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The host-microbiome axis has been implicated in promoting anti-inflammatory immune responses. Yet, the underlying molecular mechanisms of commensal-mediated IL-10 production by regulatory B cells (Bregs) are not fully elucidated. Here, we demonstrate that bacterial CpG motifs trigger the signaling downstream of TLR9 promoting IκB NS -mediated expression of Blimp-1, a transcription regulator of IL-10. Surprisingly, this effect was counteracted by the NF-κB transcription factor c-Rel. A functional screen for intestinal bacterial species identified the commensal Clostridium sporogenes, secreting high amounts of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs), as an amplifier of IL-10 production by promoting sustained mTOR signaling in B cells. Consequently, enhanced Breg functionality was achieved by combining CpG with the SCFA butyrate or the BCFA isovalerate thereby synergizing TLR- and mTOR-mediated pathways. Collectively, Bregs required two bacterial signals (butyrate and CpG) to elicit their full suppressive capacity and ameliorate T cell-mediated intestinal inflammation. Our study has dissected the molecular pathways induced by bacterial factors, which might contribute not only to better understanding of host-microbiome interactions, but also to exploration of new strategies for improvement of anti-inflammatory cellular therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lot release testing of diphtheria, tetanus and acellular pertussis vaccines traditionally relied on in vivo protection models involving challenge of laboratory animals with toxins. Meanwhile, many labs have switched to serological testing of these vaccines, which is often performed in separate in vivo assays, even if all components were formulated into one vaccine product. Here we describe the results of simultaneous serological potency determination of diphtheria (D), tetanus (T) and acellular pertussis (aP) antigens obtained following immunization of guinea pigs with multicomponent pediatric and booster vaccines from different manufacturers. The 4th World Health Organization (WHO) International Standard (IS) for diphtheria toxoid (No. 07/216) and the 4th WHO IS for tetanus toxoid (No. 08/218) were used as reference preparations. For aP, a pediatric vaccine batch containing the antigens pertussis toxoid, filamentous hemagglutinin, pertactin and fimbriae proteins type 2/3 was established as internal control. Quantification of IgG against D, T and aP antigens in guinea pig sera was performed using a hexaplex electrochemiluminescence immunoassay. We further provide proof-of-concept using experimental vaccine samples lacking or containing reduced amounts of diphtheria toxoid in the presence of full amounts of tetanus and pertussis antigens and alum adjuvant. Importantly, the assay confirmed dose-response relationships for all antigens tested and was able to detect diphtheria out-of-specification batches. The results confirmed the suitability of the protocol for combined serology batch release testing of DTaP combination vaccines as first measure towards implementation of full in vitro testing of DTaP vaccines. This report summarizes the data and the protocol used for validation prior to implementation of this method in routine batch release testing of DTaP vaccines, which led to replacement of in vivo challenge experiments in our laboratory following the 3 R (replace, reduce, refine) principle., (© 2024. The Author(s).)

Background and Objectives: Blood safety measures used by blood establishments to increase blood component safety can be validated using Transfusion-Relevant Bacterial Reference Strains (TRBRS). Ultra-cold storage conditions and manual preparation of the current TRBRS may restrict their practical use. To address this issue, the ISBT Transfusion-Transmitted Infectious Diseases Working Party's Bacterial Subgroup organized an international study to validate TRBRS in a user-friendly, lyophilised format., Materials and Methods: Two bacterial strains Klebsiella pneumoniae PEI-B-P-08 and Staphylococcus aureus PEI-B-P-63 were manufactured as lyophilised material. The lyophilised bacteria were distributed to 11 different labs worldwide to assess the robustness for enumeration, identification and determination of growth kinetics in platelet concentrates (PCs)., Results: Production of lyophilised TRBRS had no impact on the growth properties compared with the traditional format. The new format allows a direct low-quantity spiking of approximately 30 bacteria in PCs for transfusion-relevant experiments. In addition, the lyophilised bacteria exhibit long-term stability across a broad temperature range and can even be directly rehydrated in PCs without losing viability. Interlaboratory comparative study demonstrated the robustness of the new format as 100% of spiked PC exhibited growth., Conclusion: Lyophilised TRBRS provide a user-friendly material for transfusion-related studies. TRBRS in the new format have improved features that may lead to a more frequent use in the quality control of transfusion-related safety measures in the future., (© 2024 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Gut microbiota-derived metabolites play a pivotal role in the maintenance of intestinal immune homeostasis. Here, we demonstrate that the human commensal Clostridium sporogenes possesses a specific metabolic fingerprint, consisting predominantly of the tryptophan catabolite indole-3-propionic acid (IPA), the branched-chain acids (BCFAs) isobutyrate and isovalerate and the short-chain fatty acids (SCFAs) acetate and propionate. Mono-colonization of germ-free mice with C. sporogenes (CS mice) affected colonic mucosal immune cell phenotypes, including up-regulation of Il22 gene expression, and increased abundance of transcriptionally active colonic tuft cells and Foxp3 + regulatory T cells (Tregs). In DSS-induced colitis, conventional mice suffered severe inflammation accompanied by loss of colonic crypts. These symptoms were absent in CS mice. In conventional, but not CS mice, bulk RNAseq analysis of the colon revealed an increase in inflammatory and Th17-related gene signatures. C. sporogenes -derived IPA reduced IL-17A protein expression by suppressing mTOR activity and by altering ribosome-related pathways in Th17 cells. Additionally, BCFAs and SCFAs generated by C. sporogenes enhanced the activity of Tregs and increased the production of IL-22, which led to protection from colitis. Collectively, we identified C. sporogenes as a therapeutically relevant probiotic bacterium that might be employed in patients with inflammatory bowel disease (IBD).

The rise in antimicrobial resistance (AMR), and increase in treatment-refractory AMR infections, generates an urgent need to accelerate the discovery and development of novel anti-infectives. Preclinical animal models play a crucial role in assessing the efficacy of novel drugs, informing human dosing regimens and progressing drug candidates into the clinic. The Innovative Medicines Initiative-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium is establishing a validated and globally harmonized preclinical model to increase reproducibility and more reliably translate results from animals to humans. Toward this goal, in April 2021, COMBINE organized the expert workshop "Advancing toward a standardized murine model to evaluate treatments for AMR lung infections". This workshop explored the conduct and interpretation of mouse infection models, with presentations on PK/PD and efficacy studies of small molecule antibiotics, combination treatments (β-lactam/β-lactamase inhibitor), bacteriophage therapy, monoclonal antibodies and iron sequestering molecules, with a focus on the major Gram-negative AMR respiratory pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Here we summarize the factors of variability that we identified in murine lung infection models used for antimicrobial efficacy testing, as well as the workshop presentations, panel discussions and the survey results for the harmonization of key experimental parameters. The resulting recommendations for standard design parameters are presented in this document and will provide the basis for the development of a harmonized and bench-marked efficacy studies in preclinical murine pneumonia model., Competing Interests: JLH was employed by GlaxoSmithKline Pharmaceuticals. SS and SR were employed by Evotec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arrazuria, Kerscher, Huber, Hoover, Lundberg, Hansen, Sordello, Renard, Aranzana-Climent, Hughes, Gribbon, Friberg and Bekeredjian-Ding.)

Antimicrobial resistance has become one of the greatest threats to human health, and new antibacterial treatments are urgently needed. As a tool to develop novel therapies, animal models are essential to bridge the gap between preclinical and clinical research. However, despite common usage of in vivo models that mimic clinical infection, translational challenges remain high. Standardization of in vivo models is deemed necessary to improve the robustness and reproducibility of preclinical studies and thus translational research. The European Innovative Medicines Initiative (IMI)-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium, aims to develop a standardized, quality-controlled murine pneumonia model for preclinical efficacy testing of novel anti-infective candidates and to improve tools for the translation of preclinical data to the clinic. In this review of murine pneumonia model data published in the last 10 years, we present our findings of considerable variability in the protocols employed for testing the efficacy of antimicrobial compounds using this in vivo model. Based on specific inclusion criteria, fifty-three studies focusing on antimicrobial assessment against Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were reviewed in detail. The data revealed marked differences in the experimental design of the murine pneumonia models employed in the literature. Notably, several differences were observed in variables that are expected to impact the obtained results, such as the immune status of the animals, the age, infection route and sample processing, highlighting the necessity of a standardized model., Competing Interests: JLH was employed by GlaxoSmithKline Pharmaceuticals. SS and SR were employed by Evotec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arrazuria, Kerscher, Huber, Hoover, Lundberg, Hansen, Sordello, Renard, Aranzana-Climent, Hughes, Gribbon, Friberg and Bekeredjian-Ding.)

BackgroundUnavailability of vaccines endangers the overall goal to protect individuals and whole populations against infections.MethodsThe German notification system includes the publication of vaccine supply shortages reported by marketing authorisation holders (MAH), information on the availability of alternative vaccine products, guidance for physicians providing vaccinations and an unavailability reporting tool to monitor regional distribution issues.AimThis study provides a retrospective analysis of supply issues and measures in the context of European and global vaccine supply constraints.Resultsbetween October 2015 and December 2020, the 250 notifications concerned all types of vaccines (54 products). Most shortages were caused by increased demand associated with immigration in Germany in 2015 and 2016, new or extended vaccine recommendations, increased awareness, or changes in global immunisation programmes. Shortages of a duration up to 30 days were mitigated using existing storage capacities. Longer shortages, triggered by high demand on a national level, were mitigated using alternative products and re-allocation; in a few cases, vaccines were imported. However, for long lasting supply shortages associated with increased global demand, often occurring in combination with manufacturing issues, few compensatory mechanisms were available. Nevertheless, only few critical incidents were identified: (i) shortage of hexavalent vaccines endangering neonatal immunisation programmes in 2015;(ii) distribution issues with influenza vaccines in 2018; and (iii) unmet demand for pneumococcal and influenza vaccines during the coronavirus disease (COVID)-19 pandemic.ConclusionVaccine product shortages in Germany resemble those present in neighbouring EU states and often reflect increased global demand not matched by manufacturing capacities.

The periodontal microbiota is ecologically diverse and may facilitate colonization by bacteria of enteric origin ( Enterobacteriaceae , Enterococci ) and co-infections with Candida albicans , possibly producing subgingival biofilms with high antimicrobial tolerance. This retrospective surveillance study followed periodontitis-associated superinfection profiles in a large patient sample. From 2008 to 2015, biofilm samples from deep periodontal pockets were collected from a total of 16,612 German adults diagnosed with periodontitis. The presence of selected Enterobacteriaceae , Enterococci , and Candida albicans was confirmed in overnight cultures. Antimicrobial susceptibility of these clinical isolates was tested by disk diffusion with antibiotics routinely used for treatment of oral infections, e.g., amoxicillin (AML), amoxicillin/clavulanic acid (AMC), doxycycline (DO), and ciprofloxacin (CIP). The mean annual prevalence of patients harboring Enterobacteriaceae in periodontal plaques was 11.5% in total and ranged from 2.5% for Enterobacter cloacae to 3.6% for Klebsiella oxytoca , 1.1% for Klebsiella pneumoniae , 2.8% for Serratia marcescens , and 1.5% for Serratia liquefaciens . In comparison, the mean detection rates for microbiota typically found in the oral cavity were higher, e.g., 5.6% for Enterococcus spp. and 21.8% for Candida albicans . Among the Enterobacteriaceae , species harboring intrinsic resistance to AML ( Enterobacter spp., Klebsiella spp., Serratia spp.) were predominant. Non-susceptibility to AMC was observed for Serratia spp. and Enterobacter cloacae . By contrast, Enterococcus spp. only showed non-susceptibility to DO and CIP. Trends for increasing resistance were found to AML in Serratia liquefaciens and to DO in Enterococcus spp. Trend analysis showed decreasing resistance to AMC in Serratia liquefaciens and Klebsiella oxytoca ; and to DO in Serratia marcescens, liquefaciens , and Enterobacter cloacae . This study confirms the low but consistent presence of Enterobacteriaceae and Enterococci among the subgingival microbiota recovered from periodontitis specimen. Although their pathogenetic role in periodontal lesions remains unclear, their presence in the oral cavity should be recognized as a potential reservoir for development and spread of antibiotic resistance in light of antibiotic usage in oral infections.