Antibody responses in Klebsiella pneumoniae bloodstream infection: a cohort study.

Background: Klebsiella pneumonia (Kpn) is the fourth leading cause of infection-related deaths globally, yet little is known about human antibody responses to invasive Kpn. In this study, we sought to determine whether the O-specific polysaccharide (OPS) antigen, a vaccine candidate, is immunogenic in humans with Kpn bloodstream infection (BSI). We also sought to define the cross-reactivity of human antibody responses among structurally related Kpn OPS subtypes and to assess the impact of capsule production on OPS-targeted antibody binding and function. Methods: We measured plasma antibody responses to OPS (and MrkA, a fimbrial protein) in a cohort of patients with Kpn BSI and compared these with controls, including a cohort of healthy individuals and a cohort of individuals with Enterococcus BSI. We performed flow cytometry to measure the impact of Kpn capsule production on whole cell antibody binding and complement deposition, utilizing patient isolates with variable levels of capsule production and isogenic capsule-deficient strains derived from these isolates. Findings: We enrolled 69 patients with Kpn BSI. Common OPS serotypes accounted for 57/69 (83%) of infections. OPS was highly immunogenic in patients with Kpn BSI, and peak OPS-IgG antibody responses in patients were 10 to 30-fold higher than antibody levels detected in healthy controls, depending on the serotype. There was significant cross-reactivity among structurally similar OPS subtypes, including the O1v1/O1v2, O2v1/O2v2 and O3/O3b subtypes. Physiological amounts of capsule produced by both hyperencapsulated and non-hyperencapsulated Kpn significantly inhibited OPS-targeted antibody binding and function. Interpretation: OPS was highly immunogenic in patients with Kpn BSI, supporting its potential as a candidate vaccine antigen. The strong cross-reactivity observed between similar OPS subtypes in humans with Kpn BSI suggests that it may not be necessary to include all subtypes in an OPS-based vaccine. However, these observations are tempered by the fact that capsule production, even in non-highly encapsulated strains, has the potential to interfere with OPS antibody binding. This may limit the effectiveness of vaccines that exclusively target OPS. Funding: National Institute of Allergy and Infectious Diseases at the National Institutes of Health. Research in Context: Evidence before this study: Despite the potential of O-specific polysaccharide (OPS) as a vaccine antigen against Klebsiella pneumoniae (Kpn), the immunogenicity of OPS in humans remains largely unstudied, creating a significant knowledge gap with regard to vaccine development. A search of PubMed for publications up to March 18, 2024, using the terms " Klebsiella pneumoniae " and "O-specific polysaccharide" or "O-antigen" or "lipopolysaccharide" revealed no prior studies addressing OPS antibody responses in humans with Kpn bloodstream infections (BSI). One prior study 1 evaluated antibody response to a single lipopolysaccharide (which contains one subtype of OPS) in humans with invasive Kpn infection; however, in this study OPS typing of the infecting strains and target antigen were not described. Added value of this study: Our investigation into OPS immunogenicity in a human cohort marks a significant advance. Analyzing plasma antibody responses in 69 patients with Kpn BSI, we found OPS to be broadly immunogenic across all the types and subtypes examined, and there was significant cross-reactivity among structurally related OPS antigens. We also demonstrated that Kpn capsule production inhibit OPS antibody binding and the activation of complement on the bacterial surface, even in classical Kpn strains expressing lower levels of capsule.Implications of all the available evidence: While the immunogenicity and broad cross-reactivity of OPS in humans with Kpn BSI suggests it is a promising vaccine candidate, the obstruction of OPS antibody binding and engagement by physiologic levels of Kpn capsule underscores the potential limitations of an exclusively OPS-antigen based vaccine for Kpn. Our study provides insights for the strategic development of vaccines aimed at combating Kpn infections, an important antimicrobial resistant pathogen.

Vaccine value profile for Klebsiella pneumoniae.

Klebsiella pneumoniae causes community- and healthcare-associated infections in children and adults. Globally in 2019, an estimated 1.27 million (95% Uncertainty Interval [UI]: 0.91-1.71) and 4.95 million (95% UI: 3.62-6.57) deaths were attributed to and associated with bacterial antimicrobial resistance (AMR), respectively. K. pneumoniae was the second leading pathogen in deaths attributed to AMR resistant bacteria. Furthermore, the rise of antimicrobial resistance in both community- and hospital-acquired infections is a concern for neonates and infants who are at high risk for invasive bacterial disease. There is a limited antibiotic pipeline for new antibiotics to treat multidrug resistant infections, and vaccines targeted against K. pneumoniae are considered to be of priority by the World Health Organization. Vaccination of pregnant women against K. pneumoniae could reduce the risk of invasive K.pneumoniae disease in their young offspring. In addition, vulnerable children, adolescents and adult populations at risk of K. pneumoniae disease with underlying diseases such as immunosuppression from underlying hematologic malignancy, chemotherapy, patients undergoing abdominal and/or urinary surgical procedures, or prolonged intensive care management are also potential target groups for a K. pneumoniae vaccine. A 'Vaccine Value Profile' (VVP) for K.pneumoniae, which contemplates vaccination of pregnant women to protect their babies from birth through to at least three months of age and other high-risk populations, provides a high-level, holistic assessment of the available information to inform the potential public health, economic and societal value of a pipeline of K. pneumoniae vaccines and other preventatives and therapeutics. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public-private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the WHO. All contributors have extensive expertise on various elements of the K.pneumoniae VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

A combination of burn wound injury and Pseudomonas infection elicits unique gene expression that enhances bacterial pathogenicity.

Burns are a leading cause of morbidity and mortality worldwide with the most common cause of death resulting from sepsis, often from Pseudomonas aeruginosa. We previously reported that a non-lethal flame burn induced an altered host immune response. Using this model, gene expression in both the murine host and P. aeruginosa was measured using a NanoString custom probe panel. We observed differing patterns of gene expression in both host and P. aeruginosa in the skin, blood, liver, and spleen of mice that were burned and/or infected, compared to mice that were neither burned nor infected (i.e., Sham). In mice that were both burned and infected (B/I), we observed changes in gene expression in both the host and P. aeruginosa that were distinct from all other treatment conditions. These data suggest that the combination of the burned state and superimposed infection affects both host and pathogen gene expression to increase infection propensity. Gene transcription significantly changed from 6 to 24 h post-B/I in each tissue. Finally, inhibiting IL-10 signaling or co-administering arginine at the time of P. aeruginosa infection prolonged or restored survival in an otherwise 100% fatal burn and infection model. These findings suggest that disease states such as burns may differentially alter innate immune response gene expression in both a host- and pathogen-specific manner.IMPORTANCEThe interaction between an underlying disease process and a specific pathogen may lead to the unique expression of genes that affect bacterial pathogenesis. These genes may not be observed during infection in the absence of, or with a different underlying process or infection during the underlying process with a different pathogen. To test this hypothesis, we used Nanostring technology to compare gene transcription in a murine-burned wound infected with P. aeruginosa. The Nanostring probeset allowed the simultaneous direct comparison of immune response gene expression in both multiple host tissues and P. aeruginosa in conditions of burn alone, infection alone, and burn with infection. While RNA-Seq is used to discover novel transcripts, NanoString could be a technique to monitor specific changes in transcriptomes between samples and bypass the additional adjustments for multispecies sample processing or the need for the additional steps of alignment and assembly required for RNASeq. Using Nanostring, we identified arginine and IL-10 as important contributors to the lethal outcome of burned mice infected with P. aeruginosa. While other examples of altered gene transcription are in the literature, our study suggests that a more systematic comparison of gene expression in various underlying diseases during infection with specific bacterial pathogens may lead to the identification of unique host-pathogen interactions and result in more precise therapeutic interventions.

Effect of screw insertion angle and speed on the incidence of transcortical fracture development in a canine tibial diaphyseal model.

OBJECTIVE: To assess the incidence of transcortical fracture (TCF) development based on screw insertion angle and screw insertion speed. STUDY DESIGN: Cadaveric experimental study. SAMPLE POPULATION: Sixty-six canine tibiae. METHODS: Sixty-six cadaveric tibiae were randomly assigned to one of six groups that varied based on screw insertion angle relative to the pilot hole (0, 5, or 10°) and screw insertion speed (650 or 1350 revolutions per minute [rpm]). Each tibia was mounted in a custom jig. Locking self-tapping screws (3.5 mm) were inserted at varying speeds and insertion angles, based on group assignment. Orthogonal radiographs were evaluated for TCFs. Fisher's exact tests with a Bonferroni correction were performed to evaluate differences in the frequency of TCF between groups. RESULTS: In Group A (0°/650 rpm: control), a 0% TCF rate was observed (n = 0/80). Group B (5°/650 rpm) had a 3.75% TCF rate (n = 3/80). Group C (10°/650 rpm) had a 12.5% TCF rate (n = 10/80). Group D (10°/hand insertion) had a 3.75% TCF rate (n = 3/80). Group E (10°/1350 rpm) had a 17.5% TCF rate (n = 14/80). Group F (0°/1350 rpm) had a 0% TCF rate (n = 0/80). Groups C and E had the highest TCF rates with a difference in TCF rates observed between the control group and Group C (p = .001) and between the control group and Group E (p < .001). CONCLUSION: Increased screw insertion angle and insertion speed appear to be predisposing factors for TCF development in cadaveric bone. CLINICAL SIGNIFICANCE: Ensuring screw insertion is coaxial with the pilot hole and using slower screw insertion speeds may help reduce the risk of TCF development.

The effect of two platelet-rich plasma aspiration techniques on plasma cellular concentrations using a double syringe gravitational centrifugation system.

OBJECTIVE: To identify which aspiration technique increased plasma platelet concentration and which technique minimized plasma leukocyte and erythrocyte concentrations using a gravitational double-syringe platelet rich plasma (PRP) system. STUDY DESIGN: Controlled laboratory study. ANIMALS: Thirty adult dogs. METHODS: Whole blood was collected into two autologous conditioned plasma (ACP) syringes and an ethylenediaminetetraacetic acid (EDTA tube) (control samples). The ACP syringes were centrifuged for 5 min at 1500 rpm. The proximal 2 mL of plasma from one ACP syringe was deposited in an EDTA tube (preflash samples). Plasma from the second ACP syringe was withdrawn until the buffy coat was pierced, producing a "flash" of red blood cells, agitated and deposited into an EDTA tube (flash samples). Complete blood counts were performed. RESULTS: Mean plasma platelet concentrations of the control, preflash, and flash samples were 2.4 × 105 /dL, 3.3 × 105 /dL and 4.1 × 105 /dL, respectively. The mean platelet concentration of the flash samples was 7.9 × 104 /dL higher than the preflash samples (p = .005). The mean platelet concentration was lower in the control samples than the preflash (p = .002) and flash (p < .0001) samples. The median plasma leukocyte concentration of the preflash samples (0/dL) was lower than in the flash samples (2.4 × 103 /dL) (p = .001). The median plasma hematocrit value of the preflash samples (0%) was lower than in the flash samples (1.0%) (p = .002). CONCLUSION: The flash method is not necessary to produce a PRP sample. CLINICAL SIGNIFICANCE: Both methods produced PRP. However, clinicians should avoid aspirating the buffy coat when processing PRP for therapies where leukocytes and erythrocytes are contraindicated.

Hit 'em Where It Hurts: Gram-Negative Bacterial Lipopolysaccharide as a Vaccine Target.

Infections with antimicrobial-resistant (AMR) bacteria pose an increasing threat to the ability to perform surgical procedures, organ transplantation, and treat cancer among many other medical conditions. There are few new antimicrobials in the development pipeline. Vaccines against AMR Gram-negative bacteria may reduce the use of antimicrobials and prevent bacterial transmission. This review traces the origins of lipopolysaccharide (LPS)-based vaccines against Gram-negative bacteria, the role of O polysaccharides and LPS core regions as potential vaccine targets, the development of new vaccine technologies, and their application to vaccines in current development.

Extracellular RNA Sensing Mediates Inflammation and Organ Injury in a Murine Model of Polytrauma.

Severe traumatic injury leads to marked systemic inflammation and multiorgan injury. Endogenous drivers such as extracellular nucleic acid may play a role in mediating innate immune response and the downstream pathogenesis. Here, we explored the role of plasma extracellular RNA (exRNA) and its sensing mechanism in inflammation and organ injury in a murine model of polytrauma. We found that severe polytrauma-bone fracture, muscle crush injury, and bowel ischemia-induced a marked increase in plasma exRNA, systemic inflammation, and multiorgan injury in mice. Plasma RNA profiling with RNA sequencing in mice and humans revealed a dominant presence of miRNAs and marked differential expression of numerous miRNAs after severe trauma. Plasma exRNA isolated from trauma mice induced a dose-dependent cytokine production in macrophages, which was almost abolished in TLR7-deficient cells but unchanged in TLR3-deficient cells. Moreover, RNase or specific miRNA inhibitors against the selected proinflammatory miRNAs (i.e., miR-7a-5p, miR-142, let-7j, miR-802, and miR-146a-5p) abolished or attenuated trauma plasma exRNA-induced cytokine production, respectively. Bioinformatic analyses of a group of miRNAs based on cytokine readouts revealed that high uridine abundance (>40%) is a reliable predictor in miRNA mimic-induced cytokine and complement production. Finally, compared with the wild-type, TLR7-knockout mice had attenuated plasma cytokine storm and reduced lung and hepatic injury after polytrauma. These data suggest that endogenous plasma exRNA of severely injured mice and ex-miRNAs with high uridine abundance prove to be highly proinflammatory. TLR7 sensing of plasma exRNA and ex-miRNAs activates innate immune responses and plays a role in inflammation and organ injury after trauma.

Galectin-1 mediates interactions between polymorphonuclear leukocytes and vascular endothelial cells, and promotes their extravasation during lipopolysaccharide-induced acute lung injury.

The lung airway epithelial surface is heavily covered with sialic acids as the terminal carbohydrate on most cell surface glycoconjugates and can be removed by microbial neuraminidases or endogenous sialidases. By desialylating the lung epithelial surface, neuraminidase acts as an important virulence factor in many mucosal pathogens, such as influenza and S. pneumoniae. Desialylation exposes the subterminal galactosyl moieties - the binding glycotopes for galectins, a family of carbohydrate-recognition proteins playing important roles in various aspects of immune responses. Galectin-1 and galectin-3 have been extensively studied in their roles related to host immune responses, but some questions about their role(s) in leukocyte recruitment during lung bacterial infection remain unanswered. In this study, we found that both galectin-1 and galectin-3 bind to polymorphonuclear leukocytes (PMNs) and enhance the interaction of endothelial intercellular adhesion molecule-1 (ICAM-1) with PMNs, which is further increased by PMN desialylation. In addition, we observed that in vitro galectin-1 mediates the binding of PMNs, particularly desialylated PMNs, onto the endothelial cells. Finally, in a murine model for LPS-mediated acute lung injury, we observed that galectin-1 modulates PMN infiltration to the lung without altering the expression of chemoattractant cytokines. We conclude that galectins, particularly galectin-1, may function as adhesion molecules that mediate PMN-endothelial cell interactions, and modulate PMN infiltration during acute lung injury.

Short-term feeding of defatted bovine colostrum mitigates inflammation in the gut via changes in metabolites and microbiota in a chicken animal model.

BACKGROUND: Nondrug supplement strategies to improve gut health have largely focused on the effects of individual compounds to improve one aspect of gut homeostasis. However, there is no comprehensive assessment of the reproducible effects of oral, short-term, low-level colostrum supplementation on gut inflammation status that are specific to the ileum. Herein, a chicken animal model highly responsive to even mild gut inflammatory stimuli was employed to compare the outcomes of feeding a standard diet (CON) to those of CON supplemented with a centrifuge-defatted bovine colostrum (BC) or a nonfat dried milk (NFDM) control on the efficiency of nutrient use, ileal morphology, gut nitro-oxidative inflammation status, metabolites, and the composition of the microbiota. RESULTS: A repeated design, iterative multiple regression model was developed to analyze how BC affected ileal digesta-associated anti-inflammatory metabolite abundance coincident with observed changes in the ileal microbiome, mitigation of epithelial inflammation, and ileal surface morphology. An improved whole body nutrient use efficiency in the BC group (v CON and NFDM) coincided with the observed increased ileum absorptive surface and reduced epithelial cell content of tyrosine-nitrated protein (NT, biomarker of nitro-oxidative inflammatory stress). Metabolome analysis revealed that anti-inflammatory metabolites were significantly greater in abundance in BC-fed animals. BC also had a beneficial BC impact on microbiota, particularly in promoting the presence of the bacterial types associated with eubiosis and the segmented filamentous bacteria, Candidatus Arthromitus. CONCLUSION: The data suggest that an anti-inflammatory environment in the ileum was more evident in BC than in the other feeding groups and associated with an increased content of statistically definable groups of anti-inflammatory metabolites that appear to functionally link the observed interactions between the host's improved gut health with an observed increase in whole body nutrient use efficiency, beneficial changes in the microbiome and immunometabolism.

The effect of grip location on Kirschner wire bend characteristics: An in vitro study.

