Edwardsiella ictaluri T3SS effector EseN is a phosphothreonine lyase that inactivates ERK1/2, p38, JNK, and PDK1 and modulates cell death in infected macrophages.

IMPORTANCE: This work has global significance in the catfish industry, which provides food for increasing global populations. E. ictaluri is a leading cause of disease loss, and EseN is an important player in E. ictaluri virulence. The E. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions of E. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions.

Edwardsiella ictaluri T3SS Effector EseN Modulates Expression of Host Genes Involved in the Immune Response.

The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, including NF-κß (c-rel and relB), creb3L4, socs6 and foxo3a. Regulation of transcription factors leads to regulation of proinflammatory interleukins (IL-8, IL-12a, IL-15, IL-6) and cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 mRNA by WT E. ictaluri leads to decreased production of prostaglandin E2 (PGE2), which is the product of COX-2 activity. Collectively, our results indicate that E. ictaluri EseN is an important player in the modulation of host immune responses to E.ictaluri infection.

Effect of oral consumption of capsules containing Lactobacillus paracasei LPC-S01 on the vaginal microbiota of healthy adult women: a randomized, placebo-controlled, double-blind crossover study.

Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem.

Impact of a Multistrain Probiotic Formulation with High Bifidobacterial Content on the Fecal Bacterial Community and Short-Chain Fatty Acid Levels of Healthy Adults.

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio.

New insights into the relationship between taste perception and oral microbiota composition.

Fairly poor data are available on the relationship between taste perception, food preferences and oral microbiota. In the present study, we investigated the hypothesis that subjects with higher responsiveness to 6-n-propylthiuracil (PROP) might be characterized by a different taste sensitivity and tongue microbiota composition. Indeed, the bacterial metabolism may modulate/enhance the concentration of tastants near the taste receptors, modifying taste perception through a sensorial adaptation mechanism or by a broad range of microbial metabolic pathways. The detection thresholds of sweet, sour, salty and bitter, the Fungiform Papillae Density (FPD) and the composition of bacteria lining the tongue were determined in Supertasters (high PROP responsiveness, ST) and Non-tasters (low PROP responsiveness, NT). An important inter-individual variability was found for all taste stimuli and FPD between the two groups, with NT subjects showing significant higher threshold values and a lower FPD than with STs. We found five bacterial genera whose relative abundances were significantly higher in STs than NTs. This study opens new avenues of research by highlighting associations between parameters usually studied independently.

Effect of Cell Concentration on the Persistence in the Human Intestine of Four Probiotic Strains Administered through a Multispecies Formulation.

Studies devoted to evaluating the outcome of different doses of probiotics are very limited, especially for multistrain formulations. In this context, we performed an intervention study that aimed to compare the effect of the administration of two doses (7 billion and 70 billion bacterial cells) of a multistrain probiotic formulation on the persistence of the four probiotic strains that were present in the product in the fecal samples collected from healthy subjects. The overall persistence of the probiotic strains was significantly higher for the 70 billion formulation than for the 7 billion formulation. Furthermore, probiotic strains were detected earlier and for longer for the 70 billion formulation compared to those for the 7 billion formulation. All probiotic strains were recovered alive from the 70 billion preparation, whereas recovery was not possible in a few fecal samples upon administration of the 7 billion preparation. In addition, the overall number of viable probiotic cells recovered on day 14 (i.e., the last day of consumption) was significantly higher for the 70 billion formulation than that for the 7 billion formulation. Finally, we found that the viability of the probiotic cells was stable over the course of the trial independent of volunteers' handling, demonstrating good manufacturing of the product. In conclusion, this study demonstrated that strains belonging to different taxa may coexist in the human gastrointestinal tract upon ingestion of a multispecies probiotic formulation. Moreover, this study suggests that higher doses of bacterial cells in probiotic formulations may permit a higher, earlier, and longer recovery of the probiotics in the feces of healthy adults.

Survival of L. casei DG® (Lactobacillus paracasei CNCMI1572) in the gastrointestinal tract of a healthy paediatric population.

PURPOSE: Ability to survive the digestive process is a major factor in determining the effectiveness of a probiotic. In this study, the ability of the probiotic L. casei DG® (Lactobacillus paracasei CNCMI1572) to survive gastrointestinal transit in healthy children was investigated for the first time. METHODS: Twenty children aged 3-12 years received L. casei DG® as drinkable solution of 1 × 109 colony forming units (CFU), once daily for 7 consecutive days. Recovery in faecal samples was evaluated at baseline and at different time-points during and after administration. Defecation frequency, faeces consistency, digestive function and product safety were also assessed. RESULTS: Nineteen (95%) of the 20 enrolled children presented viable L. casei DG® cells in their faeces at least once during the study, with a maximum count (mean 4.3 log10 CFU/g ± 2.3) reached between day 4 and 6 from the beginning of consumption. Notably, for 11 (57.9%) of the 19 children with viable cells, L. casei DG® survived in faecal samples up to 3 days after treatment end. Defecation frequency, faeces consistency and digestive function did not change considerably during or after study treatment. Safety of the study product was very good. CONCLUSIONS: This study showed for the first time that L. casei DG® survives the gastrointestinal transit when ingested by children with a paediatric probiotic drinkable solution containing 1 × 109 CFU, and persists in the gut up to 3 days after the end of product intake, demonstrating resistance to gastric juices, hydrolytic enzymes and bile acids.

Quantitative Recovery of Viable Lactobacillus paracasei CNCM I-1572 (L. casei DG®) After Gastrointestinal Passage in Healthy Adults.

Probiotics are live microorganisms, and viability after transit through the gastrointestinal tract (GIT) is considered an inherent property of the health benefits of probiotics. The aim of the present study was to quantify the viable and total loads of Lactobacillus paracasei DG cells after passage through the GIT following the consumption of the probiotic product Enterolactis (L. casei DG®; L. paracasei CNCM I-1572; L. paracasei DG) from drinkable vials by healthy adults. We developed a novel method for discriminating and enumerating culturable L. paracasei DG cells based on the unique sticky, filamentous phenotype of this strain on MRS agar containing vancomycin and kanamycin. The identity of DG was also confirmed with strain-specific primers by colony PCR. This method was used for a recovery study of the DG strain to quantify viable cells in the fecal samples of 20 volunteers during a 1-week probiotic consumption period and a 1-week follow-up. We isolated L. paracasei DG from at least one fecal sample from all the volunteers. The highest concentration of viable DG cells [ranging from 3.6 to 6.7 log10 colony-forming unit (CFU) per gram of feces] in the feces was observed between 4 and 8 days from the beginning of Enterolactis intake and for up to 5 days after cessation of intake. As expected, the total DG count determined by real-time quantitative PCR (qPCR) was mostly higher than the viable DG cells recovered. Viable count experiments, carried out by combining ad hoc culture-based discriminative conditions and strain-specific molecular biological protocols, unambiguously demonstrated that L. paracasei DG can survive gastrointestinal transit in healthy adults when ingested as Enterolactis in drinkable vials containing no less than one billion CFU at the end of shelf life.

Melting curve analysis of a groEL PCR fragment for the rapid genotyping of strains belonging to the Lactobacillus casei group of species.

Lactobacillus casei group (Lcs) consists of three phylogenetically closely related species (L. casei, L. paracasei, and L. rhamnosus), which are widely used in the dairy and probiotic industrial sectors. Strategies to easily and rapidly characterize Lcs are therefore of interest. To this aim, we developed a method according to a technique known as high resolution melting analysis (HRMa), which was applied to a 150 bp groEL gene fragment. The analysis was performed on 53 Lcs strains and 29 strains representatives of species that are commonly present in dairy and probiotic products and can be most probably co-isolated with Lcs strains. DNA amplification was obtained only from Lcs strains, demonstrating the specificity of the groEL primers designed in this study. The HRMa clustered Lcs strains in three groups that exactly corresponded to the species of the L. casei group. A following HRMa separated the 39 L. paracasei strains in two well distinct intraspecific groups, indicating the possible existence of at least two distinct genotypes inside the species. Nonetheless, the phenotypic characterization demonstrated that the genotypes do not correspond to the two L. paracasei subspecies, namely paracasei and tolerans. In conclusion, the melting curve analysis developed in this study is demonstrably a simple, labor-saving, and rapid strategy obtain the genotyping of a bacterial isolate and simultaneously potentially confirm its affiliation to the L. casei group of species. The application of this method to a larger collection of strains may validate the possibility to use the proposed HRMa protocol for the taxonomic discrimination of L. casei group of species. In general, this study suggests that HRMa can be a suitable technique for the genetic typization of Lactobacillus strains.