Proliferation of psychrotrophic bacteria in cold-stored platelet concentrates.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PC) are stored at 20-24°C to maintain platelet functionality, which may promote growth of contaminant bacteria. Alternatively, cold storage of PC limits bacterial growth; however, data related to proliferation of psychotrophic species in cold-stored PC (CSP) are scarce, which is addressed in this study. MATERIALS AND METHODS: Eight laboratories participated in this study with a pool/split approach. Two split PC units were spiked with ~25 colony forming units (CFU)/PC of Staphylococcus aureus, Klebsiella pneumoniae, Serratia liquefaciens, Pseudomonas fluorescens and Listeria monocytogenes. One unit was stored under agitation at 20-24°C/7 days while the second was stored at 1-6°C/no agitation for 21 days. PC were sampled periodically to determine bacterial loads. Five laboratories repeated the study with PC inoculated with lyophilized inocula (~30 CFU/mL) of S. aureus and K. pneumoniae. RESULTS: All species proliferated in PC stored at 20-24°C, reaching concentrations of ≤109 CFU/mL by day 7. Psychrotrophic P. fluorescens and S. liquefaciens proliferated in CSP to ~106 CFU/mL and ~105 CFU/mL on days 10 and 17 of storage, respectively, followed by L. monocytogenes, which reached ~102 CFU/mL on day 21. S. aureus and K. pneumoniae did not grow in CSP. CONCLUSION: Psychrotrophic bacteria, which are relatively rare contaminants in PC, proliferated in CSP, with P. fluorescens reaching clinically significant levels (≥105 CFU/mL) before day 14 of storage. Cold storage reduces bacterial risk of PC to levels comparable with RBC units. Safety of CSP could be further improved by implementing bacterial detection systems or pathogen reduction technologies if storage is beyond 10 days.

A Retrospective Analysis of Combat Injury Patterns and Prehospital Interventions Associated with the Development of Sepsis.

BACKGROUND: Combat injury related wound infections are common. Untreated, these wound infections may progress to sepsis and septic shock leading to increased morbidity and mortality rates. Understanding infectious complications, patterns, progression, and correlated prehospital interventions are vital to understand the development of sepsis. We aim to analyze demographics, injury patterns, and interventions associated with sepsis in battlefield settings. MATERIALS AND METHODS: This is a secondary analysis of previously published data from the Department of Defense Trauma Registry (DoDTR) from 2007 to 2020. We searched for casualties diagnosed with sepsis (excluding line-sepsis) throughout their initial hospitalization. Regression models were used to seek associations. RESULTS: Our initial request yielded 28,950 encounters, of which 25,654 (88.6%) were adults that met inclusion, including 243 patients (0.9%) diagnosed with sepsis. Patients included US military (34%), non-North Atlantic Treaty Organization (NATO) military (33%) and humanitarian (30%) groups. Patients diagnosed with sepsis had a significantly lower survival rate than non-septic patients (78.1% vs. 95.7%, p < 0.001). There was no significant difference in administration of prehospital antibiotics between septic and the general populations (10.6% vs. 12.3%, p = 0.395). Prehospital intraosseous access (OR 1.56, 95% CI 1.27-1.91, p = 0.207) and packed red cell administration (1.63, 1.24-2.15, 0.029) were the interventions most associated with sepsis. CONCLUSIONS: Sepsis occurred infrequently in the DoDTR when evacuation from battlefield is not delayed, but despite increased intervention frequency, developing sepsis demonstrates a significant drop in survival rates. Future research would benefit from the development of risk mitigation measures.

Platelet enhancement of bacterial growth during room temperature storage: mitigation through refrigeration.

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.

Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A.

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.

Vaccination with a live attenuated Acinetobacter baumannii deficient in thioredoxin provides protection against systemic Acinetobacter infection.

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.

Pentraxin 3: an immune modulator of infection and useful marker for disease severity assessment in sepsis.

The acute phase protein pentraxin 3 (PTX3) is a pattern recognition receptor involved in regulation of the host immune response. This relatively newly discovered member of the pentraxin superfamily elicits both immunostimulatory and immunoregulatory functions preventing autoimmune pathology and orchestrated clearance of pathogens through opsonization of damage- and pathogen-associated molecular patterns (DAMP/PAMP). Thus, PTX3 has been described as a possible evolutionary precursor to immunoglobulins. While shown to provide protection against specific bacterial and fungal pathogens, persistent elevation of PTX3 levels following initial onset of infection appear to predict poor patient outcome and may contribute to disease sequelae such as tissue damage and coagulopathy. Measurement of PTX3 following onset of sepsis may improve patient risk assessment and thus be useful in guiding subsequent therapeutic interventions including steroidal anti-inflammatory and altered antibiotic therapies. In this review, we summarize the role of PTX3 in inflammatory syndromes and its utility as a marker of sepsis disease severity.

Severe Acinetobacter baumannii sepsis is associated with elevation of pentraxin 3.

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.

Genome Sequences of Four Acinetobacter baumannii-A. calcoaceticus Complex Isolates from Combat-Related Infections Sustained in the Middle East.

Acinetobacter baumannii is among the most prevalent bacterial causes of combat-related infections on the battlefield. Antibiotic resistance and a poor understanding of the protective host immune responses make treatment difficult. Here, we report the genome sequences of four clinical Acinetobacter baumannii-A. calcoaceticus complex isolates exhibiting significant differences in virulence in a mouse sepsis model.

Human parechovirus 3 causing sepsis-like illness in children from midwestern United States.

BACKGROUND: : Human parechovirus (HPeV) infections of the central nervous system (CNS) in children can be associated with severe outcomes such as neonatal sepsis-like illness, meningitis, or paralysis. We sought to determine the prevalence of HPeV CNS infections and clinical presentation in children from the United States. METHODS: : Frozen nucleic acid extracts from enterovirus-negative cerebrospinal fluid (CSF) obtained at the Children's Mercy Hospitals and Clinics, in Kansas City from 2006 (n = 242), 2007 (n = 324), and 2008 (n = 218) were tested by 2-step HPeV real-time reverse transcription polymerase chain reaction. HPeV genotype was determined by sequencing the VP3/VP1 junction. Demographic and clinical data were abstracted from medical records. RESULTS: : Overall HPeV was detected in 58/780 (7%) of tested CSF samples; 4/218 (2%) in 2006, 54/320 (17%) in 2007, and 0/242 (0%) in 2008. HPeV (17%) and enterovirus (20%) detection were comparable in 2007. HPeV-3 genotype was detected in 52/53 specimens successfully sequenced. Detection was seasonal (June-October). HPeV-3-CNS-infection occurred at a mean age of 6.6 ± 4.4 weeks and predominantly in males (71%). The most common clinical presentation was sepsis-like syndrome (66%). The most common symptoms were irritability (98%), fever (95%), and nonspecific rash (58.6%), while neurologic manifestations were rare (5%). CONCLUSIONS: : To our knowledge, this is the first multiyear prevalence report of HPeV CNS infection in the United States. HPeV CNS infection was detected mostly in male infants with sepsis-like illness during the late summer/autumn season. Routine seasonal CSF testing in infants for HPeV plus enterovirus may improve etiologic detection and clinical management of infantile sepsis-like presentations.