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Objectives: This study aims to understand the magnitude of and mechanisms underlying the development of cardiovascular events (CVEs) in patients with invasive pneumococcal disease (IPD). We aimed to identify factors that contribute to the occurrence of CVEs within 1 year after admission and discuss implications for patient care. Methods: A multicentered cohort study included adult patients from four Dutch hospitals who had a positive blood culture for Streptococcus pneumoniae and any type of clinical manifestation between 2012 and 2020. Disease characteristics and microbiological data were systematically collected from electronic patient files. The main outcome measures were the occurrence of stroke and acute coronary syndromes (ACS). Results: Of 914 eligible patients, 4.2% experienced a CVE within 1 year after admission for IPD. ACS mainly occurred in the first 2 weeks, whereas stroke developed throughout follow-up. Although ACS was positively associated with disease severity, the sole independent predictor was alcohol abuse (odds ratio [OR] 3.840, 95% confidence interval [CI] 1.108-13.303). Although stroke occurred in 6.3% of meningitis cases, the best clinical predictor of stroke was a body temperature >39.5 °C at admission (OR 3.117 [1.154-8.423]). In the adult IPD population aged

Abstract Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

SARS-CoV-2 infections elicit antibodies against the viral spike (S) and nucleocapsid (N) proteins; COVID-19 vaccines against the S-protein only. The BCG-Corona trial, initiated in March 2020 in SARS-CoV-2-naïve Dutch healthcare workers, captured several epidemic peaks and the introduction of COVID-19 vaccines during the one-year follow-up. We assessed determinants of systemic anti-S1 and anti-N immunoglobulin type G (IgG) responses using trial data. Participants were randomised to BCG or placebo vaccination, reported daily symptoms, SARS-CoV-2 test results, and COVID-19 vaccinations, and donated blood for SARS-CoV-2 serology at two time points. In the 970 participants, anti-S1 geometric mean antibody concentrations (GMCs) were much higher than anti-N GMCs. Anti-S1 GMCs significantly increased with increasing number of immune events (SARS-CoV-2 infection or COVID-19 vaccination): 104.7 international units (IU)/mL, 955.0 IU/mL, and 2290.9 IU/mL for one, two, and three immune events, respectively (p < 0.001). In adjusted multivariable linear regression models, anti-S1 and anti-N log10 concentrations were significantly associated with infection severity, and anti-S1 log10 concentration with COVID-19 vaccine type/dose. In univariable models, anti-N log10 concentration was also significantly associated with acute infection duration, and severity and duration of individual symptoms. Antibody concentrations were not associated with long COVID or long-term loss of smell/taste.

Objectives: Unnecessary and inappropriate antibiotic use is an increasing global health challenge. In limited resource settings, prophylactic antibiotics are still often used in (adeno)tonsillectomy (AT), despite evidence against their effectiveness. This study aimed to investigate the effect of prophylactic amoxicillin, given after AT in children. Methods: This is a secondary analysis from a two-center, double-blinded, randomized controlled, non-inferiority trial to study the effect of prophylactic amoxicillin on post-AT morbidity. Children aged 2-14 years with recurrent chronic tonsillitis and/or obstructive sleep apnea were randomly assigned to receive either placebo or amoxicillin for 5 days after the operation. Pre- and postoperative samples were collected for polymerase chain reaction (PCR) analyses to detect the five most important pathogens known to be common causes of tonsillitis. PCR results were compared before and after surgery as well as between placebo and amoxicillin. Results: PCR results were obtained, 109 in the amoxicillin group and 115 in the placebo group. In the amoxicillin group, 91% of patients had at least one positive PCR test before surgery and 87% after surgery. In the placebo group, the respective percentages were 92% and 90%. In both groups, a decrease in the total number of pathogens was found after surgery. Conclusion: Prophylactic amoxicillin given after AT in children did not show a clinically relevant effect with respect to the number of oropharyngeal microorganisms as compared to placebo.

Abstract Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.

Abstract Background The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. Methods We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. Results In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. Conclusion The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

Abstract Background Nasopharyngeal swabs are taken to determine the causative agent of community acquired pneumonia (CAP), while the reliability of upper respiratory tract sampling as a proxy for lower respiratory tract infections is still unclear. Methods Nasopharyngeal (NP) swabs, bronchoalveolar lavage (BAL) fluid samples and clinical data were collected from 153 hospitalized children between 3 months and 14 years of age with severe CAP, enrolled from March to June 2019. Written informed consent for the storage and use of the samples for further studies was obtained from the parents or caregivers. Putative pathogens were detected using a sensitive, high-throughput GeXP-based multiplex PCR and qPCR. Results The same bacterial species in paired samples were found in 29 (23.4%) and the same viral species in 52 (27.5%) of the patients. moderate concordance was found for Mycoplasma pneumoniae (ĸ=0.64), followed by Haemophilus influenzae (ĸ=0.42). The strongest discordance was observed for human adenovirus and also for Pseudomonas aeruginosa, the latter was exclusively detected in BAL samples. In the adenovirus cases strong concordance was associated with high viral loads in the NP swabs. Conclusion The variation in concordance in pathogen detection in the upper and lower respiratory tract of children with severe pneumonia is generally high but varies depending on the species. Novel and impactful insights are the concordance between NP and BAL detection for M. pneumoniae and H. influenzae and the strong correlation between high adenoviral loads in NP swabs and detection in BAL fluid.

Summary: Earlier studies showed that BCG vaccination improves antibody responses of subsequent vaccinations. Similarly, in older volunteers we found an increased IgG receptor-binding domain (RBD) concentration after SARS-CoV-2 infection if they were recently vaccinated with BCG. This study aims to assess the effect of BCG on the serum antibody concentrations induced by COVID-19 vaccination in a population of adults older than 60 years. Serum was collected from 1,555 participants of the BCG-CORONA-ELDERLY trial a year after BCG or placebo, and we analyzed the anti-SARS-CoV-2 antibody concentrations using a fluorescent-microsphere-based multiplex immunoassay. Individuals who received the full primary COVID-19 vaccination series before serum collection and did not test positive for SARS-CoV-2 between inclusion and serum collection were included in analyses (n = 945). We found that BCG vaccination before first COVID-19 vaccine (median 347 days [IQR 329–359]) did not significantly impact the IgG RBD concentration after COVID-19 vaccination in an older European population.

Summary: Background: The PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice. Methods: Febrile children and controls were recruited on presentation to hospital in 9 European countries 2016–2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed. Findings: Of 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92–5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07–7.59), Group A streptococcus (OR 2.73, 95% CI 1.13–6.09) and E. coli (OR 2.7, 95% CI 1.02–6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11–0.46), influenza B (OR 0.12, 95% CI 0.02–0.37) and RSV (OR 0.16, 95% CI: 0.06–0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23–0.72) and EBV (OR 0.71, 95% CI 0.56–0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively. Interpretation: Most febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics. Funding: EU Horizon 2020 grant 668303.

Bordetella pertussis (Bp), the causative agent of pertussis, continues to circulate and it’s not well understood how (sub)clinical infections shape immune memory to Bp and vaccination. Here, using a mutant Bp strain lacking antigens of the acellular pertussis vaccine, the authors show how prior exposure to Bp shapes the mucosal antibody response to booster vaccination.

Abstract A range of public health measures have been implemented to suppress local transmission of coronavirus disease 2019 (COVID-19) in Shenzhen. We examined the effect of these measures on the prevalence of respiratory pathogens in children. Clinical and respiratory pathogen data were collected for routine care from hospitalized children with acute respiratory infections in Shenzhen Children’s Hospital from July 2018 to January 2022. Nasopharyngeal swabs were collected and respiratory pathogens were detected using standardized clinical diagnostics as part of routine care. Data were analyzed to describe the effects of COVID-19 prevention procedures on other common pathogens. A total of 56,325 children under 14 years of age were hospitalized with an acute respiratory infection during the study period, 33,909 were tested from July 2018 to January 2020 (pre-lockdown), 1168 from February 2020 to May 2020 (lockdown) and 21,248 from July 2020 to January 2022 (post-lockdown). We observed a 37.3% decline of routine care in respiratory infection associated hospital admission in the 19 months’ post-lockdown vs. the 19 months’ pre-lockdown. There were 99.4%, 16.0% and 1.26% reductions measured for Mycoplasma pneumoniae, influenza virus A and adenovirus, respectively. However, a 118.7% and 75.8% rise was found for respiratory syncytial virus (RSV) and human para-influenza virus (HPIV) during the 19 months’ post-lockdown in comparison to the pre-pandemic period. The detection of RSV especially increased in toddlers after the lockdown. Lockdown measures during the COVID-19 pandemic led to a significant reduction of Mycoplasma pneumoniae, influenza virus A and adenovirus infection. In contrast, RSV and HPIV infection increased.

Summary: Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection.

Inflammation is a physiological state where immune cells evoke a response against detrimental insults. Finding a safe and effective treatment for inflammation associated diseases has been a challenge. In this regard, human mesenchymal stem cells (hMSC), exert immunomodulatory effects and have regenerative capacity making it a promising therapeutic option for resolution of acute and chronic inflammation. T cells play a critical role in inflammation and depending on their phenotype, they can stimulate or suppress inflammatory responses. However, the regulatory effects of hMSC on T cells and the underlying mechanisms are not fully elucidated. Most studies focused on activation, proliferation, and differentiation of T cells. Here, we further investigated memory formation and responsiveness of CD4+ T cells and their dynamics by immune-profiling and cytokine secretion analysis. Umbilical cord mesenchymal stem cells (UC-MSC) were co-cultured with either αCD3/CD28 beads, activated peripheral blood mononuclear cells (PBMC) or magnetically sorted CD4+ T cells. The mechanism of immune modulation of UC-MSC were investigated by comparing different modes of action; transwell, direct cell-cell contact, addition of UC-MSC conditioned medium or blockade of paracrine factor production by UC-MSC. We observed a differential effect of UC-MSC on CD4+ T cell activation and proliferation using PBMC or purified CD4+ T cell co-cultures. UC-MSC skewed the effector memory T cells into a central memory phenotype in both co-culture conditions. This effect on central memory formation was reversible, since UC-MSC primed central memory cells were still responsive after a second encounter with the same stimuli. The presence of both cell-cell contact and paracrine factors were necessary for the most pronounced immunomodulatory effect of UC-MSC on T cells. We found suggestive evidence for a partial role of IL-6 and TGFβ in the UC-MSC derived immunomodulatory function. Collectively, our data show that UC-MSCs clearly affect T cell activation, proliferation and maturation, depending on co-culture conditions for which both cell-cell contact and paracrine factors are needed.

Abstract CCR5 is the main HIV co-receptor. We aimed to (1) compare CCR5 expression on immune cells between people living with HIV (PLHIV) using combination antiretroviral therapy (cART) and HIV-uninfected controls, (2) relate CCR5 expression to viral reservoir size and (3) assess determinants of CCR5 expression. This cross-sectional study included 209 PLHIV and 323 controls. Percentages of CCR5+ cells (%) and CCR5 mean fluorescence intensity assessed by flow cytometry in monocytes and lymphocyte subsets were correlated to host factors, HIV-1 cell-associated (CA)-RNA and CA-DNA, plasma inflammation markers and metabolites. Metabolic pathways were identified. PLHIV displayed higher percentages of CCR5+ monocytes and several CD8+ T cell subsets, but lower percentages of CCR5+ naive CD4+ T cells and regulatory T cells (Tregs). HIV-1 CA-DNA and CA-RNA correlated positively with percentages of CCR5+ lymphocytes. Metabolome analysis revealed three pathways involved in energy metabolism associated with percentage of CCR5+ CD8+ T cells in PLHIV. Our results indicate that CCR5 is differently expressed on various circulating immune cells in PLHIV. Hence, cell-trafficking of CD8+ T cells and Tregs may be altered in PLHIV. Associations between energy pathways and percentage of CCR5+ CD8+ T cells in PLHIV suggest higher energy demand of these cells in PLHIV.

Abstract Knowledge about contagiousness is key to accurate management of hospitalized COVID-19 patients. Epidemiological studies suggest that in addition to transmission through droplets, aerogenic SARS-CoV-2 transmission contributes to the spread of infection. However, the presence of virus in exhaled air has not yet been sufficiently demonstrated. In pandemic situations low tech disposable and user-friendly bedside devices are required, while commercially available samplers are unsuitable for application in patients with respiratory distress. We included 49 hospitalized COVID-19 patients and used a disposable modular breath sampler to measure SARS-CoV-2 RNA load in exhaled air samples and compared these to SARS-CoV-2 RNA load of combined nasopharyngeal throat swabs and saliva. Exhaled air sampling using the modular breath sampler has proven feasible in a clinical COVID-19 setting and demonstrated viral detection in 25% of the patients.

Abstract Acellular pertussis (aP) booster vaccines are central to pertussis immunization programs, although their effectiveness varies. The Bacille Calmette-Guérin (BCG) vaccine is a prototype inducer of trained immunity, which enhances immune responses to subsequent infections or vaccinations. While previous clinical studies have demonstrated that trained immunity can protect against heterologous infections, its effect on aP vaccines in humans is unknown. We conducted a clinical study in order to determine the immunological effects of trained immunity on pertussis vaccination. Healthy female volunteers were randomly assigned to either receive BCG followed by a booster dose of tetanus-diphteria-pertussis inactivated polio vaccine (Tdap-IPV) 3 months later (BCG-trained), BCG + Tdap-IPV concurrently, or Tdap-IPV followed by BCG 3 months later. Primary outcomes were pertussis-specific humoral, T- and B-cell responses and were quantified at baseline of Tdap-IPV vaccination and 2 weeks thereafter. As a secondary outcome in the BCG-trained cohort, ex vivo leukocyte responses were measured in response to unrelated stimuli before and after BCG vaccination. BCG vaccination 3 months prior to, but not concurrent with, Tdap-IPV improves pertussis-specific Th1-cell and humoral responses, and also increases total memory B cell responses. These responses were correlated with enhanced IL-6 and IL-1β production at the baseline of Tdap-IPV vaccination in the BCG-trained cohort. Our study demonstrates that prior BCG vaccination potentiates immune responses to pertussis vaccines and that biomarkers of trained immunity are the most reliable correlates of those responses.

Abstract Background Nasopharyngeal colonisation with clinically relevant bacterial pathogens is a risk factor for severe infections, such as pneumonia and bacteraemia. In this study, we investigated the determinants of nasopharyngeal carriage in febrile patients in rural Burkina Faso. Methods From March 2016 to June 2017, we recruited 924 paediatric and adult patients presenting with fever, hypothermia or suspicion of severe infection to the Centre Medical avec Antenne Chirurgicale Saint Camille de Nanoro, Burkina Faso. We recorded a broad range of clinical data, collected nasopharyngeal swabs and tested them for the presence of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Klebsiella pneumoniae by quantitative polymerase chain reaction. Using logistic regression, we investigated the determinants of carriage and aimed to find correlations with clinical outcome. Results Nasopharyngeal colonisation with S. pneumoniae, H. influenzae and M. catarrhalis was highly prevalent and strongly dependent on age and season. Females were less likely to be colonised with S. pneumoniae (OR 0.71, p = 0.022, 95% CI 0.53–0.95) and M. catarrhalis (OR 0.73, p = 0.044, 95% CI 0.54–0.99) than males. Colonisation rates were highest in the age groups

Objectives: To explore the negative predictive value (NPV) of C-reactive protein (CRP) at admission to exclude complicated disease manifestations of pneumococcal disease.Methods: A Dutch multicentre retrospective cohort study was conducted between 01-01-2012 and 30-06-2020. Adults with positive blood cultures for Streptococcus pneumoniae, whose CRP was measured at admission and whose infection focus was known, were included. Electronic medical and microbiological records were reviewed.Results: Of the 832 bacteraemic patients enrolled, 30% had complicated manifestations of pneumococcal disease; most frequent were pleural effusion (8.9%), pleural empyema (5.4%) and meningitis (7.5%). Compared to solitary pneumonia, patients with pleural effusion and empyema presented with higher CRP levels. Although low CRP levels did not exclude complicated disease in general, a CRP level < 114 mg/L at admission could reliably exclude empyema among adult pneumonia patients with an NPV of 93% and a specificity of 26%. However, in cases where pleural fluid was present, CRP levels were mostly > 114 mg/L, such that suspicion of empyema could only be ruled out in a minority of cases (10%).Conclusions: Complicated manifestations are prevalent in adult pneumococcal bacteraemia. Low blood CRP levels can reliably exclude the development of pulmonary empyema. Practical value may be largest in settings without thoracic imaging at hand.

There has been limited research on the role of the mucosal immune response to SARS-CoV-2 infection. Here, the authors perform a prospective observational household study and find that mucosal antibody responses are associated with decreased viral load and faster resolution of systemic symptoms.

ABSTRACT The nasopharyngeal commensal Streptococcus pneumoniae can become invasive and cause metastatic infection. This requires the pneumococcus to have the ability to adapt, grow, and reside in diverse host environments. Therefore, we studied whether the likelihood of severe disease manifestations was related to pneumococcal growth kinetics. For 383 S. pneumoniae blood isolates and 25 experimental mutants, we observed highly reproducible growth curves in nutrient-rich medium. The derived growth features were lag time, maximum growth rate, maximum density, and stationary-phase time before lysis. First, the pathogenicity of each growth feature was probed by comparing isolates from patients with and without marked preexisting comorbidity. Then, growth features were related to the propensity of causing severe manifestations of invasive pneumococcal disease (IPD). A high maximum bacterial density was the most pronounced pathogenic growth feature, which was also an independent predictor of 30-day mortality (P = 0.03). Serotypes with an epidemiologically higher propensity for causing meningitis displayed a relatively high maximum density (P

Abstract Background Knowledge on the etiology of LRTIs is essential for improvement of the clinical diagnosis and accurate treatment. Molecular detection methods were applied to identify a broad range of bacterial and viral pathogens in a large set of bronchial alveolar lavage (BAL) fluid samples. The patterns of detected pathogens were correlated to the clinical symptoms. Methods BAL fluid samples and clinical data were collected from 573 hospitalized children between 1 month and 14 years of age with LRTIs, enrolled from January to December 2018. Pathogens were detected using standardized clinical diagnostics, with a sensitive, high-throughput GeXP-based multiplex PCR and with multiplex qPCR. Data were analyzed to describe the correlation between the severity of respiratory tract disease and the pathogens identified. Results The pathogen detection rate with GeXP-based PCR and multiplex qPCR was significantly higher than by clinical routine diagnostics (76.09% VS 36.13%,χ2 = 8.191, P = 0.004). The most frequently detected pathogens in the BAL fluid were human adenovirus (HADV)(21.82%), Mycoplasma pneumoniae (20.24%), human rhinovirus (13.96%), Streptococcus pneumoniae (8.90%) and Haemophilus influenzae (8.90%). In 16.4% of the cases co-detection with two or three different pathogens was found. Viral detection rates declined with age, while atypical pathogen detection rates increased with age. Oxygen supply in the HADV and Influenza H1N1 infected patients was more frequent (49.43%) than in patients infected with other pathogens. Conclusion Broad range detection of viral and bacterial pathogens using molecular methods is a promising and implementable approach to improve clinical diagnosis and accurate treatment of LRTI in children.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.

Streptococcus suis is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for S. suis infection. We created Tn-Seq libraries in a virulent S. suis strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in ex vivo models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in S. suis pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs.

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of C. albicans by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG.

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

Background: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection. Methods: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens. Findings: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively. Interpretation: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. Fund: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303. Keywords: Targeted mass spectrometry, Multiple reaction monitoring (MRM), Complement system, Infectious disease, C-reactive protein (CRP), Clusterin

Abstract Background Patients with X-linked agammaglobulinemia (XLA) are protected against invasive bacterial infections due to IgG replacement therapy, but are still at higher risk for mucosal infections of the gut and respiratory tract. This might be explained by to the lack of IgA and IgM, as these antibodies are especially important for protection against invading bacterial pathogens on the mucosal surface. Methods In an attempt to eliminate a chronic norovirus infection in a patient with X-linked agammaglobulinemia, fresh frozen plasma (FFP) was given two times a week for 3 weeks. At each visit, pre- and post-FFP infusion serum and saliva was collected to determine IgG-, IgA- and IgM-concentrations and serum half-life was calculated. Functionality of the immunoglobulins pre- and post-FFP infusion in both serum and saliva was tested by measuring complement activation, agglutination and killing of non-typeable Haemophilus influenzae (NTHi). Results Administration of FFP failed to eradicate the chronic norovirus infection. Serum IgA and IgM half-life was 4.2 ± 0.3 and 3.8 ± 0.3 days, respectively. The presence of serum IgM was associated with increased complement binding and complement-mediated killing of NTHi. IgA in saliva was detectable post-FFP and was associated with increased agglutination of NTHi. IgM in saliva was not detectable. Conclusions We conclude that FFP treatment, although ineffective in clearing a chronic norovirus infection in this single patient, might be beneficial to prevent or eliminate bacterial infections in XLA patients by increasing IgM dependent complement-mediated killing in serum and IgA dependent bacterial agglutination on the mucosal surface.

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/β receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-β-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-β in contrast to adults. The distinct IFN-β-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.

Several bacteria in the human gut microbiome have been associated with colorectal cancer (CRC) by high-throughput screens. In some cases, molecular mechanisms have been elucidated that drive tumorigenesis, including bacterial membrane proteins or secreted molecules that interact with the human cancer cells. For most gut bacteria, however, it remains unknown if they enhance or inhibit cancer cell growth. Here, we screened bacteria-free supernatants (secretomes) and inactivated cells of over 150 cultured bacterial strains for their effects on cell growth. We observed family-level and strain-level effects that often differed between bacterial cells and secretomes, suggesting that different molecular mechanisms are at play. Secretomes of Bacteroidaceae, Enterobacteriaceae, and Erysipelotrichaceae bacteria enhanced cell growth, while most Fusobacteriaceae cells and secretomes inhibited growth, contrasting prior findings. In some bacteria, the presence of specific functional genes was associated with cell growth rates, including the virulence genes TcdA, TcdB in Clostridiales and FadA in Fusobacteriaceae, which both inhibited growth. Bacteroidaceae cells that enhanced growth were enriched for genes of the cobalamin synthesis pathway, while Fusobacteriaceae cells that inhibit growth were enriched for genes of the ethanolamine utilization pathway. Together, our results reveal how different gut bacteria have wide-ranging effects on cell growth, contribute a better understanding of the effects of the gut microbiome on host cells, and provide a valuable resource for identifying candidate target genes for potential microbiome-based diagnostics and treatment strategies.

Abstract Background While almost all infants are infected with respiratory syncytial virus (RSV) before the age of 2 years, only a small percentage develops severe disease. Previous studies suggest that the nasopharyngeal microbiome affects disease development. We therefore studied the effect of the nasopharyngeal microbiome on viral load and mucosal cytokine responses, two important factors influencing the pathophysiology of RSV disease. To determine the relation between (i) the microbiome of the upper respiratory tract, (ii) viral load, and (iii) host mucosal inflammation during an RSV infection, nasopharyngeal microbiota profiles of RSV infected infants (

The aetiology of Kawasaki disease (KD), an acute inflammatory disorder of childhood, remains unknown despite various triggers of KD having been proposed. Host ‘omic profiles offer insights into the host response to infection and inflammation, with the interrogation of multiple ‘omic levels in parallel providing a more comprehensive picture. We used differential abundance analysis, pathway analysis, clustering, and classification techniques to explore whether the host response in KD is more similar to the response to bacterial or viral infections at the transcriptomic and proteomic levels through comparison of ‘omic profiles from children with KD to those with bacterial and viral infections. Pathways activated in patients with KD included those involved in anti-viral and anti-bacterial responses. Unsupervised clustering showed that the majority of KD patients clustered with bacterial patients on both ‘omic levels, whilst application of diagnostic signatures specific for bacterial and viral infections revealed that many transcriptomic KD samples had low probabilities of having bacterial or viral infections, suggesting that KD may be triggered by a different process not typical of either common bacterial or viral infections. Clustering based on the transcriptomic and proteomic responses during KD revealed three clusters of KD patients on both ‘omic levels, suggesting heterogeneity within the inflammatory response during KD. The observed heterogeneity may reflect differences in the host response to a common trigger, or variation dependent on different triggers of the condition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.

Streptococcus pneumoniae infections lead to high morbidity and mortality rates worldwide. Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but have a limited range of protection, which encourages the development of a broadly protective protein-based alternative. We and others have shown that immunization with pneumococcal lipoproteins that lack the lipid anchor protects against colonization. Since immunity against S. pneumoniae is mediated through Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects of a lipid modification on the induced immune responses in either intranasally or subcutaneously vaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrA strongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhanced antibody concentrations and different induction kinetics. The induced humoral immune response was modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-type immunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderate but consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins, indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus, protein lipidation represents a promising approach for the development of a serotype-independent pneumococcal vaccine.

Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic.

Patients with primary immunoglobulin deficiency have lower immunoglobulin levels or decreased immunoglobulin function, which makes these patients more susceptible to bacterial infection. Most prevalent are the selective IgA deficiencies (~1:3,000), followed by common variable immune deficiency (~1:25,000). Agammaglobulinemia is less common (~1:400,000) and is characterized by very low or no immunoglobulin production resulting in a more severe disease phenotype. Therapy for patients with agammaglobulinemia mainly relies on prophylactic antibiotics and the use of IgG replacement therapy, which successfully reduces the frequency of invasive bacterial infections. Currently used immunoglobulin preparations contain only IgG. As a result, concurrent IgA and IgM deficiency persist in a large proportion of agammaglobulinemia patients. Especially patients with IgM deficiency remain at risk for recurrent infections at mucosal surfaces, which includes the respiratory tract. IgA and IgM have multiple functions in the protection against bacterial infections at the mucosal surface. Because of their multimeric structure, both IgA and IgM are able to agglutinate bacteria efficiently. Agglutination allows for entrapment of bacteria in mucus that increases clearance from the respiratory tract. IgA is also important for blocking bacterial adhesion by interfering with bacterial adhesion receptors. IgM in its place is very well capable of activating complement, therefore, it is thought to be important in complement-mediated protection at the mucosal surface. The purpose of this Mini Review is to highlight the latest advances regarding IgA- and IgM-enriched immunoglobulin replacement therapy. We describe the different IgA- and IgM-enriched IgG formulations, their possible modes of action and potential to protect against respiratory tract infections in patients with primary immunoglobulin deficiencies.

IntroductionThe effects of bacterial infections on the response to subsequent viral infections are largely unknown. This is important to elucidate to increase insight into the pathophysiology of bacterial and viral co-infections, and to assess whether bacterial infections may influence the course of viral infections.MethodsHealthy male subjects received either bacterial endotoxin [Escherichia coli-derived lipopolysaccharide (LPS), 2 ng/kg, n = 15] or placebo (n = 15) intravenously, followed by intranasal Fluenz (live-attenuated influenza vaccine) 1 week later.ResultsLPS administration resulted in increased plasma cytokine levels and development of endotoxin tolerance in vivo and ex vivo, illustrated by attenuated cytokine production upon rechallenge with LPS. Following Fluenz administration, infectivity for the Fluenz A/B strains was similar between the LPS–Fluenz and placebo–Fluenz groups (13/15 subjects in both groups). Also, the Fluenz-induced increase in temperature and IL-6, G-CSF and IP-10 concentrations in nasal wash were similar between both groups.ConclusionWhile endotoxemia profoundly attenuates the immune response upon a second LPS challenge, it does not influence the Fluenz-induced immune response. These results suggest immune suppression after bacterial infection does not alter the response to a subsequent viral infection.

The incidence of severe Haemophilus influenza infections, such as sepsis and meningitis, has declined substantially since the introduction of the H. influenzae serotype b vaccine. However, the H. influenzae type b vaccine fails to protect against nontypeable H. influenzae strains, which have become increasingly frequent causes of invasive disease, especially among children and the elderly. We summarize recent literature supporting the emergence of invasive nontypeable H. influenzae and describe mechanisms that may explain its increasing prevalence over the past 2 decades.

Advanced age is associated with chronic low-grade inflammation, which is usually referred to as inflammaging. Elderly are also known to have an altered gut microbiota composition. However, whether inflammaging is a cause or consequence of an altered gut microbiota composition is not clear. In this study, gut microbiota from young or old conventional mice was transferred to young germ-free (GF) mice. Four weeks after gut microbiota transfer immune cell populations in spleen, Peyer’s patches, and mesenteric lymph nodes from conventionalized GF mice were analyzed by flow cytometry. In addition, whole-genome gene expression in the ileum was analyzed by microarray. Gut microbiota composition of donor and recipient mice was analyzed with 16S rDNA sequencing. Here, we show by transferring aged microbiota to young GF mice that certain bacterial species within the aged microbiota promote inflammaging. This effect was associated with lower levels of Akkermansia and higher levels of TM7 bacteria and Proteobacteria in the aged microbiota after transfer. The aged microbiota promoted inflammation in the small intestine in the GF mice and enhanced leakage of inflammatory bacterial components into the circulation was observed. Moreover, the aged microbiota promoted increased T cell activation in the systemic compartment. In conclusion, these data indicate that the gut microbiota from old mice contributes to inflammaging after transfer to young GF mice.

Males and females are known to have gender-specific differences in their immune system and gut microbiota composition. Whether these differences in gut microbiota composition are a cause or consequence of differences in the immune system is not known. To investigate this issue, gut microbiota from conventional males or females was transferred to germ-free (GF) animals of the same or opposing gender. We demonstrate that microbiota-independent gender differences in immunity are already present in GF mice. In particular, type I interferon signaling was enhanced in the intestine of GF females. Presumably, due to these immune differences bacterial groups, such as Alistipes, Rikenella, and Porphyromonadaceae, known to expand in the absence of innate immune defense mechanism were overrepresented in the male microbiota. The presence of these bacterial groups was associated with induction of weight loss, inflammation, and DNA damage upon transfer of the male microbiota to female GF recipients. In summary, our data suggest that microbiota-independent gender differences in the immune system select a gender-specific gut microbiota composition, which in turn further contributes to gender differences in the immune system.

ABSTRACT To improve our understanding about the severity of invasive pneumococcal disease (IPD), we investigated the association between the genotype of Streptococcus pneumoniae and disease outcomes for 349 bacteremic patients. A pneumococcal genome-wide association study (GWAS) demonstrated a strong correlation between 30-day mortality and the presence of the phage-derived gene pblB, encoding a platelet-binding protein whose effects on platelet activation were previously unknown. Platelets are increasingly recognized as key players of the innate immune system, and in sepsis, excessive platelet activation contributes to microvascular obstruction, tissue hypoperfusion, and finally multiorgan failure, leading to mortality. Our in vitro studies revealed that pblB expression was induced by fluoroquinolones but not by the beta-lactam antibiotic penicillin G. Subsequently, we determined pblB induction and platelet activation by incubating whole blood with the wild type or a pblB knockout mutant in the presence or absence of antibiotics commonly administered to our patient cohort. pblB-dependent enhancement of platelet activation, as measured by increased expression of the α-granule protein P-selectin, the binding of fibrinogen to the activated αIIbβ3 receptor, and the formation of platelet-monocyte complex occurred irrespective of antibiotic exposure. In conclusion, the presence of pblB on the pneumococcal chromosome potentially leads to increased mortality in patients with an invasive S. pneumoniae infection, which may be explained by enhanced platelet activation. This study highlights the clinical utility of a bacterial GWAS, followed by functional characterization, to identify bacterial factors involved in disease severity. IMPORTANCE The exact mechanisms causing mortality in invasive pneumococcal disease (IPD) patients are not completely understood. We examined 349 patients with IPD and found in a bacterial genome-wide association study (GWAS) that the presence of the phage-derived gene pblB was associated with mortality in the first 30 days after hospitalization. Although pblB has been extensively studied in Streptococcus mitis, its consequence for the interaction between platelets and Streptococcus pneumoniae is largely unknown. Platelets are important in immunity and inflammation, and excessive platelet activation contributes to microvascular obstruction and multiorgan failure, leading to mortality. We therefore developed this study to assess whether the expression of pblB might increase the risk of death for IPD patients through its effect on enhanced platelet activation. This study also shows the value of integrating extensive bacterial genomics and clinical data in predicting and understanding pathogen virulence, which in turn will help to improve prognosis and therapy.

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that mainly causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. However, the underlying mechanisms involved in this process are poorly elucidated. In this study, we used a transposon mutant library to identify bacterial genes involved in biofilm formation. The growth and biofilm formation of 4,172 transposon mutants were determined, and the involvement of the identified genes in biofilm formation was validated in in vitro experiments. Here, we present experimental data showing that increased bacterial lysis, through interference with peptidoglycan synthesis, results in elevated levels of extracellular DNA, which increased biofilm formation. Interestingly, similar results were obtained with subinhibitory concentrations of β-lactam antibiotics, known to interfere with peptidoglycan synthesis, but such an effect does not appear with other classes of antibiotics. These results indicate that treatment with β-lactam antibiotics, especially for β-lactam-resistant NTHi isolates, might increase resistance to antibiotics by increasing biofilm formation. IMPORTANCE Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of β-lactam antibiotics, which are often prescribed to treat H. influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when β-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for β-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds.