A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values

Cholera remains a significant public health burden worldwide, and better methods for monitoring cholera incidence would enhance the effectiveness of public health interventions. The serum bactericidal assay (SBA) has been used extensively for Vibrio cholerae vaccine assessments and serosurveillance. Current SBA approaches for V. cholerae rely on colony enumeration or optical density (OD600nm) readings to measure viable bacteria following complement-mediated lysis. These methods provide titer values that are constrained to discrete dilution values and rely on bacterial outgrowth, which is time consuming and prone to variation. Detection of bacterial proteins following complement-mediated lysis presents a faster and potentially less variable alternative approach independent of bacterial outgrowth. Here, we present an SBA that measures luciferase luminescence driven by lysis-released adenylate kinase. This approach is faster and less variable than growth-dependent SBAs and directly measures continuous titer values. This novel SBA method can potentially be applied to other bacteria of interest.

Tenascin-C Deficiency Is Associated With Reduced Bacterial Outgrowth During Klebsiella pneumoniae-Evoked Pneumosepsis in Mice

Tenascin C (TNC) is an extracellular matrix glycoprotein that recently emerged as an immunomodulator. TNC-deficient (TNC−/−) mice were reported to have a reduced inflammatory response upon systemic administration of lipopolysaccharide, the toxic component of gram-negative bacteria. Here, we investigated the role of TNC during gram-negative pneumonia derived sepsis. TNC+/+ and TNC−/− mice were infected with Klebsiella pneumoniae via the airways and sacrificed 24 and 42 h thereafter for further analysis. Pulmonary TNC protein levels were elevated 42 h after infection in TNC+/+ mice and remained undetectable in TNC−/− mice. TNC−/− mice showed modestly lower bacterial loads in lungs and blood, and a somewhat reduced local—but not systemic—inflammatory response. Moreover, TNC−/− and TNC+/+ mice did not differ with regard to neutrophil recruitment, lung pathology or plasma markers of distal organ injury. These results suggest that while TNC shapes the immune response during lipopolysaccharide-induced inflammation, this role may be superseded during pneumosepsis caused by a common gram-negative pathogen.

The impact of RAGE inhibition in animal models of bacterial sepsis: a systematic review and meta-analysis

Objective To evaluate the impact of inhibition of the receptor for advanced glycation end products (RAGE) on the outcome of bacterial sepsis in animal models. Methods Relevant publications were identified by systematic searches of PubMed, ISI Web of Science and Elsevier-Scopus databases. Results A total of Eleven studies with moderate quality were selected for analysis. A meta-analysis of survival rates revealed a significant advantage of RAGE inhibition in comparison with controls (HR 0.67, 95% CI 0.52–0.86). This effect was most pronounced in polymicrobial infection (HR 0.28, 95% CI 0.14–0.55), followed by Gram positive (G+) bacterial infection (HR 0.70, 95% CI 0.50–0.97) and Gram negative (G−) bacterial infection (HR 0.89, 95% CI 0.58–1.38). For G+ bacterial infection, RAGE inhibition decreased bacterial outgrowth and dissemination, inflammatory cell influx, plasma cytokine levels, and pulmonary injury. Conclusions RAGE inhibition appears to have a beneficial impact on the outcome of sepsis in animal models, although there are discrepancies between different types of infection.

Coagulation factor XI improves host defence during murine pneumonia-derived sepsis independent of factor XII activation

SummaryBacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.Supplementary Material to this article is available online at www.thrombosis-online.com.

Hierarchical effects of pro-inflammatory cytokines on the post-influenza susceptibility to pneumococcal coinfection

In the course of influenza A virus (IAV) infections, a secondary bacterial infection frequently leads to serious respiratory conditions provoking high hospitalization and death tolls. Although abundant pro-inflammatory responses have been reported as key contributing factors for these severe dual infections, the relative contribution of cytokines remain largely unclear.In the current study, mathematical modelling on in vivo experimental data highlight IFN-γ as a decisive candidate responsible for impaired bacterial clearance, thereby promoting bacterial growth and systemic dissemination during acute IAV infection. Moreover, we found a time-dependent detrimental role of IL-6 in curtailing bacterial outgrowth which was however not as distinct as for IFN-γ. Importantly, our results furthermore challenge current beliefs that the TNF-α response or the increased availability of nutrients modulated by IAV infection have a central role to the bacterial outgrowth. Ultimately, our findings contribute to a detailed understanding of the mechanisms underlying impaired bacterial clearance following influenza infection.

Enhanced inflammation in aged mice following infection with Streptococcus pneumoniae is associated with decreased IL-10 and augmented chemokine production

Streptococcus pneumoniae is the most common cause of severe pneumonia in the elderly. However, the impact of aging on the innate inflammatory response to pneumococci is poorly defined. We compared the innate immune response in old vs. young adult mice following infection with S. pneumoniae. The accumulation of neutrophils recovered from bronchoalveolar lavage fluid and lung homogenates was increased in aged compared with young adult mice, although bacterial outgrowth was similar in both age groups, as were markers of microvascular leak. Aged mice had similar levels of IL-1β, TNF, IFN-γ, IL-17, and granulocyte colony-stimulating factor following S. pneumoniae infection, compared with young mice, but increased levels of the chemokines CXCL9, CXCL12, CCL3, CCL4, CCL5, CCL11, and CCL17. Moreover, levels of IL-10 were significantly lower in aged animals. Neutralization of IL-10 in infected young mice was associated with increased neutrophil recruitment but no decrease in bacterial outgrowth. Furthermore, IL-10 neutralization resulted in increased levels of CCL3, CCL5, and CXCL10. We conclude that aging is associated with enhanced inflammatory responses following S. pneumoniae infection as a result of a compromised immunomodulatory cytokine response.

The endothelial protein C receptor impairs the antibacterial response in murine pneumococcal pneumonia and sepsis

SummaryPneumococcal pneumonia is a frequent cause of gram-positive sepsis and has a high mortality. The endothelial protein C receptor (EPCR) has been implicated in both the activation of protein C (PC) and the anti-inflammatory actions of activated (A)PC. The aim of this study was to determine the role of the EPCR in murine pneumococcal pneumonia and sepsis. Wild-type (WT), EPCR knockout (KO) and Tie2-EPCR mice, which overexpress EPCR on the endothelium, were infected intranasally (pneumonia) or intravenously (sepsis) with viable Streptococcus pneumoniae and euthanised at 24 or 48 hours after initiation of the infection for analyses. Pneumonia did not alter constitutive EPCR expression on pulmonary endothelium but was associated with an influx of EPCR positive neutrophils into lung tissue. In pneumococcal pneumonia EPCR KO mice demonstrated diminished bacterial growth in the lungs and dissemination to spleen and liver, reduced neutrophil recruitment to the lungs and a mitigated inflammatory response. Moreover, EPCR KO mice displayed enhanced activation of coagulation in the early phase of disease. Correspondingly, in pneumococcal sepsis EPCR KO mice showed reduced bacterial growth in lung and liver and attenuated cytokine release. Conversely, EPCR-overexpressing mice displayed higher bacterial outgrowth in lung, blood, spleen and liver in pneumococcal sepsis. In conclusion, EPCR impairs antibacterial defense in both pneumococcal pneumonia and sepsis, which is associated with an enhanced pro-inflammatory response.

Antibodies Mediate Formation of Neutrophil Extracellular Traps in the Middle Ear and Facilitate Secondary Pneumococcal Otitis Media

ABSTRACTOtitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) andStreptococcus pneumoniae(the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth ofS. pneumoniaein the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.