPs) on 2nd instar nymphs of D. citri under laboratory and greenhouse conditions was evaluated. Mortality was recorded 24, 48, and 72 h after application on D. citri nymphs under both laboratory and greenhouse conditions. The laboratory results showed that AgNPs caused 97.84 and 100% mortality at 32 and 64 ppm, respectively, 72 h after treatment. In the greenhouse, AgNPs caused 78.69 and 80.14% mortality using 64 and 128 ppm 72 h after application. This research is the first to evaluate the green synthesis AgNPs on D. citri and are a promising strategy to control the pest.

Lower plasma melatonin levels in non-hypoxic premature newborns associated with neonatal pain.

We analyzed plasma melatonin levels in different groups of preterm newborns without hypoxia and their relationship with several perinatal variables like gestational age or neonatal pain. Prospective cohort study of preterm newborns (PTNB) without perinatal hypoxia, Apgar > 6 at 5 min, and oxygen needs on the third day of life. We compared melatonin levels at day 3 of life in different groups of non-hypoxic preterm infants (Student's t-tests, Mann-Whitney U, and chi2) and analyzed the relationship of melatonin with GA, birth weight, neonatal pain (Premature Infant Pain Profile (PIPP) scale), caffeine treatment, parenteral nutrition, or the development of free radical diseases (correlation study, linear regression) and factors associated with moderate/intense pain and free radical diseases (logistic regression analysis). Sixty-one preterm infants with gestational age (GA) of 30.7 ± 2.0 weeks with no oxygen requirements at day 3 of life were studied with plasma melatonin levels of 33.8 ± 12.01 pg/ml. Preterm infants weighing < 1250 g at birth had lower plasma melatonin levels (p = 0.05). Preterm infants with moderate or severe pain (PPIPP > 5) have lower melatonin levels (p = 0.01), and being preterm with PIPP > 5 is associated with lower plasma melatonin levels (p = 0.03). Being very preterm (GA < 32 GS), having low weight for gestational age (LWGA), receiving caffeine treatment, or requiring parenteral nutrition did not modify melatonin levels in non-hypoxic preterm infants (p = NS). Melatonin on day 3 of life in non-hypoxic preterm infants is not associated with later development of free radical diseases (BPD, sepsis, ROP, HIV, NEC). CONCLUSION: We observed that preterm infants with moderate to severe pain have lower melatonin levels. These findings are relevant because they reinforce the findings of other authors that melatonin supplementation decreases pain and oxidative stress in painful procedures in premature infants. Further studies are needed to evaluate whether melatonin could be used as an analgesic in painful procedures in preterm infants. TRIAL REGISTRATION: Trial registration was not required since this was an observational study. WHAT IS KNOWN: • Melatonin is a potent antioxidant and free radical scavenger in newborns under stress conditions: hypoxia, acidosis, hypotension, painful procedures, or parenteral nutrition. • Pain stimulates the production of melatonin. • Various studies conclude that melatonin administration decreases pain during the neonatal period. WHAT IS NEW: • Non-hypoxic preterm infants with moderate to severe pain (PIPP>5) have lower levels of melatonin. • Administration of caffeine and treatment with parenteral nutrition do not modify melatonin levels in non-hypoxic preterm infants.

Influence of the medical treatment schedule in new diagnoses patients with heart failure and reduced ejection fraction.

AIMS: Heart failure (HF) guidelines recommend treating all patients with HF and reduced ejection fraction (HFrEF) with quadruple therapy, although they do not establish how to start it. This study aimed to evaluate the implementation of these recommendations, analyzing the efficacy and safety of the different therapeutic schedules. METHODS AND RESULTS: Prospective, observational, and multicenter registry that evaluated the treatment initiated in patients with newly diagnosed HFrEF and its evolution at 3 months. Clinical and analytical data were collected, as well as adverse reactions and events during follow-up. Five hundred and thirty-three patients were included, selecting four hundred and ninety-seven, aged 65.5 ± 12.9 years (72% male). The most frequent etiologies were ischemic (25.5%) and idiopathic (21.1%), with a left ventricular ejection fraction of 28.7 ± 7.4%. Quadruple therapy was started in 314 (63.2%) patients, triple in 120 (24.1%), and double in 63 (12.7%). Follow-up was 112 days [IQI 91; 154], with 10 (2%) patients dying. At 3 months, 78.5% had quadruple therapy (p < 0.001). There were no differences in achieving maximum doses or reducing or withdrawing drugs (< 6%) depending on the starting scheme. Twenty-seven (5.7%) patients had any emergency room visits or admission for HF, less frequent in those with quadruple therapy (p = 0.02). CONCLUSION: It is possible to achieve quadruple therapy in patients with newly diagnosed HFrEF early. This strategy makes it possible to reduce admissions and visits to the emergency room for HF without associating a more significant reduction or withdrawal of drugs or significant difficulty in achieving the target doses.

