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Supplementary Table S2-S4. Pathway enrichment analysis by DAVID and GSEA in the ileum and colon of antibiotic treated mice.

1. Supplementary Materials and Methods Table S1. List of the antibodies used for flow cytometry 2. Supplementary Figures Supplementary Fig. S1. Impact of antibiotic treatment on trastuzumab efficacy and the tumor microenvironment. Supplementary Fig. S2. Depletion of commensal microbiota by antibiotic cocktail (ABX) treatment and fecal microbiota transplantation. Supplementary Fig. S3. Impact of vancomycin on trastuzumab efficacy in Î"16HER2 transgenic FVB mice. Supplementary Fig. S4. Differentially abundant bacteria in the gut microbiota of antibiotic-treated mice. Supplementary Fig. S5. Short-chain fatty acids (SCFAs) quantification in fecal samples from antibiotic-treated mice. Supplementary Fig. S6. Analysis of intratumor and stromal cell staining in tumors of antibiotic-treated mice. Supplementary Fig. S7. Impact of antibiotic treatment on tumor immune infiltrate. Supplementary Fig. S8. Analysis of intratumor and stromal cell staining in tumors from FMT mice. Supplementary Fig. S9. Impact of vancomycin treatment on intestinal and systemic immune features. Supplementary Fig. S10. Impact of IL12p70 and CD4+ cell depletion on trastuzumab antitumor efficacy and on tumor immune infiltrate. Supplementary Fig. S11. Causal role of human commensal bacteria in immune-mediated trastuzumab antitumor efficacy. 3. Supplementary References

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B–positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response.Significance:Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937

An immunosuppressive microenvironment in lung concurs to pre-malignant lesions progression to cancer. Here, we explore if perturbing lung microbiota, which contribute to immunosuppression, by antibiotics or probiotic aerosol interferes with lung cancer development in a mouse carcinogen-induced tumor model. Urethane-injected mice were vancomycin/neomycin (V/N)-aerosolized or live or dead L. rhamnosus GG (L.RGG)-aerosolized, and tumor development was evaluated. Transcriptional profiling of lungs and IHC were performed. Tumor nodules number, diameter and area were reduced by live or heat-killed L.RGG, while only a decrease in nodule diameter was observed in V/N-treated lungs. Both L.RGG and V/N reduced Tregs in the lung. In L.RGG-treated groups, the gene encoding the joining chain (J chain) of immunoglobulins was increased, and higher J chain protein and IgA levels were observed. An increased infiltration of B, NK and myeloid-derived cells was predicted by TIMER 2.0. The Kaplan–Meier plotter revealed an association between high levels of J chain mRNA and good prognosis in lung adenocarcinoma patients that correlated with increased B and CD4 T cells and reduced Tregs and M2 macrophages. This study highlights L.RGG aerosol efficacy in impairing lung cancer growth by promoting local immunity and points to this non-invasive strategy to treat individuals at risk of lung cancer.

A novel method was developed for virus-like particle (VLP) extraction and characterization from biological soil crust (BSC) after microbial community reactivation. The method consisted of a single cell analysis by flow cytometry to monitor viable cells in BSC reactivated under controlled hydration, temperature, and light/dark exposure. Then, VLPs were extracted from reactivated BSCs, followed by viral DNA extraction and shotgun metagenomic analysis. The hydrated BSC under light/dark conditions showed the highest number of viable cells, and this condition was optimal for VLPs isolation. Taxonomic composition showed that families of the order of Caudovirales (Podoviridae, Myoviridae and Syphoviridiae) were the most abundant double strand DNA phages while Microviridiae were the most abundant single strand DNA phages. The isolated VLPs also carried sequences of relevant bacterial inhabiting soil. The functional categories of “phages, prophages, transposable elements, plasmids” and “clustering-base subsystem” were abundant (38 and 12%, respectively). All these data suggest viral predation as a key factor in shaping and maintaining bacterial diversity in the BSCs.

Although the clinical benefit of trastuzumab for the management of HER2-positive breast carcinomas (BCs) has been largely demonstrated, many women, even those reported to have tumors potentially most sensitive to trastuzumab (e.g., HER2-enriched by PAM50 and with infiltrating immune cells) do not respond to this agent. The relevance of immunity in the cytotoxic mechanism of action of trastuzumab supports the notion that the anti-tumor effect of this monoclonal antibody depends on host immune system activity. Since gut commensal bacteria reportedly contribute to the development and maintenance of the immune system and have immunomodulatory effects, we investigated whether a relationship between gut microbiota composition and the response to trastuzumab exists in patients with HER2-positive BC. Stool samples were collected from 18 patients with primary HER2-positive BCs before the outset of neoadjuvant trastuzumab-based chemotherapy and analyzed by 16S rRNA gene profiling using Illumina Miseq platform. Gut microbiota β-diversity analysis by UniFrac algorithm revealed a heterogeneous microbiota composition mainly due to differences in the relative abundance of Bacteroides, Faecalibacterium and a genus belonging to Ruminococcaceae family. Unsupervised analysis identified two microbiota clusters that significantly discriminated patients according to pathological complete response (pCR) (p=0.0128, by Fisher test). No association between microbiota clusters and PAM50 molecular classification of tumor biopsies, as evaluated by gene expression, was observed. Moreover, HER2-enriched cases were distributed in the two microbiota clusters based on trastuzumab response. Differences in the intestinal microbiota between responsive (R) and non-responsive (NR) patients were assessed by LEfSe analysis: a significant higher and lower abundance of Clostridiales taxonomic order and Bacteroides taxonomic genus, respectively, was foundin R as compared to NR patients. Immune genes expressed in tumor biopsies that were found significantly correlated with these bacteria mainly belong to innate and adaptive immune response and cellular response to tumor necrosis factor pathways. To investigate the causal role of gut microbiota in trastuzumab benefit, FVB mice bearing syngeneic mammary carcinoma overexpressing human HER2 were transplanted with fecal material from R and NR patients after intestinal flora depletion by the use of an antibiotic cocktail. The mouse response to trastuzumab treatment recapitulated the response observed in patients from which stool derived. The obtained results support the contribution of gut microbiota in trastuzumab activity by influencing tumor immune microenvironment, independently of tumor intrinsic molecular characteristics. These data represent the proof-of-concept that manipulating gut microbes in resistant patients could be a new strategy to improve the response to trastuzumab. Supported by AIRC Citation Format: Elda Tagliabue, Martina Di Modica, Giorgio Gargari, Viola Regondi, Arianna Bonizzi, Stefania Arioli, Fabio Corsi, Simone Guglielmetti, Tiziana Triulzi. Commensal gut microbiota influences efficacy of trastuzumab in patients with HER2-positive breast carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-32.

Probiotic microorganisms may benefit the host by influencing diverse physiological processes, whose nature and underlying mechanisms are still largely unexplored. Animal models are a unique tool to understand the complexity of the interactions between probiotic microorganisms, the intestinal microbiota, and the host. In this regard, in this pilot study, we compared the effects of 5-day administration of three different probiotic bacterial strains (Bifidobacterium bifidum MIMBb23sg, Lactobacillus helveticus MIMLh5, and Lacticaseibacillus paracasei DG) on three distinct murine intestinal sites (ileum, cecum, and colon). All probiotics preferentially colonized the cecum and colon. In addition, probiotics reduced in the ileum and increased in the cecum and colon the relative abundance of numerous bacterial taxonomic units. MIMBb23sg and DG increased the inducible nitric oxide synthase (iNOS) in the ileum, which is involved in epithelial homeostasis. In addition, MIMBb23sg upregulated cytokine IL-10 in the ileum and downregulated the cyclooxygenase COX-2 in the colon, suggesting an anti-inflammatory/regulatory activity. MIMBb23sg significantly affected the expression of the main gene involved in serotonin synthesis (TPH1) and the gene coding for the serotonin reuptake protein (SERT) in the ileum and colon, suggesting a potential propulsive effect toward the distal part of the gut, whereas the impact of MIMLh5 and DG on serotonergic genes suggested an effect toward motility control. The three probiotics decreased the expression of the permeability marker zonulin in gut distal sites. This preliminary in vivo study demonstrated the safety of the tested probiotic strains and their common ability to modulate the intestinal microbiota. The probiotics affected host gene expression in a strain-specific manner. Notably, the observed effects in the gut were site dependent. This study provides a rationale for investigating the effects of probiotics on the serotonergic system, which is a topic still widely unexplored.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigen- esis. We identified two anti-tumourigenic strains of the microbiota--Faecalibaculum rodentium and its human homologue, Holdemanella biformis--that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymeth- ane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endog- enous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

Yeasts are responsible for spoilage of fruit juices and beverages because of their low pH and high sugar content. Their control is generally guaranteed by thermal treatments that however can affect organoleptic and nutritional properties. A strategy to reduce thermal damage is the combination of mild treatments with natural antimicrobials. This work studied the effect of citral on thermal inactivation of a Saccharomyces cerevisiae strain isolated from spoiled beverages, also in relation to medium pH, through plate counting and flow cytometry (FCM), to assess cell viability and membrane integrity. The results of treatments at 60 °C for 30 min showed that sublethal concentrations of citral increased death kinetics, especially at lower pH. Conversely, FCM analysis performed on cells treated at 60 °C for 10 min evidenced higher mortality at pH 6.0, while at pH 4.0 the population was mainly injured. This different behavior was likely due to higher membrane permeability observed at pH 6.0. No recovery of injured cells to a viable status was observed after 3 h of incubation. Since the fate of these cells in foods is a great concern, these results are helpful to increase knowledge about the physiological response of S. cerevisiae and to optimize strategies for food stability.

Enterococci isolated from different sites of an urban wastewater treatment plant (consisting of three horizontal subsurface flow constructed wetlands) were investigated. One-hundred-thirty isolates were identified at species level and tested for resistance to eleven antibiotics, by microdilution method, and their clonal relatedness was established by SmaI-PFGE analysis. Results highlighted the persistence of enterococcal population in all effluents and the dominance of E. faecium species. A high incidence of antibiotic resistance against erythromycin, chloramphenicol, rifampicin and ampicillin was observed, with 120 strains (93%) showing a multi-drug-resistance. Numerous pulso-types with a unique pattern were detected indicating a high diversity within enterococcal population. The recurrence of some pulso-types in different effluents was disclosed and, within the same pulso-types, different resistance patterns were observed. Comparing the MIC values of strains from inlet and outlet, different trends were observed, highlighting a certain variability among constructed wetlands in affecting the antibiotic resistance among enterococcal population.

Exopolysaccharides production by 3 ropy strains of Lactobacillus delbrueckii subsp. bulgaricus of dairy origin was evaluated in synthetic medium by combining different approaches: impedometric measurements, fluorescent microscopy and flow cytometry analyses. The evaluation of ΔE by impedometric measurement (E%max-E%40h) allowed the detection of EPS production in synthetic medium, but the differences in EPS production kinetic was highlighted by flow cytometry analysis and fluorescent microcopy. This approach enabled us to unravel the diversity in EPS synthesis and release into the laboratory medium during the growth of the strains. Our results showed that the maximum EPS production occurred after 8 h of incubation, when cells were in late exponential growth phase. Furthermore, flow cytometry analysis revealed that only part of the cell population could be identified as EPS producer or as EPS-bounded cell. Therefore, the combined approach used, allowed us to define at the same time the kinetics of EPS production and release by three strains belonging to the same species and, highlight that the production of EPS depends also on the number of EPS-producing cells within the same population. This approach could be useful for the selection of strains to be used as starter cultures in dairy products where EPS production is considered an important feature.

We appreciate the intent by Salminen et al. to clarify and reorganize the nomenclature regarding the use of inactivated bacteria and their products as health-promoting factors (Salminen, S. et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 18, 649–667 (2021))1. Nonetheless, we found several major caveats in the consensus statement (Box 1) that might generate ambiguity.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B–positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. Significance: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients. See related commentary by Sharma, p. 1937

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., This study aims to assess whether a lactobacillus of vaginal origin can benefit the eubosis of the vaginal mucosa of childbearing age woman upon oral intake.

The combined effect of thymol, carvacrol and mild heat treatments against Listeria monocytogenes Scott A was assessed in buffered system by plate counting and flow cytometry (FCM). The susceptibility of cells increased when heat treatment was combined with terpenes. The data modelling with Bigelow and Weibull equations showed that the latter was more appropriate to describe the inactivation kinetics. After treatments, cells were no longer able to form colonies on plates; nevertheless, FCM indicated that most of the cells were damaged rather than dead. Treated cells were not able to recover the damage after 6 h. This opens the question if they could recover in a food matrix. FCM can be a helpful technique to better comprehend the physiological state of microorganisms. In the perspective of industrial applications, studies based on predictive microbiology, as well as a deeper comprehension of the action mechanisms of antimicrobials, play a key role for process optimization.

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. 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. 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. 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.

In the present study, 110 enterococci were isolated from two Sicilian cheese types, Ragusano PDO and Pecorino Siciliano. Isolates, firstly identified by MALDI-TOF/MS and a multiplex PCR assay, were tested for susceptibility to the most relevant clinical antibiotics. Clonal relationships among isolates were evaluated by pulsed-field-gel electrophoresis (PFGE) analysis and the presence of vanA and vanB genes, in vancomycin resistant enterococci (VRE), was investigated. Overall, E. faecalis, E. durans (35% for each species) and E. faecium (28%) were the major identified species. Different occurrence between cheese types was revealed. Most isolates from Ragusano PDO cheese were identified as E. durans (46%) and/or E. faecalis (43%), while E. faecium (605) was mainly detected in Pecorino Siciliano cheese. High incidence of resistance (97% of total strains) was detected for rifampicin, erythromycin and ampicillin. Moreover, 83 isolates (75%) exhibited multidrug-resistant phenotypes and the one VRE (vanB) isolate was identified as E. durans. PFGE analysis clustered isolates into 22 genotypes and the presence of the same PFGE types, for both E. durans and E. faecalis, in the two cheese types, suggest the link between enterococci and geographical area of production. Results of present study raise concerns about possible role of dairy enterococci as reservoirs of antibiotic resistance.

Aims To characterize airborne virus-like particles isolated from two cheese production plants in order to reveal their complexity in terms of viral communities and microbial genes potentially mobilized by viruses. Methods and results Airborne virus-like particles have been isolated from Grana Padano and Gorgonzola PDO cheese production plants and ripening cellars. A shotgun metagenomics analysis of the isolated viromes highlighted a high complexity of the viral communities both in terms of viral taxonomy and phage-host associations. Bacterial reads in each of the viromes were confirmed to be abundant and their taxonomy appeared to be associated with the environmental parameters and the technological processes that characterize the sampling area. Antibiotic resistance genes have been identified in each virome thus confirming that phages could be involved in the mobilization of antimicrobial resistances among bacterial populations. Interestingly human viruses were also identified even if the contamination source was not revealed. Conclusions The environmental conditions, which are imposed by the technology of the dairy process, seam to shape the viral populations as a consequence of the adaptation of microbial taxa to those environments. The identification of sequences belonging to Legionella pneumophila and to the human papillomavirus, raised some considerations about the safety of cheese-ripening cellars. Significance and impact of the study In conclusion, the analysis of the dairy airborne viromes, has revealed a high complexity of the viral communities even if the environments where the samples were collected were confined environments. Metagenomics of airborne viral population could be a promising monitoring tool for the biological characterization of dairy environments.

Plant polyphenolic compounds are considered a promising source for new antibacterial agents. In this study, we evaluated the antimicrobial activity of a collection of resveratrol-derived monomers and dimers screened as single molecules against a panel of nine foodborne pathogens. The results demonstrated that two monomers (i.e., pterostilbene 2 and (E)-3-hydroxy-4′,5-dimethoxystilbene 9) and three dimers (i.e., δ-viniferin 10, viniferifuran 14 and dehydro-δ-viniferin 15) were endowed with significant antibacterial activity against gram-positive bacteria. The exposure of gram-positive foodborne pathogens to 100 µg/mL of 2, 9 and 15 induced severe cell membrane damage, resulting in the disruption of the phospholipid bilayer. The most promising dimeric compound, dehydro-δ-viniferin 15, was tested against Listeria monocytogenes, resulting in a loss of cultivability, viability and cell membrane potential. TEM analysis revealed grave morphological modifications on the cell membrane and leakage of intracellular content, confirming that the cell membrane was the principal biological target of the tested derivative.

The growing commercial interest in multi-strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi-strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single-cell analysis was used to evaluate the cell viability, and beta-galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi-strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production. IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

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. Electronic supplementary material The online version of this article (10.1007/s00394-018-1860-5) contains supplementary material, which is available to authorized users.

Summary: Pulmonary immunological tolerance to inhaled particulates might create a permissive milieu for lung metastasis. Lung microbiota contribute to pulmonary tolerance; here, we explored whether its manipulation via antibiotic or probiotic aerosolization favors immune response against melanoma metastasis. In lungs of vancomycin/neomycin-aerosolized mice, a decrease in bacterial load was associated with reduced regulatory T cells and enhanced T cell and NK cell activation that paralleled a significant reduction of melanoma B16 lung metastases. Reduction of metastases also occurred in lungs transplanted with bacterial isolates from antibiotic-treated lungs. Aerosolized Lactobacillus rhamnosus strongly promoted immunity against B16 lung metastases as well. Furthermore, probiotics or antibiotics improved chemotherapy activity against advanced B16 metastases. Thus, we identify a role for lung microbiota in metastasis and show that its targeting via aerosolization is a therapy that can prevent metastases and enhance responses to chemotherapy. : Le Noci et al. reveal that modulation of pulmonary microbiota by antibiotic or probiotic aerosolization decreases tumor growth in the lung. Antibiotic treatment induces a reduction of immunosuppressive cells in the lung, while probiotic administration promotes maturation of resident antigen-presenting cells. Keywords: lung microbiota, cancer immunosurveillance, aerosolization, antibiotics, probiotics, mouse models, immunosuppression and/or suppressor cells

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity. Moreover, the stability of this production system was ascertained by up to 7 days of continuous working. These results suggest the suitability of the proposed method for a large-scale production of the desired FOS mixture, in view of a foreseeable use as a novel prebiotic preparation.