Personalized microbiome-driven effects of non-nutritive sweeteners on human glucose tolerance.

Non-nutritive sweeteners (NNS) are commonly integrated into human diet and presumed to be inert; however, animal studies suggest that they may impact the microbiome and downstream glycemic responses. We causally assessed NNS impacts in humans and their microbiomes in a randomized-controlled trial encompassing 120 healthy adults, administered saccharin, sucralose, aspartame, and stevia sachets for 2 weeks in doses lower than the acceptable daily intake, compared with controls receiving sachet-contained vehicle glucose or no supplement. As groups, each administered NNS distinctly altered stool and oral microbiome and plasma metabolome, whereas saccharin and sucralose significantly impaired glycemic responses. Importantly, gnotobiotic mice conventionalized with microbiomes from multiple top and bottom responders of each of the four NNS-supplemented groups featured glycemic responses largely reflecting those noted in respective human donors, which were preempted by distinct microbial signals, as exemplified by sucralose. Collectively, human NNS consumption may induce person-specific, microbiome-dependent glycemic alterations, necessitating future assessment of clinical implications.

Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation.

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.

Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features.

Empiric probiotics are commonly consumed by healthy individuals as means of life quality improvement and disease prevention. However, evidence of probiotic gut mucosal colonization efficacy remains sparse and controversial. We metagenomically characterized the murine and human mucosal-associated gastrointestinal microbiome and found it to only partially correlate with stool microbiome. A sequential invasive multi-omics measurement at baseline and during consumption of an 11-strain probiotic combination or placebo demonstrated that probiotics remain viable upon gastrointestinal passage. In colonized, but not germ-free mice, probiotics encountered a marked mucosal colonization resistance. In contrast, humans featured person-, region- and strain-specific mucosal colonization patterns, hallmarked by predictive baseline host and microbiome features, but indistinguishable by probiotics presence in stool. Consequently, probiotics induced a transient, individualized impact on mucosal community structure and gut transcriptome. Collectively, empiric probiotics supplementation may be limited in universally and persistently impacting the gut mucosa, meriting development of new personalized probiotic approaches.

Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by Autologous FMT.

Probiotics are widely prescribed for prevention of antibiotics-associated dysbiosis and related adverse effects. However, probiotic impact on post-antibiotic reconstitution of the gut mucosal host-microbiome niche remains elusive. We invasively examined the effects of multi-strain probiotics or autologous fecal microbiome transplantation (aFMT) on post-antibiotic reconstitution of the murine and human mucosal microbiome niche. Contrary to homeostasis, antibiotic perturbation enhanced probiotics colonization in the human mucosa but only mildly improved colonization in mice. Compared to spontaneous post-antibiotic recovery, probiotics induced a markedly delayed and persistently incomplete indigenous stool/mucosal microbiome reconstitution and host transcriptome recovery toward homeostatic configuration, while aFMT induced a rapid and near-complete recovery within days of administration. In vitro, Lactobacillus-secreted soluble factors contributed to probiotics-induced microbiome inhibition. Collectively, potential post-antibiotic probiotic benefits may be offset by a compromised gut mucosal recovery, highlighting a need of developing aFMT or personalized probiotic approaches achieving mucosal protection without compromising microbiome recolonization in the antibiotics-perturbed host.

Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations.

The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.

The microbiome in anti-cancer therapy.

The commensal microbiome constitutes an important modulator of host physiology and risk of disease, including cancer development and progression. Lately, the microbiome has been suggested to modulate the efficacy of anti-cancer treatment. Examples include chemotherapy and total body irradiation-induced barrier function disruption, leading to microbial efflux that drives activation of anti-tumorigenic T cells; Microbiome-driven release of reactive oxygen species contributing to the efficacy of platinum salts; and microbiome-induced immune priming promoting the anti-tumor effects of alkylating chemotherapy and immune checkpoint inhibitors. Furthermore, selected commensals are able to colonize solid tumors. This 'tumor microbiome' may further impact local tumor responses to treatment and potentially be harnessed for tumor-specific targeting and therapeutic delivery. In this review, we present recent advances in understanding of the intricate role of microbiome in modulating efficacy of a number of anti-cancer treatments, and discuss how anti-cancer treatment approaches utilizing the tumor microbiome may enhance oncological treatment efficacy.

Lactobacillus spp. inhibit the growth of Cronobacter sakazakii ATCC 29544 by altering its membrane integrity.

The effect of the cell-free culture supernatants (CFCSs) from different Lacobacillus spp. on growth ability of Cronobacter sakazakii ATCC 29544 was investigated by time-killing studies. The antimicrobial effect was evaluated using crude and 2.5 × concentrated CFCSs. Most of the CFCSs showed a dose-dependent antimicrobial activity, with the greatest C. sakazakii growth inhibition exerted by the CFCS 2.5 × of Lactobacillus casei rhamnosus ATCC 7469. Indeed, C. sakazakii growth was completely inhibited after 4 h of incubation with the crude CFCSs of L. casei rhamnosus and Lactobacillus acidophilus and after only 2 h using the related 2.5 × CFCSs. The flow cytometric analysis revealed that CFCSs altered the permeability of C. sakazakii cell membrane, showing 55% of live cells after 30 min of treatment with 2.5 × CFCSs of L. casei rhamnosus and L. acidophilus, reaching 1% of live cells after 2 h of exposure. The CFCSs of L. casei rhamnosus and L. acidophilus have showed anti-Cronobacter activity, determining a progressively inhibition of C. sakazakii growth as result of alterations in its membrane permeability.

Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams.

Vibrio parahaemolyticus is part of the natural microflora of estuarine and coastal marine waters and can be also present in seafood, especially shellfish and bivalve molluscs. In this study we compared the reference cultural method ISO 6887-3 with two molecular methods, multiplex PCR and real-time PCR, for the detection of two distinct genetic markers (tlh species-specific gene and tdh virulence gene) of V. parahaemolyticus in bivalve mollusc. The analyses were performed on clams inoculated with V. parahaemolyticus ATCC 43996 at T0 and after a 3 and 6 h of pre-enrichment in alkaline saline peptone water. Counts on agar plates were largely inaccurate, probably due to other Vibrio species grown on the TCBS selective agar. Multiplex PCR assays, performed using primers pairs for tdh and tlh genes, showed a detection limit of 104 CFU/g of shell stock within 6 h of pre-enrichment, respecting however the action level indicated by the National Seafood Sanitation Program guideline. Detection by tdh gene in real-time PCR reached the definitely highest sensitivity in shorter times, 101 CFU/g after 3 h of pre-enrichment, while the sensitivity for the tlh gene was not promising, detecting between 105 and 106 CFU/g after 6 h of pre-enrichment. Our findings provide a rapid routine method of detection of V. parahaemolyticus based on tdh gene by real-time PCR for commercial seafood analysis to identify the risk of gastrointestinal diseases.

In vitro activity of Carvacrol against titanium-adherent oral biofilms and planktonic cultures.

OBJECTIVE: The aim of this study was to test the effect of Carvacrol against oral pathogens and their preformed biofilms on titanium disc surface. METHODS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm inhibitory concentration (BIC) were performed to evaluate Carvacrol antibacterial activity, while flow cytometry (FCM) was used to verify the Carvacrol effect on esterase activity and membrane permeability. Carvacrol was tested in vitro on single- and multi-species biofilms formed on titanium disc by Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277 or Fusobacterium nucleatum ATCC 25586, in different combinations, comparing its effect to that of chlorhexidine. RESULTS: The pathogens were sensitive to Carvacrol with MICs and MBCs values of 0.25 % and 0.50 % and BICs of 0.5 % for S. mutans ATCC 25175 and 1 % for P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586. FCM analysis showed that treatment of planktonic cultures with Carvacrol caused an increase of damaged cells and a decrement of bacteria with active esterase activity. Moreover, Carvacrol demonstrated greater biofilm formation preventive property compared to chlorhexidine against titanium-adherent single- and multi-specie biofilms, with statistically significant values. CONCLUSIONS: Carvacrol showed inhibitory activity against the tested oral pathogens and biofilm formation preventive property on their oral biofilm; then, it could be utilized to control and prevent the colonization of microorganisms with particular significance in human oral diseases. CLINICAL RELEVANCE: This natural compound may be proposed in daily hygiene formulations or as an alternative agent supporting traditional antimicrobial protocols to prevent periodontal diseases in implanted patients.

CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state.

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46-48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.

Phage therapy in noncommunicable diseases.

Bacteriophages have potential as suppressors of disease-contributing commensal bacteria.

Microbiome-phage interactions in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) constitutes a group of auto-inflammatory disorders that impact the gastrointestinal tract and other systemic organs. The gut microbiome contributes to IBD pathology through multiple mechanisms. Bacteriophages (hereafter termed phages) are viruses that are able to specifically infect bacteria. Considered as part of the gut microbiome, phages may impact the bacterial community structure in various clinical contexts. Additionally, exogenous phage administration may represent a means of suppressing IBD-associated pathobionts; however, the utilization of phage therapy remains at an early developmental phase. OBJECTIVES: Herein, we summarize the latest advances in understanding endogenous phage impacts on the gut microbiome in a healthy gut and in IBD. We highlight the prospect of phage utilization as a targeted mode of pathobiont eradication, for preventing and treating IBD manifestations and complications. SOURCES: Selected peer-reviewed publications regarding the role of phages in a healthy gut and in IBD, published between 2013 and 2022. CONTENT: The human gut microbiome is increasingly suggested to play a significant role in the onset and progression of multiple non-communicable diseases such as IBD. Several studies suggest that this effect may be mediated by discrete disease-contributing commensals. However, the eradication of such pathogenic bacteria remains a daunting unmet task. Altered community structure in IBD may be influenced by blooms of phages within the gut bacterial ecosystem. Moreover, combinations of phages specifically targeting disease-contributing pathobiont strain clades may be harnessed as potential eradication treatment preventing and treating IBD, while bearing minimal adverse impacts on the surrounding bacterial microbiome. IMPLICATIONS: Understanding the endogenous phage-gut commensal interactions in a healthy gut and in IBD may enable phage utilization in precision gut microbiome editing, towards treating IBD and other non-communicable microbiome-associated diseases. Nevertheless, developing phage combination-mediated IBD pathobiont eradication treatment modalities will likely necessitate better strain-level bacterial target identification and resolution of treatment-related challenges, such as phage delivery, off-target effects, and bacterial resistance.

Antagonistic activity of Lactobacillus acidophilus ATCC 4356 on the growth and adhesion/invasion characteristics of human Campylobacter jejuni.

The aim of this research was to determine the potential probiotic activity of Lactobacillus acidophilus ATCC 4356 against several human Campylobacter jejuni isolates. The ability to inhibit the pathogen's growth was evaluated by co-culture experiments as well as by antimicrobial assays with cell-free culture supernatant (CFCS), while interference with adhesion/invasion to intestinal Caco-2 cells was studied by exclusion, competition, and displacement tests. In the co-culture experiments L. acidophilus ATCC 4356 strain reduced the growth of C. jejuni with variable percentages of inhibition related to the contact time. The CFCS showed inhibitory activity against C. jejuni strains, stability to low pH, and thermal treatment and sensitivity to proteinase K and trypsin. L. acidophilus ATCC 4356 was able to reduce the adhesion and invasion to Caco-2 cells by most of the human C. jejuni strains. Displacement and exclusion mechanisms seem to be the preferred modalities, which caused a significant reduction of adhesion/invasion of pathogens to intestinal cells. The observed inhibitory properties of L. acidophilus ATCC 4356 on growth ability and on cells adhesion/invasion of C. jejuni may offer potential use of this strain for the management of Campylobacter infections.

Uniportal VATS S9 segmentectomy: The ligamentum-based approach.

Isolated resection of the lateral-basal segment (S9) is uncommon, and it is considered one of the most complex anatomic segmentectomies. First, the segmental arterial and venous supply is located deeply in the lung parenchyma, making the dissection difficult. Second, the cuboidal shape of the lateral basilar segment hampers the identification of the intersegmental plane. Although identifying the segmental arterial branches is easier from a fissure-based technique, the ligamentum-based approach emerges as a valid and safe alternative in cases of a fused fissure.

Phages and their potential to modulate the microbiome and immunity.

Bacteriophages (hence termed phages) are viruses that target bacteria and have long been considered as potential future treatments against antibiotic-resistant bacterial infection. However, the molecular nature of phage interactions with bacteria and the human host has remained elusive for decades, limiting their therapeutic application. While many phages and their functional repertoires remain unknown, the advent of next-generation sequencing has increasingly enabled researchers to decode new lytic and lysogenic mechanisms by which they attack and destroy bacteria. Furthermore, the last decade has witnessed a renewed interest in the utilization of phages as therapeutic vectors and as a means of targeting pathogenic or commensal bacteria or inducing immunomodulation. Importantly, the narrow host range, immense antibacterial repertoire, and ease of manipulating phages may potentially allow for their use as targeted modulators of pathogenic, commensal and pathobiont members of the microbiome, thereby impacting mammalian physiology and immunity along mucosal surfaces in health and in microbiome-associated diseases. In this review, we aim to highlight recent advances in phage biology and how a mechanistic understanding of phage-bacteria-host interactions may facilitate the development of novel phage-based therapeutics. We provide an overview of the challenges of the therapeutic use of phages and how these could be addressed for future use of phages as specific modulators of the human microbiome in a variety of infectious and noncommunicable human diseases.

Outcome of parapneumonic empyema managed surgically or by fibrinolysis: a multicenter study.

BACKGROUND: Parapneumonic empyema (PPE) management remains debated. Here we present the outcome of a comparable population with PPE treated over a 4-year period in two Thoracic Surgery University Centers with different approaches: one with an early "surgical" and the other with a "fibrinolytic" approach. METHODS: All operable patients with PPE managed in both centers between January 2014 and January 2018 were reviewed. Patients with persistent pleural effusion/loculations following drainage were managed by a "surgical" approach in one center and by "fibrinolytic" approach in the other. For each patient, we recorded the age, sex, hospital stay, morbidity/mortality and change in pleural opacity on chest X-ray before and at the end of the treatment. RESULTS: During the study period, 66 and 93 patients underwent PPE management in the "surgical" and "fibrinolytic" centers respectively. The population characteristics were comparable. Infection was controlled in all patients. In the "fibrinolytic" group, 20 patients (21.5%) underwent an additional drain placement while 12 patients (12.9%) required surgery to correct PPE. In the "surgical" group, 4 patients (6.1%) developed postoperative arrhythmia while 2 patients (3%) underwent a second surgery to evacuate a hemothorax. Median drainage {3 [2-4] vs. 5 [4-7] days} and hospital {7 [5-10] vs. 11 [7-19] days} durations were significantly lower in the "surgical" compared to the "fibrinolytic" center. Pleural opacity regression with therapy was significantly more important in the "surgical" compared to the "fibrinolytic" group (-22%±18% vs. -16%±17%, P=0.035). CONCLUSIONS: Surgical management of PPE was associated with shorter chest tube and hospital duration and better pleural space control. Prospective randomized studies are mandatory.

Our Microbiome: On the Challenges, Promises, and Hype.

The microbiome field is increasingly raising interest among scientists, clinicians, biopharmaceutical entities, and the general public. Technological advances from the past two decades have enabled the rapid expansion of our ability to characterize the human microbiome in depth, highlighting its previously underappreciated role in contributing to multifactorial diseases including those with unknown etiology. Consequently, there is growing evidence that the microbiome could be utilized in medical diagnosis and patient stratification. Moreover, multiple gut microbes and their metabolic products may be bioactive, thereby serving as future potential microbiome-targeting or -associated therapeutics. Such therapies could include new generation probiotics, prebiotics, fecal microbiota transplantations, postbiotics, and dietary modulators. However, microbiome research has also been associated with significant limitations, technical and conceptual challenges, and, at times, "over-hyped" expectations that microbiome research will produce quick solutions to chronic and mechanistically complex human disorders. Herein, we summarize these challenges and also discuss some of the realistic promises associated with microbiome research and its applicability into clinical application.

Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae.

This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae. The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of two-armed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes. Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.

Is repeated pulmonary metastasectomy justified?

Recurrence after pulmonary metastasectomy (PM) is frequent, but it is unclear to whom repeated pulmonary metastasectomy (RPM) offers highest benefits. Retrospective analysis of oncological and post-operative outcomes of consecutive patients who underwent PM from 2003 to 2018. Overall survival (OS) and disease-free interval (DFI) were calculated. Cox regression was used to identify variables influencing OS and DFI. In total, 264 patients (female/male: 114/150; median age: 62 years) underwent PM for colorectal cancer (32%), sarcoma (19%), melanoma (16%) and other primary tumors (33%). Pulmonary metastasectomy was approached by video-assisted thoracic surgery (VATS) in 73% and pulmonary resection was realized by non-anatomical resection in 76% of cases. The overall median follow-up time was 33 months (IQR 16-56 months) and overall 5-year survival rate was 62%. Local or distant recurrences were observed in 172 patients (65%) and RPM could be performed in 66 patients (25%) for a total of 116 procedures. RPM was realized by VATS in 49% and pulmonary resection by wedge in 77% of cases. In RPM patients, the 5-year survival rate after first PM was 79%. Post-operative cardio-pulmonary complication rate (13% vs. 12%; p = 0.8) and median length of stay (4 vs. 5 days; p = 0.2) were not statistically different between first PM and RPM. Colorectal cancer (HR 0.56), metachronous metastasis (HR 0.48) and RPM (HR 0.5) were associated with better survival. In conclusion, our results suggest that RPM offers favorable survival rates without increasing post-operative morbidity.