OBJECTIVE: To determine the effect of Kirschner wire (K-wire) grip location on bend angle, bend radius, and torque when performing a Z-bend technique. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Ten samples at each of five grip locations for each of three K-wire diameters. METHODS: K-wires of three diameters (0.9, 1.1, 1.6 mm) were drilled into PVC pipe, and a Jacob's chuck was used to bend the wires at five periodic grip locations (distance from the bone model). Torque, bend angle, and bend radius were determined for each sample. Outcome variables were statistically analyzed by grip location to determine significant relationships. RESULTS: A grip location of 2.0 cm in the 0.9 mm K-wire group minimized bend angle (mean ± SD: 75.92° ± 0.81) and bend radius (2.89 mm ± 0.08). A grip location of 3.0 cm in the 1.1 mm K-wire group minimized bend angle (72.88° ± 0.98) and bend radius (2.47 mm ± 0.20). A grip location of 3.0 cm minimized bend angle (74.38° ± 1.93) and bend radius (2.71 mm ± 0.27) in the 1.6 mm K-wire group. Torque at these grip locations for the 0.9, 1.1, and 1.6 mm K-wires was 6.50 N-m ± 0.0, 11.00 N-m ± 0.0, and 19.05 N-m ± 0.16, respectively. CONCLUSION: Bend angle and bend radius can be minimized by bending K-wires at specific grip locations, though torque is not minimized at these locations. Clinical significance These findings provide an evidence-based recommendation of where surgeons should grip K-wires when bending them.

Altered sialidase expression in human myeloid cells undergoing apoptosis and differentiation.

To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.

Genome sequencing of Pseudomonas aeruginosa strain M2 illuminates traits of an opportunistic pathogen of burn wounds.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen and one of the most prevalent organisms isolated from burn wounds worldwide. Pseudomonas aeruginosa strain M2 (O5 serotype, type B flagella) is utilized for examining the murine model associated with burns. Pseudomonas aeruginosa M2 is similar in lethality to common laboratory P. aeruginosa strains when infecting CD-1 mice. Conversely, we recently showed that, relative to these strains, P. aeruginosa M2-infected mice are more susceptible to sepsis and demonstrate a 6-log reduction in LD50 from subcutaneous infection at the infection site directly after 10% total body surface area burn. To better understand this striking phenotypic difference from other P. aeruginosa strains employed in burn models, we sequenced the P. aeruginosa M2 genome. A total of 4,136,641 read pairs were obtained, providing an average genome coverage of 97.5X; subsequent assembly yielded a draft genome with 187 contigs comprising 6,360,304 bp with a G + C content of 66.45%. Genome-based phylogeny estimation of 92 P. aeruginosa strains placed P. aeruginosa M2 with P. aeruginosa-12-4-4(59), a nonairway clinical strain isolated from the blood culture of a burn patient. Phylogenomic analyses identified genes shared between P. aeruginosa M2 and P. aeruginosa 14, another strain exhibiting increased lethality in thermal tissues, as well as P. aeruginosa M2 unique genes with diverse functions like degradation of toxic aromatic compounds, iron scavenging, swarming motility and biofilm formation, defense against invasive DNA, and host assault. Predicted lateral gene transfers illuminate proteins heretofore uncharacterized for roles in P. aeruginosa biology. Our work yields a rich resource for assessing P. aeruginosa genes required for increased lethality in burn tissue seroma.

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. RESULTS: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). CONCLUSIONS: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

A Nonlethal Full-Thickness Flame Burn Produces a Seroma Beneath the Forming Eschar, Thereby Promoting Pseudomonas aeruginosa Sepsis in Mice.

The World Health Organization estimates ~180,000 deaths occur annually from burn-related injuries. Many victims who survive the initial burn trauma succumb to bacterial infections that lead to sepsis during treatment. Although advancements in burn care continue to improve in high-income countries due to their burn centers and advanced research, low and middle-income countries continue to see high frequencies of burn injuries and burn-related deaths due to secondary infections. Bacterial-derived sepsis is the most life-threatening danger for people that survive burn injuries. Here we provide evidence for the first time that a subeschar seroma forms postburn even in the absence of infection in mice. The seroma fills with a volume estimated at 500 µL of fluid, 25% of the blood supply, free of red blood cells. The seroma fluid supports robust Pseudomonas aeruginosa (PA) growth and contains inflammatory cytokines and chemokines, which recruit immature neutrophils and monocytes to the seroma in the absence of endothelial breakdown. These immune cells fail to contain PA expansion and dissemination. This recruitment of monocytes and immature neutrophils may result in sequestering these critical immune cells away from other tissues during a pivotal time during bacterial dissemination, promoting PA-mediated sepsis.

Effect of mGluR2 positive allosteric modulation on frontostriatal working memory activation in schizophrenia.

Negative symptoms and cognitive deficits contribute strongly to disability in schizophrenia, and are resistant to existing medications. Recent drug development has targeted enhanced NMDA function by increasing mGluR2/3 signaling. However, the clinical utility of such agents remains uncertain, and markers of brain circuit function are critical for clarifying mechanisms and understanding individual differences in efficacy. We conducted a double-blind, placebo-controlled, randomized cross-over (14 day washout) pilot study evaluating adjunctive use of the mGluR2 positive allosteric modulator AZD8529 (80 mg daily for 3 days), in chronic stable patients with schizophrenia (n = 26 analyzed). We focused on 3 T fMRI response in frontostriatal regions during an n-back working memory task, testing the hypothesis that AZD8529 produces fMRI changes that correlate with improvement in negative symptoms and cognition. We found that AZD8529 did not produce significant group-average effects on symptoms or cognitive accuracy. However, AZD8529 did increase n-back fMRI activation in striatum (p < 0.0001) and anterior cingulate/paracingulate (p = 0.002). Greater drug-versus-placebo effects on caudate activation significantly correlated with greater reductions in PANSS negative symptom scores (r = -0.42, p = 0.031), and exploratory correlations suggested broader effects across multiple symptom domains and regions of interest. These findings demonstrate that fMRI responses to mGluR2 positive modulation relate to individual differences in symptom reduction, and could be pursued for future biomarker development. Negative clinical results at the group level should not lead to premature termination of investigation of this mechanism, which may benefit an important subset of individuals with schizophrenia. Imaging biomarkers may reveal therapeutic mechanisms, and help guide treatment toward specific populations.

MUC1 ectodomain is a flagellin-targeting decoy receptor and biomarker operative during Pseudomonas aeruginosa lung infection.

We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.

Evaluation of the clinical value of routine radiographic examination during convalescence for tibial plateau-leveling osteotomy.

OBJECTIVE: To determine how frequently routine follow-up radiographic findings would result in a change to the postoperative plan following tibial plateau-leveling osteotomy (TPLO) in dogs. STUDY DESIGN: Retrospective study SAMPLE POPULATION: Short-term group: 100 cases; intermediate-term group: 50 cases. METHODS: Medical records of 100 consecutive cases meeting the inclusion criteria were reviewed (the short-term group). The cases had no owner-perceived issues and underwent routinely prescribed radiographic follow up between 40 and 60 postoperative days after TPLO performed by one experienced surgeon. Complications identified on physical examination (PE) and radiographic examination (RE) were recorded, along with any changes to the postoperative plan. Medical records of 50 consecutive cases that had short-term and intermediate-term (≥180 days) REs and PEs were reviewed similarly (intermediate-term group). RESULTS: Fifty-one cases in the short-term group had no complications on PE or RE. Forty-nine dogs were diagnosed with minor complications (patellar ligament desmitis, patella or fibula fracture, gait abnormalities): 42 on RE only; 6 on PE and RE; 1 on PE only. Exercise restriction was extended for 2 weeks in 2 cases with radiographic patellar ligament desmitis. Two cases in the intermediate-term group had minor complications at intermediate-term RE. No new PE or RE complications developed between short-term and intermediate-term evaluations. CONCLUSION: At routine rechecks of dogs with no owner-perceived issues after TPLO, 49% had minor complications but only 2% were deemed significant enough to alter patient management. The likelihood of new radiographic complications developing after short-term evaluation is low. CLINICAL SIGNIFICANCE: Routine radiographic recheck examinations rarely altered the postoperative plan in TPLO cases with unremarkable clinical recoveries.

Using Adjuvants to Drive T Cell Responses for Next-Generation Infectious Disease Vaccines.

Using adjuvants to drive features of T cell responses to vaccine antigens is an important technological challenge in the design of new and improved vaccines against infections. Properties such as T helper cell function, T cell memory, and CD8+ T cell cytotoxicity may play critical roles in optimal and long-lived immunity through vaccination. Directly manipulating specific immune activation or antigen delivery pathways with adjuvants may selectively augment desired T cell responses in vaccination and may improve the effectiveness and durability of vaccine responses in humans. In this review we outline recently studied adjuvants in their potential for antigen presenting cell and T cell programming during vaccination, with an emphasis on what has been observed in studies in humans as available.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.