Lethal Bacteremia Research Articles

Fitness factor genes conserved within the multi-species core genome of Gram-negative Enterobacterales species contribute to bacteremia pathogenesis.

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

Effect of copper nanoparticles green-synthesized using Ocimum basilicum against Pseudomonas aeruginosa in mice lung infection model

Abstract The frequency of lung infection induced by multi-drug resistant strains of Pseudomonas aeruginosa has significantly risen, primarily due to the inadequate effectiveness of powerful chemotherapeutic methods. This study demonstrates that the Ocimum basilicum aqueous extract and copper nanoparticles (CuNPs) exhibited significant antioxidant and anti-infectious properties under in vivo conditions. To analyze the characteristics of the CuNPs synthesized from the reaction between copper nitrate solution and the aqueous O. basilicum extract, various techniques such as energy dispersive X-ray analysis, field emission scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, and transmission electron microscopy were employed. The in vivo study encompasses the assessment of P. aeruginosa lethal dose in mice and the disease manifestation analysis, which comprises reduction in body weight, hypothermia, bacteremia, and other parameters, over a 48 h infection period. The infected mice exhibited a notable decrease in body temperature, measuring at 25°C after 48 h, compared to the initial temperature of 39°C. Additionally, a 30% reduction in weight was seen at the conclusion of the study. To assess the effectiveness of CuNPs on lung infection caused by the calculated lethal dose and bacteremia, histopathology analysis was employed. The bacterial load in the CuNPs group was determined to be 0.5 Log10CFU/mL on Day 8, indicating a notable decrease from the initial measurement of 1.5 Log10CFU/mL on Day 1. The histopathological findings revealed a widespread and sporadic buildup of alveolar space inflammatory cells, with infiltrates observed throughout all lung sections in infected mice. Enhanced lung histology was observed in the group of animal treated with reduced exudates noted at 200 µg/kg. CuNPs demonstrated inhibitory effects on the growth of P. aeruginosa at 8 µg/mL, while at 16 µg/mL, they effectively eradicated P. aeruginosa. The research unequivocally demonstrates the efficacy of CuNPs extract in combating lung infections induced by P. aeruginosa at 200 µg/kg. The recent survey aims to further explore the biomedical characteristics of these CuNPs in order to develop a powerful treatment against this dangerous pathogen.

Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

298. A Novel Murine Model of Neonatal Staphylococcus aureus Colonization Induces Changes in the CD4 T cell Compartment and Protects from Lethal Bacteremia

Abstract Background Staphylococcus aureus is a leading infectious cause of invasive bacterial infection in the United States. The pathogen’s capacity to cause disease is due, in part, to its ability to asymptomatically colonize the human host. Classically, the nose has been presumed to be the primary colonization site, but there is increasing evidence that the gastrointestinal tract may be a fundamental niche occupied during S. aureus colonization. There remain significant deficits in preclinical modeling of invasive S. aureus infection; Laboratory mice are commonly employed for modeling of S. aureus infection, but are rarely colonized by or pre-exposed to S. aureus. This contrasts with the human interaction with the pathogen, where a vast majority of humans demonstrate a specific adaptive response to S. aureus and a plurality of patients have lifelong colonization. To overcome this deficit, we designed a novel neonatal mouse model of asymptomatic intestinal S. aureus colonization. Methods Neonatal mice were subjected to oral gavage with S. aureus at day 10 of life; colonization was followed by fecal S. aureus titer. Infection outcomes were compared between mock- and S. aureus colonized mice. Results Mice from internal breeding and external vendors had no detectable S. aureus in the nasopharynx or feces at all time points tested. We found that adult mice are unable to be durably colonized by S. aureus despite multiple methods of inoculation. We identified that neonatal mice have a narrow, early window of susceptibility when they can be durably colonized, demonstrating robust fecal titers of S. aureus sustained through adulthood. Intestinal exposure to S. aureus shifted the nature of the colonic T cell compartment to encompass a greater proportion of CD4 T regulatory cells and a decreased frequency of GATA3+ CD4 cells. Finally, colonized mice demonstrated protection from severe infection, mimicking what is seen in humans. Conclusion Our findings support the hypothesis that gastrointestinal colonization influences the development of systemic immunity against S. aureus. This model will allow exploration into the mechanism of protective immunity against S. aureus and may inform the development of novel therapeutic and vaccination approaches to mitigate staphylococcal disease. Disclosures All Authors: No reported disclosures

Dissemination of Gram-positive bacteria to the lung of newborn mice increases local IL-6 and TNFα levels in lethal bacteremia

Neonatal bacteremia remains the major cause of infectious diseases–related death, especially in preterm newborns. Gram-positive bacteria are the main causative agent of neonatal bacteremia and exhibit a high risk of causing pneumonia and/or meningitis. The pathogenesis of bacteremia in preterm newborn is poorly understood. Current neonatal models of bacterial infection have been used to study the disease mechanisms; however, these studies employed mice of several days of age that could be less comparable to the bacteremia in preterm infants. In this study, we infected intravenously 0-day-old BALB/c mice with different inocula of Staphylococcus aureus, Streptococcus agalactiae or Enterococcus faecalis. We found that the mortality of the newborn mice was inoculum-dependent and also bacterial species-dependent. We observed bacterial burden in the lung, liver, brain, kidney and spleen of the infected animals. The lung was the tissue with the greatest bacterial burden and cellular infiltration in animals infected with the three bacteria evaluated. We found increased production of IL-6 and TNFα in the lung from newborn mice at 3 days post-infection. This neonatal model shows bacterial dissemination to the lung and will be useful for promote a better understanding of the pathophysiology of neonatal pneumonia.

1065. Efficacy of Cefiderocol in Experimental Stenotrophomonas maltophilia Pneumonia in Persistently Neutropenic Rabbits

Background Stenotrophomonas maltophilia causes lethal pneumonia, bacteremia, and sepsis in immunocompromised patients. As a standard of care, trimethoprim-sulfamethoxazole (T/S) is considered to be the first-line therapy for Stenotrophomonas pneumonia. Cefiderocol (CFDC) is a new parenteral siderophore cephalosporin that is transported through the outer cell membrane as a siderophore mimic that then inhibits Gram-negative cell wall biosynthesis. CFDC has potent activity in vitro against S. maltophilia; however, little is known about its in vivo activity against Stenotrophomonas pneumonia in immunocompromised hosts. We therefore studied CFDC in comparison to TS in the persistently neutropenic rabbit model of Stenotrophomonas pneumonia. This rabbit model, in contrast to conventional murine models, reflects the human pattern of infection more accurately over time.MethodsWe initially studied the plasma pharmacokinetics of CFDC in non-infected and infected animals. Stenotrophomonas pneumonia was established by direct endotracheal inoculation of S. maltophilia 1×1010 CFUs for tracheobronchial colonization that evolved into bronchopneumonia. Experimental groups consisted of CFDC, T/S, and untreated controls (UC). Rabbits received CFDC at 120 mg/kg IV Q8h and T/S at 5 mg/kg IV Q12h. Profound persistent neutropenia was maintained with cytosine arabinoside. Treatment was continued for 10 days.ResultsThere were no significant differences between non-infected and infected rabbits in CFDC pharmacokinetics. Rabbits treated with CFDC and T/S demonstrated significant decreases of residual pulmonary and BAL bacterial burden vs UC (p≤0.001). CFDC achieved full clearance of S. maltophilia from lung tissue and BAL. This antibacterial activity coincided with significant reduction of lung weights (marker of organism-mediated pulmonary injury) in the CFDC group vs T/S and UC (p< 0.01). Survival was prolonged in the CFDC treatment group with 87% survival in comparison to that of T/S (25%) and UC (0%) (p< 0.01). Table 1. Efficacy of Cefiderocol in Experimental Stenotrophomonas maltophilia Pneumonia in Persistency Neutropenic Rabbits ConclusionCefiderocol is highly active in treatment of experimental S. maltophilia pneumonia in persistently neutropenic rabbits, thus laying the foundation for future clinical investigations against this lethal infection. Disclosures Naoki Ishibashi, MD, Shionogi, Inc. (Employee) Benjamin Georgiades, n/a, Shionogi, Inc. (Consultant) Roger Echols, MD, Shionogi (Consultant) Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support) Yoshinori Yamano, PhD, Shionogi (Employee) Thomas J. Walsh, MD, PhD (hon), Scynexis (Consultant, Grant/Research Support)Shionogi (Consultant, Grant/Research Support)

Loss of Dihydroxyacid Dehydratase Induces Auxotrophy in Bacillus anthracis.

Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that ΔilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to nonsupplemented wild type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and ΔilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis, and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts. IMPORTANCE Anthrax disease, caused by B. anthracis, can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.

Histone deacetylase 6 inhibition improves survival in a swine model of lethal hemorrhage, polytrauma, and bacteremia.

Trauma is the leading cause of death for young Americans. Nonspecific histone deacetylase inhibitors, such as valproic acid, have been shown to improve survival in preclinical models of lethal trauma, hemorrhage, and sepsis. The doses needed to achieve a survival benefit are higher than Food and Drug Administration-approved doses, and the nonspecificity raises concerns about unintended adverse effects. The isoform-specific histone deacetylase 6 inhibitor, ACY-1083, has been found to be as efficacious as valproic acid in a rodent model of hemorrhagic shock. We hypothesized that ACY-1083 treatment would improve survival in a swine model of lethal hemorrhage, polytrauma, and bacteremia. Swine were subjected to 45% blood volume hemorrhage, brain injury, femur fracture, rectus crush, splenic and liver lacerations, and colon injury. After 1 hour of shock (mean arterial pressure, 30-35 mm Hg), animals were randomized to normal saline resuscitation (control) or normal saline plus ACY-1083 30 mg/kg treatment (n = 5/group). After 3 hours (simulating delayed evacuation), packed red blood cells and antibiotics were administered, the colon injury was repaired, and the abdomen was closed. Animals were then monitored for another 4 hours. Survival was assessed using Kaplan-Meier and log-rank test. This combination of injuries was lethal. All animals became bacteremic, in addition to the severe hemorrhagic shock. Survival in the control group was 0%, and ACY-1083 treatment increased survival to 80% (p = 0.019). There was no difference in the brain lesion size between the groups. A single dose of ACY-1083 markedly improves survival in an otherwise lethal model of polytrauma, hemorrhagic shock, and bacteremia.

Isolation and Characterization of a New Phage Infecting Elizabethkingia anophelis and Evaluation of Its Therapeutic Efficacy in vitro and in vivo.

Elizabethkingia spp. are a group of non-fermentative, Gram-negative, catalase-positive, and non-motile bacilli. They can cause meningitis in neonates and immunosuppressed patients, and lead to high mortality. Considering the rising trend of drug resistance among bacteria pathogens, bacteriophage (phage) therapy is a potential alternative to antibiotics for treating multidrug-resistant bacterial infections. However, so far, no phages specific for Elizabethkingia spp. have been reported. Using a clinically isolated Elizabethkingia anophelis as the host, the phage TCUEAP1 was isolated from wastewater of Hualien Tzu Chi hospital. The phage particle of TCUEAP1 under electron microscopy was revealed to belong to the siphoviridae family, with a head size of 47 nm, and a tail dimension 12 nm in diameter and 172 nm in length. The one-step growth analysis showed that the latent period of TCUEAP1 was about 40 min with a rise period lasting about 20 min, yielding a burst size of approximately 10 PFU/cell. The adsorption rate of TCUEAP1 reached about 70% in 20 min. Using 20 isolates of Elizabethkingia spp. to test the host range of TCUEAP1, it displayed narrow spectrum infecting three strains of E. anophelis, but forming spot lysis on 16 strains. The sequence result showed that the genome of TCUEAP1 is a double-stranded DNA of 49,816 bp, containing 73 predicted open reading frames. Further genomic analysis showed TCUEAP1 to be a new phage with no resemblance to publicly available phage genomes. Finally, in a mouse intraperitoneal infection model, at 6 h after the bacterial injection, TCUEAP1 decreased the bacterial load by fivefold in blood. Also, TCUEAP1 rescued 80% of mice heavily infected with E. anophelis from lethal bacteremia. We hope that the isolation and characterization of TCUEAP1, the first phage infecting Elizabethkingia spp., can promote more studies of the phages targeting this newly emerging bacterial pathogen.

The PlyB Endolysin of Bacteriophage vB_BanS_Bcp1 Exhibits Broad-Spectrum Bactericidal Activity against Bacillus cereus Sensu Lato Isolates.

Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereussensu lato group species, including B. cereussensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereussensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereussensu lato isolates tested (n = 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium.IMPORTANCE Organisms of the Bacillus cereussensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereussensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereussensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.

Protection against Staphylococcus aureus bacteremia-induced mortality depends on ILC2s and eosinophils.

The dysregulated, unbalanced immune response of sepsis results in a mortality exceeding 20%, yet recent findings by our group indicate that patients with allergic, type 2-mediated immune diseases are protected from developing sepsis. We evaluated CD4+ Th cell polarization among patients with Staphylococcus aureus bacteremia and confirmed that survivors had a higher percentage of circulating Th2 cells but lower frequencies of Th17 cells and neutrophils early in the course of infection. To establish the mechanism of this protection, we used a mouse model of lethal S. aureus bacteremia and found that intratracheal pretreatment with the type 2-initiating cytokine IL-33 activated pulmonary type 2 innate lymphoid cells (ILC2s) and promoted eosinophilia. In addition, stimulation of type 2 immunity before lethal infection suppressed the pulmonary neutrophilic response to S. aureus. Mice lacking functional ILC2s did not respond to IL-33 and were not protected from lethal bacteremia, but treatment of these mice with the type 2 cytokines IL-5 and IL-13 rescued them from death. Depletion of eosinophils abrogated IL-33-mediated protection, indicating that eosinophilia is also necessary for the survival benefit. Thus, we have identified a potentially novel mechanism by which type 2 immunity can balance dysregulated septic inflammatory responses, thereby clarifying the protective benefit of type 2 immune diseases on sepsis mortality.

Therapeutic Potential of a Scorpion Venom-Derived Antimicrobial Peptide and Its Homologs Against Antibiotic-Resistant Gram-Positive Bacteria.

The alarming rise in the prevalence of antibiotic resistance among pathogenic bacteria poses a unique challenge for the development of effective therapeutic agents. Antimicrobial peptides (AMPs) have attracted a great deal of attention as a possible solution to the increasing problem of antibiotic-resistant bacteria. Marcin-18 was identified from the scorpion Mesobuthus martensii at both DNA and protein levels. The genomic sequence revealed that the marcin-18 coding gene contains a phase-I intron with a GT-AG splice junction located in the DNA region encoding the N-terminal part of signal peptide. The peptide marcin-18 was also isolated from scorpion venom. A protein sequence homology search revealed that marcin-18 shares extremely high sequence identity to the AMPs meucin-18 and megicin-18. In vitro, chemically synthetic marcin-18 and its homologs (meucin-18 and megicin-18) showed highly potent inhibitory activity against Gram-positive bacteria, including some clinical antibiotic-resistant strains. Importantly, in a mouse acute peritonitis model, these peptides significantly decreased the bacterial load in ascites and rescued nearly all mice heavily infected with clinical methicillin-resistant Staphylococcus aureus from lethal bacteremia. Peptides exerted antimicrobial activity via a bactericidal mechanism and killed bacteria through membrane disruption. Taken together, marcin-18 and its homologs have potential for development as therapeutic agents for treating antibiotic-resistant, Gram-positive bacterial infections.

Quorum‐sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4-mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.

Severe deficiency in vaccine and immune responses to Streptococcus peumoniae of growth hormone (GH) deficient mice with GHRH gene ablation

Event Abstract Back to Event Severe deficiency in vaccine and immune responses to Streptococcus peumoniae of growth hormone (GH) deficient mice with GHRH gene ablation Khalil Farhat1, 2, Gwennaëlle Bodart3, Anne-Simone Parent3, Roberto Salvatori4, Henri J. Martens2 and Vincent Geenen1* 1 Université de Liège, Belgium 2 GIGA Inflammation, Infection et Immunité, Université de Liège, Belgium 3 Centre Hospitalier Universitaire de Liège, Belgium 4 The Johns Hopkins Hospital, Johns Hopkins Medicine, United States The precise impact of the somatotrope axis upon the immune system is still highly debated. From neurons in the hypothalamic arcuate nucleus, growth hormone (GH) releasing hormone (GHRH) controls the entire GHRH/GH/IGF-1 somatotrope axis. We have previously shown that mice with generalized ablation of growth hormone (GH) releasing hormone (GHRH) gene (Ghrh-/-) have normal thymus and T-cell development, but present a marked spleen atrophy and relative B-cell lymphopenia. Therefore, in this study we have investigated vaccinal and anti-infectious responses of Ghrh-/- mice against Streptococcus (S) pneumoniae, a pathogen carrying T-independent antigens. Ghrh-/- mice were unable to trigger production of specific IgM after vaccination with either native pneumococcal polysaccharides (PPS, PPV23) or protein-PPS conjugate (PCV13). Human GH supplementation (Genotonorm 2µg/g for 5 weeks) of Ghrh-/- mice restored IgM response to PPV23 vaccine but not to PCV13 suggesting that GH could exert a specific impact on the spleen marginal zone that is strongly implicated in T-independent response against pneumococcal polysaccharides. After an intranasal administration of a sublethal dose of S. pneumoniae, wild type (WT) completely cleared bacteria after 24 h, as expected. In marked contrast, Ghrh-/- mice exhibited a dramatic susceptibility to S. pneumoniae infection with a time-dependent increase in lung bacterial load and a lethal bacteremia already after 24 h. Lungs of infected Ghrh-/- mice were massively infiltrated by inflammatory macrophages and neutrophils, while lung B cells were markedly decreased. The inflammatory transcripts signature was also significantly elevated in Ghrh-/- mice. By flow cytometry analysis, the percentage of spenic marginal and follicular B cells was signifantly decreased in the spleen of infected KO mice. The distribution of a special subset of splenic macrophages identified by an anti-macrophage receptor with collagenous structure (MARCO) was also also severely affected in the spleen of infected Ghrh-/- mice. To verify whether Ghrh-/- mice have a general susceptibility to pathogens, we infected WT and KO mice with a thymus-dependent antigen: the H1N1 murine influenza virus. Infected animals exhibit a restrained mobility from day 4 to day 10, associated with a rapid respiration and fever that reach the lowest level at day 10. No difference in body weight was observed between WT and KO mice after a sublethal dose of H1N1, and KO even gained more weight after 16 days. During a supralethal H1N1 infection, both strains reached death limit point at day 6 for Ghrh-/- mice or day 8 for WT mice. This animal model unambiguously demonstrates that the somatotrope GHRH/GH/IGF1 axis plays a vital and unsuspected role in vaccine and immunological defense against S. pneumoniae. In a translational perspective, the data indicate that spleen development and response to anti-pneumococcus vaccines could be cautiously monitored in children with isolated GH deficiency, from congenital and genetic origin in particular. Acknowledgements This study was supported by grants from Association of Specialization and Scientific Guidance (Lebanon), F.R.S-FNRS, Fonds Léon Fredericq, Wallonia and W-B Federation. VG is Research director at F.R.S-FNRS. We are grateful to L. Duwez, F. Olivier, G. Lambert and Pr P. Drion from the GIGA Animal Facility for animal management, as well as the Cell Imaging and Flow Cytometry GIGA Technological Platforms. We also thank Pfizer for kindly providing Genotonorm (recombinant hGH).

Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2

The increase in the prevalence of multidrug-resistant Acinetobacter baumannii (MDRAB) strains is a serious public health concern. Antimicrobial peptides (AMPs) are a possible solution to this problem. In this study, we examined whether AMPs could be derived from phage endolysins. We synthesized four AMPs based on an amphipathic helical region in the C-terminus of endolysin LysAB2 encoded by the A. baumannii phage ΦAB2. These peptides showed potent antibacterial activity against A. baumannii (minimum inhibitory concentration, 4–64 μM), including some MDR and colistin-resistant A. baumannii. Of the four peptides, LysAB2 P3, with modifications that increased its net positive charge and decreased its hydrophobicity, showed high antibacterial activity against A. baumannii but little haemolytic and no cytotoxic activity against normal eukaryotic cells. The results of electron microscopy experiments and a fluorescein isothiocyanate staining assay indicated that this peptide killed A. baumannii through membrane permeabilization. Moreover, in a mouse intraperitoneal infection model, at 4 h after the bacterial injection, LysAB2 P3 decreased the bacterial load by 13-fold in ascites and 27-fold in blood. Additionally, LysAB2 P3 rescued sixty percent of mice heavily infected with A. baumannii from lethal bacteremia. Our results confirmed that bacteriophage endolysins are a promising resource for developing effective AMPs.

Transdermally administered proline-arginine-rich host defense peptides show systemic efficacy in a lethal mouse bacteremia model.

Host defense peptides are preferably administered as topical therapeutic agents. We have investigated whether peptide A3-APO can enter the circulation when applied to the ear skin. Efficacy of peptide monotherapy as transdermal administration option was assessed in a systemic mouse Acinetobacter baumannii model. A3-APO reduced mortality and demonstrated a statistically significant reduction of blood bacterial counts, regardless whether it was administered prior or after bacterial challenge. The peptidic metabolite of A3-APO was efficacious when applied to the ear or tail.

In vivo Lipopolysaccharide Tolerance Recruits CD11b+ Macrophages to the Liver with Enhanced Bactericidal Activity and Low Tumor Necrosis Factor-Releasing Capability, Resulting in Drastic Resistance to Lethal Septicemia

Objectives: In vivo lipopolysaccharide (LPS) tolerance on bacterial infection was investigated, focusing on liver macrophages. Methods: LPS tolerance was induced by intraperitoneal injections with 5 μg/kg of LPS for 3 consecutive days, and then mice were intravenously infected with Escherichia coli. Results: All LPS-primed mice survived lethal bacterial infection. Drastic enhancement of bactericidal activity of liver macrophages strongly contributed to bacterial clearance. Although LPS-primed mice produced substantial amounts of tumor necrosis factor (TNF) inside the liver, TNF efflux into the systemic circulation was markedly suppressed. These mice showed a dramatic increase in CD11b⁺ monocyte- derived macrophages in the liver. The CD11b⁺ macrophages that increased in LPS-primed mice were those with strong phagocytic/bactericidal activity and an upregulated expression of Fcγ receptor I, but the subfraction with a potent TNF-producing capacity and poor phagocytic activity diminished. The adoptive transfer of CD11b⁺ macrophages from LPS-primed mice to control mice increased survival after bacterial infection and reduced the elevation of plasma TNF. LPS priming did not affect the CD68⁺ resident Kupffer cells, and CD68⁺ Kupffer cell-depleted mice still exhibited LPS tolerance with strong resistance to bacteremia. Conclusions: LPS tolerance recruits CD11b⁺ macrophages to the liver with enhanced bactericidal activity, which plays a central role in resistance to lethal bacteremia.

Differential expression and roles of Staphylococcus aureus virulence determinants during colonization and disease.

ABSTRACTStaphylococcus aureus is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of sdrC, fnbA, fhuD, sstD, and hla was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens.

Isotype switching increases efficacy of antibody protection against staphylococcal enterotoxin B-induced lethal shock and Staphylococcus aureus sepsis in mice.

ABSTRACTStaphylococcal enterotoxin B (SEB) is a potent toxin that is produced by Staphylococcus aureus strains and is classified as a category B select agent. We have previously shown that monoclonal antibody (MAb) 20B1, a murine anti-SEB IgG1, successfully treats SEB-induced lethal shock (SEBILS) and bacteremia that is caused by SEB-producing S. aureus. In this study, we have generated two isotype switch variants of the original IgG1 MAb 20B1, an IgG2a and IgG2b, both bearing the same variable region sequence, and compared their neutralizing and protective activity in in vitro and in vivo assays, respectively. All 3 isotypes demonstrated comparable affinity to SEB and comparable 50% inhibitory concentrations (IC50s) in T cell proliferation assays. In vivo, however, the IgG2a isotype variant of 20B1 exhibited significantly greater protection than IgG1 or IgG2b in murine SEB intoxication and S. aureus sepsis models. Protection was associated with downmodulation of inflammatory host response. Our data demonstrate that changing the isotype of already protective MAbs, without affecting their antigen specificity or sensitivity, can result in an enhancement of their protective ability. Isotype selection, therefore, should be carefully considered in the development of toxin-neutralizing MAbs and the design of antibody therapeutics.

Anthrax SET Protein: A POTENTIAL VIRULENCE DETERMINANT THAT EPIGENETICALLY REPRESSES NF-κB ACTIVATION IN INFECTED MACROPHAGES

Toxins play a major role in the pathogenesis of Bacillus anthracis by subverting the host defenses. However, besides toxins, B. anthracis expresses effector proteins, whose role in pathogenesis are yet to be investigated. Here we present that suppressor-of-variegation, enhancer-of-zeste, trithorax protein from B. anthracis (BaSET) methylates human histone H1, resulting in repression of NF-κB functions. Notably, BaSET is secreted and undergoes nuclear translocation to enhance H1 methylation in B. anthracis-infected macrophages. Compared with wild type Sterne, delayed growth kinetics and altered septum formation were observed in the BaSET knock-out (BaΔSET) bacilli. Uncontrolled BaSET expression during complementation of the BaSET gene in BaΔSET partially restored growth during stationary phase but resulted in substantially shorter bacilli throughout the growth cycle. Importantly, in contrast to Sterne, the BaΔSET B. anthracis is avirulent in a lethal murine bacteremia model of infection. Collectively, BaSET is required for repression of host transcription as well as proper B. anthracis growth, making it a potentially unique virulence determinant.