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ObjectivesIntraductal papillary mucinous neoplasms (IPMNs) are pancreatic cysts that can progress to invasive pancreatic cancer. Associations between oncogenesis and oral microbiome alterations have been reported. This study aims to investigate a potential intracystic pancreatic microbiome in a pancreatic cystic neoplasm (PCN) surgery patient cohort.DesignPaired cyst fluid and plasma were collected at pancreatic surgery from patients with suspected PCN (n=105). Quantitative and qualitative assessment of bacterial DNA by qPCR, PacBio sequencing (n=35), and interleukin (IL)-1β quantification was performed. The data were correlated to diagnosis, lesion severity and clinical and laboratory profile, including proton-pump inhibitor (PPI) usage and history of invasive endoscopy procedures.ResultsIntracystic bacterial 16S DNA copy number and IL-1β protein quantity were significantly higher in IPMN with high-grade dysplasia and IPMN with cancer compared with non-IPMN PCNs. Despite high interpersonal variation of intracystic microbiota composition, bacterial network and linear discriminant analysis effect size analyses demonstrated co-occurrence and enrichment of oral bacterial taxa including Fusobacterium nucleatum and Granulicatella adiacens in cyst fluid from IPMN with high-grade dysplasia. The elevated intracystic bacterial DNA is associated with, but not limited to, prior exposure to invasive endoscopic procedures, and is independent from use of PPI and antibiotics.ConclusionsCollectively, these findings warrant further investigation into the role of oral bacteria in cystic precursors to pancreatic cancer and have added values on the aetiopathology as well as the management of pancreatic cysts.

Pancreatic cystic neoplasms (PCNs) are a highly prevalent disease of the pancreas. Among PCNs, Intraductal Papillary Mucinous Neoplasms (IPMNs) are common lesions that may progress from low-grade dysplasia (LGD) through high-grade dysplasia (HGD) to invasive cancer. Accurate discrimination of IPMN-associated neoplastic grade is an unmet clinical need. Targeted (semi)quantitative analysis of 100 metabolites and >1000 lipid species were performed on peri-operative pancreatic cyst fluid and pre-operative plasma from IPMN and serous cystic neoplasm (SCN) patients in a pancreas resection cohort (n = 35). Profiles were correlated against histological diagnosis and clinical parameters after correction for confounding factors. Integrated data modeling was used for group classification and selection of the best explanatory molecules. Over 1000 different compounds were identified in plasma and cyst fluid. IPMN profiles showed significant lipid pathway alterations compared to SCN. Integrated data modeling discriminated between IPMN and SCN with 100% accuracy and distinguished IPMN LGD or IPMN HGD and invasive cancer with up to 90.06% accuracy. Free fatty acids, ceramides, and triacylglycerol classes in plasma correlated with circulating levels of CA19-9, albumin and bilirubin. Integrated metabolomic and lipidomic analysis of plasma or cyst fluid can improve discrimination of IPMN from SCN and within PMNs predict the grade of dysplasia.

Opportunistic bacteria in apical periodontitis (AP) may pose a risk for systemic dissemination. Mucosal-associated invariant T (MAIT) cells are innate-like T cells with a broad and potent antimicrobial activity important for gut mucosal integrity. It was recently shown that MAIT cells are present in the oral mucosal tissue, but the involvement of MAIT cells in AP is unknown. Here, comparison of surgically resected AP and gingival tissues demonstrated that AP tissues express significantly higher levels of Vα7.2-Jα33, Vα7.2-Jα20, Vα7.2-Jα12, Cα and tumour necrosis factor (TNF), interferon (IFN)-γ and interleukin (IL)-17A transcripts, resembling a MAIT cell signature. Moreover, in AP tissues the MR1-restricted MAIT cells positive for MR1–5-OP-RU tetramer staining appeared to be of similar levels as in peripheral blood but consisted mainly of CD4+ subset. Unlike gingival tissues, the AP microbiome was quantitatively impacted by factors like fistula and high patient age and had a prominent riboflavin-expressing bacterial feature. When merged in an integrated view, the examined immune and microbiome data in the sparse partial least squares discriminant analysis could identify bacterial relative abundances that negatively correlated with Vα7.2-Jα33, Cα, and IL-17A transcript expressions in AP, implying that MAIT cells could play a role in the local defence at the oral tissue barrier. In conclusion, we describe the presence of MAIT cells at the oral site where translocation of oral microbiota could take place. These findings have implications for understanding the immune sensing of polymicrobial-related oral diseases.

Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.

ABSTRACT We report the draft whole-genome sequence of Rothia nasimurium isolated from a porcine tonsil. The genome encodes a nonribosomal peptide synthetase predicted to produce valinomycin, a cyclic dodecadepsipeptide ionophore. Previously, valinomycin was known to be produced only by Streptomyces species and isolates belonging to the Bacillus pumilus group.

Increasing evidence suggests that the gut microbiome may modulate the efficacy of cancer immunotherapy. In a B cell lymphoma patient cohort from five centers in Germany and the United States (Germany, n  = 66; United States, n  = 106; total, n  = 172), we demonstrate that wide-spectrum antibiotics treatment (‘high-risk antibiotics’) prior to CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is associated with adverse outcomes, but this effect is likely to be confounded by an increased pretreatment tumor burden and systemic inflammation in patients pretreated with high-risk antibiotics. To resolve this confounding effect and gain insights into antibiotics-masked microbiome signals impacting CAR-T efficacy, we focused on the high-risk antibiotics non-exposed patient population. Indeed, in these patients, significant correlations were noted between pre-CAR-T infusion Bifidobacterium longum and microbiome-encoded peptidoglycan biosynthesis, and CAR-T treatment-associated 6-month survival or lymphoma progression. Furthermore, predictive pre-CAR-T treatment microbiome-based machine learning algorithms trained on the high-risk antibiotics non-exposed German cohort and validated by the respective US cohort robustly segregated long-term responders from non-responders. Bacteroides , Ruminococcus , Eubacterium and Akkermansia were most important in determining CAR-T responsiveness, with Akkermansia also being associated with pre-infusion peripheral T cell levels in these patients. Collectively, we identify conserved microbiome features across clinical and geographical variations, which may enable cross-cohort microbiome-based predictions of outcomes in CAR-T cell immunotherapy. Conserved microbiome features across clinical and geographical variations may enable microbiome-based predictions of outcomes in CD19-targeted CAR-T cell immunotherapy

Background Epstein-Barr virus and Cytomegalovirus reactivations frequently occur after allogeneic stem cell transplantation (SCT). Methods Here we investigated the role of immune cell reconstitution in the onset and subsequent severity of EBV- and CMV-reactivation. To this end, 116 patients were prospectively sampled for absolute T cell (CD4 and CD8), B-cell (CD19) and NK-cell (CD16 and CD56) numbers weekly post-SCT during the first 3 months and thereafter monthly until 6 months post-SCT. Viral load was monitored in parallel. Results In contrast to the general belief, we found that early T-cell reconstitution does not play a role in the onset of viral reactivation. CMV reactivation in the first 7 weeks after SCT however resulted in higher absolute CD8 + T-cell numbers 6 months post-SCT in patients with high-level reactivation, many of which were CMV-specific. Interestingly, rapid reconstitution of CD4 + T-cells, as well as NK cells and the presence of donor KIR3DL1, are associated with the absence of CMV-reactivation after SCT, suggestive of a protective role of these cells. In contrast, EBV-reactivations were not affected in any way by the level of immune reconstitution after SCT. Conclusion In conclusion, these data suggest that CD4 + T-cells and NK cells, rather than CD8 + T-cells, are associated with protection against CMV-reactivation.

The increasing prevalence of antibiotic resistance in pathogenic bacteria and the potential future implications for human and animal morbidity and mortality, health-care costs and economic losses pose an urgent worldwide problem. As a result, exploration of alternative strategies to combat antibiotic resistant bacteria have intensified over the last decades. The use of naturally occurring antimicrobial peptides (AMPs) has received considerable scientific and commercial interest due to their antimicrobial activity and plethora of possible therapeutic applications. The work described in this thesis focused on the study of AMPs and other bioactive molecules produced by bacteria as potential alternatives to prevent or treat infections with pathogenic bacteria. In the first part of the thesis, the antimicrobial potency of eukaryotic AMPs and the responses they elicit in bacteria were studied. Chapter 2 described the potential for a selection of frog skin AMPs and analogues for development into novel topical therapeutics to treat skin infections caused by antibiotic resistant bacteria. We showed that several natural AMPs and analogues displayed promising growth-inhibiting activity against multi-drug resistant pathogenic bacteria whereas these AMPs or analogues did not inhibit growth of probiotic or commensal strains. In Chapter 3 the generic and adaptive resistance mechanisms in Streptococcus suis upon exposure to sub-lethal concentrations of a model cationic AMP, the human cathelicidin LL-37, were studied. We found significantly altered expression of genes associated with two-component signalling, membrane transport and carbohydrate uptake, as well as upregulation of genes involved in adhesion, pilus formation, and resistance against bacteriocins or small peptides. The second part of this thesis aimed to increase knowledge about the role of the porcine microbiota in S. suis abundance, with the purpose to identify commensal bacteria that displayed strong and selective antagonism against this pathogen, possibly via the production of AMPs. In Chapter 4 we identified bacterial taxa that show strong positive or negative correlations with the abundance of S. suis and other porcine pathogens. The results indicated that colonisation by S. suis was correlated with a group of other opportunist pathogens, including species belonging to the genera Actinobacillus, Actinomyces, Pasteurella and Streptococcus which are are known to cause respiratory disease. The results also revealed that the microbiota from the tonsil or small intestine contain bacteria that might display (in) direct antagonism against S. suis. Based on this result, we screened the tonsillar and small intestinal microbiota for isolates that could specifically inhibit the growth of S. suis. Chapter 5 described the isolation and identification of a Staphylococcus pasteuri isolate producing two α-helical peptides that possess narrow-spectrum antimicrobial activity against multiple serotypes of S. suis. In Chapter 6 we characterized a Rothia nasimurium strain from the palatine tonsil that produces the non-ribosomally synthesized ionophore antibiotic valinomycin, and investigated the effects of this cyclic peptide on other bacteria and host cells. Chapter 7 summarizes and discusses the key results of the research described in this thesis and describes possible directions for future research. This thesis increased the understanding of the role of host- and microbiota-derived biologically active small molecules in microbe-microbe and microbe-host interplay. Such knowledge may contribute to the development of novel therapeutic solutions to treat antibiotic resistant bacteria, such as beneficial microbial communities (i.e. next-generation probiotics) or biotechnological applications of natural or modified AMPs.