Targeted depletion of pks+ bacteria from a fecal microbiota using specific antibodies.

The pks island is one of the most prevalent pathogenicity islands among the Escherichia coli strains that colonize the colon of colorectal carcinoma (CRC) patients. This pathogenic island encodes the production of a nonribosomal polyketide-peptide named colibactin, which induces double-strand breaks in DNA molecules. Detection or even depletion of this pks-producing bacteria could help to understand the role of these strains in the context of CRC. In this work, we performed a large-scale in silico screening of the pks cluster in more than 6,000 isolates of E. coli. The results obtained reveal that not all the pks-detected strains could produce a functional genotoxin and, using antibodies against pks-specific peptides from surface cell proteins, a methodology for detection and depletion of pks+ bacteria in gut microbiotas was proposed. With our method, we were able to deplete a human gut microbiota of this pks+ strains, opening the door to strain-directed microbiota modification and intervention studies that allow us to understand the relation between these genotoxic strains and some gastrointestinal diseases. IMPORTANCE The human gut microbiome has also been hypothesized to play a crucial role in the development and progression of colorectal carcinoma (CRC). Between the microorganisms of this community, the Escherichia coli strains carrying the pks genomic island were shown to be capable of promoting colon tumorigenesis in a colorectal cancer mouse model, and their presence seems to be directly related to a distinct mutational signature in patients suffering CRC. This work proposes a novel method for the detection and depletion of pks-carrying bacteria in human gut microbiotas. In contrast to methods based on probes, this methodology allows the depletion of low-abundance bacterial strains maintaining the viability of both targeted and non-targeted fractions of the microbiota, allowing the study of the contribution of these pks-carrying strains to different diseases, such as CRC, and their role in other physiological, metabolic or immune processes.

Towards the isolation of more robust next generation probiotics: The first aerotolerant Bifidobacterium bifidum strain.

This work reports on the first described aerotolerant Bifidobacterium bifidum strain, Bifidobacterium bifidum IPLA60003, which has the ability to form colonies on the surface of agar plates under aerobic conditions, a weird phenotype that to our knowledge has never been observed in B. bifidum. The strain IPLA60003 was generated after random UV mutagenesis from an intestinal isolate. It incorporates 26 single nucleotide polymorphisms that activate the expression of native oxidative-defense mechanisms such as the alkyl hydroxyperoxide reductase, the glycolytic pathway and several genes coding for enzymes involved in redox reactions. In the present work, we discuss the molecular mechanisms underlying the aerotolerance phenotype of B. bifidum IPLA60003, which will open new strategies for the selection and inclusion of probiotic gut strains and next generation probiotics into functional foods.

Immunomagnetic Capture of Faecalibacterium prausnitzii Selectively Modifies the Fecal Microbiota and Its Immunomodulatory Profile.

Faecalibacterium represents one of the most abundant bacterial groups in the human intestinal microbiota of healthy adults and can represent more than 10% of the total bacterial population, Faecalibacterium prausnitzii being the only recognized species up to the past year. Reduction in the abundance of F. prausnitzii in the human gut has been linked to several human disorders, such as Crohn's disease. In this study, we developed a strategy to modify the relative abundance of F. prausnitzii in fecal microbiotas as a means of evaluating its contribution to the immunomodulatory effect of intestinal microbiotas with different F. prausnitzii contents using a peripheral blood mononuclear cell (PBMC) model. We used a polyclonal antibody against the surface of F. prausnitzii M21 to capture the bacterium from synthetic and human fecal microbiotas using immunoseparation techniques. As a proof-of-principle study, the levels of immunomodulation exerted by microbiotas of healthy donors (HDs) with different relative abundances of F. prausnitzii, achieved with the above-mentioned immunoseparation technique, were evaluated in a PBMC model. For this purpose, PBMCs were cocultivated with the modified microbiotas or a pure culture of F. prausnitzii and, subsequently, the microbiota of Crohn's donors was added to the coculture. The cytokine concentration was determined, showing that our experimental model supports the anti-inflammatory effects of this bacterium. IMPORTANCE There is increasing interest in deciphering the contribution of gut microbiota species to health and disease amelioration. The approach proposed herein provides a novel and affordable strategy to probe deeply into microbiota-host interactions by strategically modifying the relative abundance of specific gut microbes, hence facilitating the study of their contribution to a given trait of the microbiota.

Convergence of flow cytometry and bacteriology. Current and future applications: a focus on food and clinical microbiology.

Since its development in the 1960s, flow cytometry (FCM) was quickly revealed a powerful tool to analyse cell populations in medical studies, yet, for many years, was almost exclusively used to analyse eukaryotic cells. Instrument and methodological limitations to distinguish genuine bacterial signals from the background, among other limitations, have hampered FCM applications in bacteriology. In recent years, thanks to the continuous development of FCM instruments and methods with a higher discriminatory capacity to detect low-size particles, FCM has emerged as an appealing technique to advance the study of microbes, with important applications in research, clinical and industrial settings. The capacity to rapidly enumerate and classify individual bacterial cells based on viability facilitates the monitoring of bacterial presence in foodstuffs or clinical samples, reducing the time needed to detect contamination or infectious processes. Besides, FCM has stood out as a valuable tool to advance the study of complex microbial communities, or microbiomes, that are very relevant in the context of human health, as well as to understand the interaction of bacterial and host cells. This review highlights current developments in, and future applications of, FCM in bacteriology, with a focus on those related to food and clinical microbiology.

Benefits of Kangaroo Mother Care on the Physiological Stress Parameters of Preterm Infants and Mothers in Neonatal Intensive Care.

It is well documented that the stress of separation of mother and baby can lead to short-term physiological instability as well as neurological, sociological or psychological consequences that may last a lifetime. OBJECTIVE: The goal was to estimate the effect of kangaroo mother care (KMC) on physiological and biochemical parameters of preterm infant stress and maternal stress in neonatal intensive care. METHODS: The investigation involved 112 preterm infants. Two groups were compared according to the mean duration of KMC during 12 days of study: the KMC group (mean duration more than 90 min daily) and the control group (less than 90 min). RESULTS: Kangaroo mother care for more than 90 min on average per day in preterm infants is associated 12 days after the intervention with lower mean cortisol levels (p = 0.02), greater weight gain and less need for parenteral nutrition in preterm infants, as well as less postpartum depression (p = 0.02) and lower cortisol levels (p = 0.002) in the mothers of preterm infants. CONCLUSIONS: This study suggests that KMC can be used to improve the stress of preterm infants and their mothers, and that the greater weight gain observed in these preterm infants could contribute to a shorter average hospital stay and lower healthcare expenditure.

Cardiopatía asociada a infección por VIH / HIV-associated heart disease

Resumen La infección por VIH continúa representando un problema sanitario de primer orden en el mundo. El aumento de la esperanza de vida gracias a la terapia antirretroviral ha aumentado la prevalencia de la enfermedad de manera importante. La infección por VIH es una causa importante de cardiopatía adquirida, en especial en relación con el desarrollo de cardiopatía isquémica (manifestación cardiovascular más frecuente en los países desarrollados en la actualidad). Se presenta el caso de una paciente de 42 años, con infección por VIH, sin factores de riesgo cardiovascular clásicos, quien presentó un síndrome coronario agudo de alto riesgo. Se discuten la etiopatogenia de la cardiopatía isquémica asociada a la infección por VIH y sus aspectos particulares diagnósticos y terapéuticos.

Abstract Human immunodeficiency virus (HIV) infection continues to be a leading health problem worldwide. Increased life expectancy due to antiretroviral therapy has significantly increased the prevalence of the disease. Human immunodeficiency virus infection is an important cause of acquired heart disease, especially the development of ischemic heart disease (the most common cardiovascular manifestation in developed countries today). We present the case of a 42-year-old patient with HIV infection with no classical cardiovascular risk factors who developed a high-risk acute coronary syndrome. We discuss the etiopathogenesis of HIV-associated ischemic heart disease and its particular diagnostic and therapeutic aspects.

Unravelling the immunomodulatory role of apple phenolic rich extracts on human THP-1- derived macrophages using multiplatform metabolomics.

Apples represent a significant source of dietary phenolic compounds with evidenced anti-inflammatory and immunomodulatory activities. Nevertheless, the effect of the whole apple matrix on human macrophages is unknown. In this context, our study attempts to evaluate the effect of apple-derived phenolic compounds-rich extracts (pulp, peel and leaf) on IL-1ß production in THP-1-differentiated macrophages and derived metabolic alterations through untargeted metabolomics. Our results have showed that apple pulp treatment inhibited the release of the pro-inflammatory cytokine IL-1ß induced by LPS in THP-1 macrophages by ELISA analysis. Metabolomics demonstrate that different proportions of phenolic compounds led to differential alterations in the metabolism of THP-1 macrophages. Indeed, apple extracts promoted alterations in lipid, carbohydrate, amino acid and vitamins as well as cofactors metabolism. Specifically, leaf extracts were characterized by alteration of galactose metabolism while the extracts derived from the fruit showed predominant alterations in lipids metabolism. All extracts mimicked the response observed under normal conditions in LPS-stimulated macrophages, inhibiting LPS response. Thus, the phenolic enriched extracts from apples will be a good source of natural compounds with a beneficial effect against inflammation, and they may be applied as a food supplement and/or functional ingredient for the treatment of inflammatory diseases.

Efficacy and safety of abciximab versus tirofiban in addition to ticagrelor in STEMI patients undergoing primary percutaneous intervention.

Platelet glycoprotein IIb/IIIa inhibitors (GPIs) have been part of the adjuvant treatment of acute coronary syndrome for years. However, real-life data regarding the efficacy and safety of GPIs under the current indications are lacking in the setting of potent platelet inhibition. The objectives were to assess the efficacy and safety of abciximab versus tirofiban in patients with ST-elevation acute myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI) and pretreated with ticagrelor, and to identify independent predictor factors of efficacy, bleeding and platelet drop. Three hundred sixty-two patients were divided by GPI administered. Clinical, laboratory, angiographic and outcome characteristics were compared. The primary objective was a composite efficacy endpoint (death from any cause, nonfatal myocardial infarction and nonfatal stroke) at 30 days. The secondary objectives were its individual components, safety (bleeding) and the impact on platelet count during hospital stay. The composite efficacy endpoint was similar in the abciximab and tirofiban groups (6.1% vs 7.3%; p = .632). There were also no differences in cardiovascular death (2.5% vs 2.4%; p = .958), nonfatal myocardial infarction (3% vs 4.3%; p = .521) and nonfatal stroke (0.5% vs 1.8%; p = .332). Tirofiban administration was associated with a higher incidence of bleeding (11.6% vs 22%; p = .008) with no differences in BARC ≥ 3b bleeding (3.6 vs 2.5%; p = .760). In STEMI patients undergoing PPCI with ticagrelor, abciximab and tirofiban had similar rates in the composite efficacy endpoint at 30 days. The 30-day bleeding rate was significantly higher in the tirofiban group. Tirofiban administration was an independent predictor of both bleeding and platelet count drop.

Microbiota-Derived ß-Amyloid-like Peptides Trigger Alzheimer's Disease-Related Pathways in the SH-SY5Y Neural Cell Line.

Here, we present the first in silico and in vitro evidence of Aß-like peptides released from meaningful members of the gut microbiome (mostly from the Clostridiales order). Two peptides with high homology to the human Aß peptide domain were synthesized and tested in vitro in a neuron cell-line model. Gene expression profile analysis showed that one of them induced whole gene pathways related to AD, opening the way to translational approaches to assess whether gut microbiota-derived peptides might be implicated in the neurodegenerative processes related to AD. This exploratory work opens the path to new approaches for understanding the relationship between the gut microbiome and the triggering of potential molecular events leading to AD. As microbiota can be modified using diet, tools for precise nutritional intervention or targeted microbiota modification in animal models might help us to understand the individual roles of gut bacteria releasing Aß-like peptides and therefore their contribution to this progressive disease.

Ruminococcoides bili gen. nov., sp. nov., a bile-resistant bacterium from human bile with autolytic behavior.

A strictly anaerobic, resistant starch-degrading, bile-tolerant, autolytic strain, IPLA60002T, belonging to the family Ruminococcaceae, was isolated from a human bile sample of a liver donor without hepatobiliary disease. Cells were Gram-stain-positive cocci, and 16S rRNA gene and whole genome analyses showed that Ruminococcus bromii was the phylogenetically closest related species to the novel strain IPLA60002T, though with average nucleotide identity values below 90 %. Biochemically, the new isolate has metabolic features similar to those described previously for gut R. bromii strains, including the ability to degrade a range of different starches. The new isolate, however, produces lactate and shows distinct resistance to the presence of bile salts. Additionally, the novel bile isolate displays an autolytic phenotype after growing in different media. Strain IPLA60002T is phylogenetically distinct from other species within the genus Ruminococcus. Therefore, we propose on the basis of phylogenetic, genomic and metabolic data that the novel IPLA60002T strain isolated from human bile be given the name Ruminococcoides bili gen. nov., sp. nov., within the new proposed genus Ruminococcoides and the family Ruminococcaceae. Strain IPLA60002T (=DSM 110008T=LMG 31505T) is proposed as the type strain of Ruminococcoides bili.

Determination of Bile Salt Hydrolase Activity in Bifidobacteria.

Bile salt hydrolase (BSH) activity is a desirable trait in putative probiotic bacteria, such as those belonging to the Bifidobacterium genus. On the one hand, bile salt hydrolysis is considered to represent a bile detoxification mechanism for gut commensal bacteria and thus the presence of this activity was believed to be a predictor of bile tolerance of putative probiotic strains. On the other hand, it has recently been revealed that chemical modifications of the bile acid pool performed by the gut microbiota strongly impact on host health. This explains the increasing interest to investigate the role played by bile-modifying enzymes of gut commensals on lowering cholesterol levels, on modulating gut inflammation or on influencing the development of cancer or metabolic disorders. This chapter compiles qualitative and quantitative methods to analyse BSH activity in bifidobacteria, though they could be adapted to other bacterial groups of interest.

Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages.

Dihydrochalcones, phlorizin (PZ) and its aglycone phloretin (PT), have evidenced immunomodulatory effects through several mechanisms. However, the differential metabolic signatures that lead to these properties are largely unknown. Since macrophages play an important role in the immune response, our study aimed to characterise human THP-1 macrophages under PZ and PT exposure. A multiplatform-based untargeted metabolomics approach was used to reveal metabolites associated with the anti-inflammatory mechanisms triggered by the dihydrochalcones in LPS-stimulated macrophages, for the first time. Results showed differential phenotypic response in macrophages for all treatments. Dihydrochalcone treatment in LPS-stimulated macrophages mimics the response under normal conditions, suggesting inhibition of LPS response. Antagonistic effects of dihydrochalcones against LPS was mainly observed in glycerophospholipid and sphingolipid metabolism besides promoting amino acid biosynthesis. Moreover, PT showed greater metabolic activity than PZ. Overall, the findings of this study yielded knowledge about the mechanisms of action PZ and PT at metabolic level in modulating inflammatory response in human cells.

Precision modification of the human gut microbiota targeting surface-associated proteins.

This work describes a new procedure that allows the targeted modification of the human gut microbiota by using antibodies raised against bacterial surface-associated proteins specific to the microorganism of interest. To this end, a polyclonal antibody recognising the surface-associated protein Surface Layer Protein A of Lactobacillus acidophilus DSM20079T was developed. By conjugating this antibody with fluorescent probes and magnetic particles, we were able to specifically identify this bacterium both in a synthetic, and in real gut microbiotas by means of a flow cytometry approach. Further, we demonstrated the applicability of this antibody to deplete complex human gut microbiotas from L. acidophilus in a single step. L. acidophilus was found to interact with other bacteria both in synthetic and in real microbiotas, as reflected by its concomitant depletion together with other species. Further optimization of the procedure including a trypsin step enabled to achieve the selective and complete isolation of this species. Depleting a single species from a gut microbiota, using antibodies recognizing specific cell surface elements of the target organism, will open up novel ways to tackle research on the specific immunomodulatory and metabolic contributions of a bacterium of interest in the context of a complex human gut microbiota, including the investigation into therapeutic applications by adding/depleting a key bacterium. This represents the first work in which an antibody/flow-cytometry based application enabled the targeted edition of human gut microbiotas, and represents the basis for the design of precision microbiome-based therapies.

Computational Approach to the Systematic Prediction of Glycolytic Abilities: Looking Into Human Microbiota.

Glycoside hydrolases are responsible for the enzymatic deconstruction of complex carbohydrates. Most of the families are known to conserve the catalytic machinery and molecular mechanisms. This work introduces a new method to predict glycolytic abilities in sequenced genomes and thus, gain a better understanding of how to target specific carbohydrates and identify potentially interesting sources of specialised enzymes. Genome sequences are aligned to those of organisms with expertly curated glycolytic abilities. Clustering of homology scores helps identify organisms that share common abilities and the most promising organisms regarding specific glycolytic abilities. The method has been applied to members of the bacterial families Ruminococcaceae (39 genera), Eubacteriaceae (11 genera) and Lachnospiraceae (59 genera), which hold major representatives of the human gut microbiota. The method predicted the potential presence of glycoside hydrolases in 1701 species of these genera. Here, the validity and practical usefulness of the method is discussed based on the predictions obtained for members of the genus Ruminococcus. Results were consistent with existing literature and offer useful, complementary insights to comparative genomics and physiological testing. The implementation of the Gleukos web portal (http://sing-group.org/gleukos) offers a public service to those interested in targeting microbial carbohydrate metabolism for biotechnological and health applications.

Addressing the Covid-19 pandemic and future public health challenges through global collaboration and a data-driven systems approach.

Covid-19 has already taught us that the greatest public health challenges of our generation will show no respect for national boundaries, will impact lives and health of people of all nations, and will affect economies and quality of life in unprecedented ways. The types of rapid learning envisioned to address Covid-19 and future public health crises require a systems approach that enables sharing of data and lessons learned at scale. Agreement on a systems approach augmented by technology and standards will be foundational to making such learning meaningful and to ensuring its scientific integrity. With this purpose in mind, a group of individuals from Spain, Italy, and the United States have formed a transatlantic collaboration, with the aim of generating a proposed comprehensive standards-based systems approach and data-driven framework for collection, management, and analysis of high-quality data. This framework will inform decisions in managing clinical responses and social measures to overcome the Covid-19 global pandemic and to prepare for future public health crises. We first argue that standardized data of the type now common in global regulated clinical research is the essential fuel that will power a global system for addressing (and preventing) current and future pandemics. We then present a blueprint for a system that will put these data to use in driving a range of key decisions. In the context of this system, we describe and categorize the specific types of data the system will require for different purposes and document the standards currently in use for each of these categories in the three nations participating in this work. In so doing, we anticipate some of the challenges to harmonizing these data but also suggest opportunities for further global standardization and harmonization. While we have scaled this transnational effort to three nations, we hope to stimulate an international dialogue with a culmination of realizing such a system.

Exopolysaccharide Producing Bifidobacterium animalis subsp. lactis Strains Modify the Intestinal Microbiota and the Plasmatic Cytokine Levels of BALB/c Mice According to the Type of Polymer Synthesized.

Bacteria-host interactions are mediated by different microbial associated molecular patterns which are most often surface structures such as, among others, exopolysaccharides (EPSs). In this work, the capability of two isogenic EPS-producing Bifidobacterium animalis subsp. lactis strains to modulate the gut microbiota of healthy mice, was assessed. Each strain produces a different type of polymer; the ropy strain S89L synthesized a rhamnose-rich, high-molecular weight EPS in highest abundance than the non-ropy DMS10140 one. BALB/c mice were orally fed for 10 days with milk-bifidobacterial suspensions and followed afterward for 7 post-intervention days (wash-out period). The colonic content of mice was collected in several sampling points to perform a metataxonomic analysis. In addition, the influence of specific microbial clades, apparently stimulated by the ropy and non-ropy strains, on mouse plasmatic cytokine levels was investigated through hierarchical association testing. Analysis of 16S rRNA gene sequences showed that the abundance of Firmicutes phylum significantly increased 7 days after cessing the treatment with both strains. The relative abundance of Alloprevotella genus also rose, but after shorter post-treatment times (3 days for both DMS10140 and S89L strains). Some bacterial clades were specifically modulated by one or another strain. As such, the non-ropy DMS10140 strain exerted a significant influence on Intestinomonas genus, which increased after 4 post-administration days. On the other hand, feeding with the ropy strain S89L led to an increase in sequences of Faecalibaculum genus at 4 post-treatment days, while the abundance of Erysipelotrichaceae and Lactobacillaceae families increased for prolonged times. Association testing revealed that several lactobacilli and bifidobacterial significantly stimulated by ropy S89L strain were positively associated with the levels of certain cytokines, including IL-5 and IL-27. These results highlight relevant changes in mice gut microbiota produced after administration of the ropy S89L strain that were associated to a potential immune modulation effect.

Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line.

In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis-CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway.