RESUMO
INTRODUCTION: Due to high risk of septic transfusion reactions arising from bacterial contamination, US Food and Drug Administration regulations currently limit platelet storage to 5 days at room temperature (RT). However, blood culturing methods can take up to 7 days to detect bacteria, allowing transfusion of potentially contaminated units. Thus, cold storage (CS) may be a viable means of extending shelf life and improving safety. STUDY DESIGN AND METHODS: Platelets and fresh plasma (FP) were collected by apheresis from healthy donors, aliquoted, and challenged with Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Staphylococcus epidermidis. Aliquots were then stored at either RT or CS. RESULTS: Significant (p < 0.05) bacterial growth was detected at RT for most bacteria as early as Day 1 after collection, with peak growth occurring between Days 3 and 4. Growth remained static during CS. Additionally, platelets appeared to enhance bacterial replication with growth significantly lower (p < 0.05) in FP relative to RT-stored platelets. Lactic acid promoted bacterial growth when added to FP at RT. Bacterial challenge also resulted in significantly increased platelet activation (p < 0.05) and significantly reduced platelet function (p < 0.05) in RT storage relative to uninfected controls by Day 5 after collection. Conversely, CS ablated bacteria growth, limited platelet metabolism, and preserved platelet function throughout the study. CONCLUSION: These data suggest that CS presents an attractive alternative to RT to both extend storage life and reduce the risk of transfusion-related sepsis.
Assuntos
Bactérias/crescimento & desenvolvimento , Plaquetas , Preservação de Sangue , Desinfecção/métodos , Refrigeração , Plaquetas/metabolismo , Plaquetas/microbiologia , Humanos , Sepse/metabolismo , Sepse/microbiologia , Sepse/prevenção & controle , Fatores de TempoRESUMO
Multidrug-resistant Acinetobacter baumannii is among the most common causes of infectious complications associated with combat-related trauma in military personnel serving overseas. However, little is currently known about its pathogenesis. While the gastrointestinal (GI) tract has been found to be a major reservoir for A. baumannii, as well as to potentially contribute to development of multidrug resistance, no studies have addressed the mechanisms involved in gut colonization. In this study, we address this critical gap in knowledge by first assessing the interaction between secretory IgA (SIgA), the principal humoral immune defense on mucosal surfaces, and the A. baumannii clinical isolate Ci79. Surprisingly, SIgA appeared to enhance A. baumannii GI tract colonization, in a process mediated by bacterial thioredoxin A (TrxA), as evidenced by reduction of bacterial attachment in the presence of TrxA inhibitors. Additionally, a trxA targeted deletion mutant (ΔtrxA) showed reduced bacterial burdens within the GI tract 24 h after oral challenge by in vivo live imaging, along with loss of thiol-reductase activity. Surprisingly, not only was GI tract colonization greatly reduced but the associated 50% lethal dose (LD50) of the ΔtrxA mutant was increased nearly 100-fold in an intraperitoneal sepsis model. These data suggest that TrxA not only mediates A. baumannii GI tract colonization but also may contribute to pathogenesis in A. baumannii sepsis following escape from the GI tract under conditions when the intestinal barrier is compromised, as occurs with cases of severe shock and trauma.IMPORTANCEAcinetobacter baumannii is an emerging bacterial pathogen recently classified as a serious threat to U.S. and global health by both the Centers for Disease Control and Prevention and the World Health Organization. It also is one of the leading causes of combat-related infections associated with injured military personnel serving overseas. Little is known regarding mechanisms of gastrointestinal tract colonization despite this site being shown to serve as a reservoir for multidrug-resistant (MDR) A. baumannii isolates. Here, we establish that secretory IgA, the major immunoglobulin of mucosal surfaces, promotes A. baumannii GI tract colonization via bacterial thioredoxin A as evidenced through significant reduction in colonization in IgA-deficient animals. Additionally, bacterial colonization and mortality were significantly reduced in animals challenged with a thioredoxin A-deficient A. baumannii mutant. Combined, these data suggest that thioredoxin A is a novel virulence factor, for which antithioredoxin therapies could be developed, for this important multidrug-resistant pathogen.
Assuntos
Acinetobacter baumannii/fisiologia , Aderência Bacteriana , Trato Gastrointestinal/microbiologia , Imunoglobulina A Secretora/metabolismo , Fatores Imunológicos/metabolismo , Tiorredoxinas/metabolismo , Fatores de Virulência/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidade , Animais , Modelos Animais de Doenças , Deleção de Genes , Camundongos Endogâmicos C57BL , Oxirredução , Sepse/microbiologia , Sepse/patologia , Análise de Sobrevida , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/genéticaRESUMO
Multi-drug resistant Acinetobacter baumannii (MDR-Ab), an opportunistic pathogen associated with nosocomial and combat related infections, has a high mortality due to its virulence and limited treatment options. Deletion of the thioredoxin gene (TrxA) from a clinical isolate of MDR-Ab resulted in a 100-fold increase in 50% lethal dose (LD50) in a systemic challenge murine model. Thus, we investigated the potential use of this attenuated strain as a live vaccine against MDR-Ab. Mice were vaccinated by subcutaneous (s.c.) injection of 2×105 CFU of the ΔtrxA mutant, boosted 14days later with an equivalent inoculum, and then challenged 30days post-vaccination by i.p. injection with 10 LD50 of the wild type (WT) Ci79 strain. Efficacy of vaccination was evaluated by monitoring MDR-Ab specific antibody titers and cytokine production, observing pathology and organ burdens after WT challenge, and measuring levels of serum pentraxin-3, a molecular correlate of A. baumannii infection severity, before and after challenge. Mice vaccinated with ΔtrxA were fully protected against the lethal challenge of WT. However, minimal immunoglobulin class switching was observed with IgM predominating. Spleens harvested from vaccinated mice exhibited negligible levels of IL-4, IFN-γ and IL-17 production when stimulated with UV-inactivated WT Ci79. Importantly, tissues obtained from vaccinated mice displayed reduced pathology and organ burden compared to challenged non-vaccinated mice. Additionally, serum pentraxin-3 concentrations were not increased 24h after challenge in vaccinated mice, correlating with reduction of WT MDR-Ab infection in ΔtrxA immunized mice. Furthermore, passive immunization with ΔtrxA-immune sera provided protection against lethal systemic Ci79 challenge. Collectively, the defined live attenuated ΔtrxA strain is a vaccine candidate against emerging MDR Acinetobacter infection.
Assuntos
Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/imunologia , Vacinas Bacterianas/imunologia , Tiorredoxinas/genética , Infecções por Acinetobacter/patologia , Acinetobacter baumannii/genética , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Proteína C-Reativa/análise , Citocinas/imunologia , Deleção de Genes , Imunidade nas Mucosas , Imunização Passiva , Switching de Imunoglobulina , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos C57BL , Sepse/prevenção & controle , Componente Amiloide P Sérico/análise , Baço/imunologia , Vacinas Atenuadas/imunologia , Fatores de Virulência/genéticaRESUMO
Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo. A. baumannii strain CI 79 exhibited significantly (P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD50) of 10(5) CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis.