Acute intraperitoneal infection with a hypervirulent Acinetobacter baumannii isolate in mice.

Acinetobacter baumannii infection has become a major cause of healthcare-associated infection and a critical pathogen in the WHO antimicrobial resistance research and development priority list. Catheter-related septicemia is one of the major clinical manifestations of A. baumannii infection associated with high morbidity and mortality. In this study, we used a clinical A. baumannii strain (LAC-4) that is hypervirulent to immunocompetent C57BL/6 and BALB/c mice and established a mouse model of intraperitoneal (i.p.) A. baumannii infection. Our study showed that i.p. LAC-4 infection of C57BL/6 and BALB/c mice induces a lethal or sublethal infection with high bacterial burdens in peritoneal cavity, blood and tissues and the infected mice either succumbed to the infection within 24 hours or completely recovered from the infection. The infection induces acute peritoneal recruitment of neutrophils and other innate immune cells, and the local and systemic production of proinflammatory cytokines and chemokines (IL-1ß, IL-5, IL-6, TNF-α, RANTES, MIP-1ß, MCP-1, KC and IL-10). Mechanistic studies suggest an important role of macrophages in the host innate defense in this model in that in vitro stimulation of peritoneal macrophages with killed LAC-4 induced a similar pattern of cytokine/chemokine responses to those in the infected mice, and depletion of peritoneal macrophages rendered the mice significantly more susceptible to the infection. Thus, this mouse infection model will provide an alternative and useful tool for future pathogenesis studies of A. baumannii-associated septicemia and identification and characterization of important virulence factors, as well as serve as a surrogate model for rapid evaluation of novel therapeutics and vaccines for this emerging infectious agent.

Mouse Models of Acinetobacter baumannii Infection.

This unit describes basic protocols for infecting mice through intranasal and intraperitoneal routes with Acinetobacter baumannii to induce associated pneumonia and sepsis, the two most common manifestations of clinical infections with this pathogen. By selecting the appropriate protocols and bacterial strains of different virulence, these mouse models provide an opportunity to study the infection pathogenesis and host-immune responses, and to evaluate the efficacies of prophylactic and therapeutic anti-A. baumannii candidates. © 2017 by John Wiley & Sons, Inc.

Host resistance to intranasal Acinetobacter baumannii reinfection in mice.

Acinetobacter baumannii is a major causative agent of healthcare-associated infection and develops multidrug resistance rapidly. However, little is known in the host defense mechanisms against this infection. In this study, we examined if mice recovered from a previous intranasal A. baumannii infection (recovered mice) are fully protected against a subsequent reinfection. We found that, despite the presence of specific serum IgG and mucosal IgA responses prior to the reinfection, the recovered mice were only marginally better protected against intranasal challenge with low doses of homologous or heterologous A. baumannii strains than the naïve mice. Post-challenge immune and inflammatory (cells and cytokines) responses were generally comparable between recovered and naïve mice although the recovered mice produced significantly higher amounts of IFN-γ and IL-17 and had higher percentages and numbers of resident lung CD44(hi)CD62L(-)CD4(+) and CD19(+) B lymphocytes. Taken together, our results suggest that mice recovered from a previous A. baumannii infection remain susceptible to reinfection, indicating the complexity of immune protection mechanism for this Gram-negative, multidrug-resistant emerging pathogen.

Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice.

Multidrug-resistant Acinetobacter baumannii has become an important causative agent of healthcare associated infections. Hospital- and community-acquired pneumonia is the most common clinical manifestation of A. baumannii infection worldwide and is often associated with high mortality. Most experimental vaccine studies to date have evaluated vaccines against systemic A. baumannii infections following systemic immunization. We recently demonstrated that a mouse model of respiratory A. baumannii infection using the strain LAC-4 results in disease progression that is similar to that observed in humans. Here we used this model in conjunction with an inactivated whole cell vaccine to evaluate the feasibility of developing protective mucosal vaccines against respiratory A. baumannii infection and to investigate the potential mechanism of protection of such vaccines. Our results showed that intranasal immunization with formalin-killed whole cells of the LAC-4 strain elicited mucosal and systemic antigen-specific immune responses, and protected mice against lethal intranasal or intraperitoneal challenges. Compared to naïve mice, immunized mice had significantly fewer bacteria in their lungs, and the pathogen was barely detectable in blood and spleens at 24h post challenge, indicating the ability of immunized mice to control extrapulmonary dissemination of the pathogen. Mechanistic studies using gene-deficient mice, neutropenic mice, or passive immunization showed that B cells and neutrophils, but not FcRγ, played crucial roles in the protection against respiratory A. baumannii challenge of intranasally immunized mice whereas passive transfer of hyperimmune sera only prolonged the survival time of challenged mice by 48 h. These results provide immunological insights for the rational design of novel mucosal vaccines to protect against respiratory A. baumannii infection and demonstrate the feasibility to develop such vaccines.

Tolerance to cocaine in brain stimulation reward following continuous cocaine infusions.

This study examined tolerance to cocaine's threshold-lowering effect in brain stimulation reward (BSR) following continuous cocaine infusions and secondly, used the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) to determine NO's involvement in the development of cocaine tolerance. Animals were continuously infused with saline or cocaine (30 mg/kg per day) via osmotic minipump for 14 days and injected daily with saline or L-NAME (30 mg/kg, i.p.) following BSR testing. Saline-treated animals continuously infused with saline showed stable BSR thresholds across the 14-day infusion period. Saline-treated animals continuously infused with cocaine showed markedly lowered BSR thresholds on Day 1 followed by a progressive increase in BSR thresholds across the infusion period - indicating the development of tolerance. L-NAME-treated animals continuously infused with cocaine showed stimulation thresholds that were not significantly different from saline-treated animals continuously infused with cocaine. A cocaine challenge injection (10 mg/kg, i.p.) administered 3 and again at 10 days following minipump removal revealed that saline-treated animals continuously infused with saline showed lowered BSR thresholds. Saline-treated animals continuously infused with cocaine displayed lowered BSR thresholds that were not significantly different from saline-infused animals. L-NAME treated animals continuously infused with cocaine showed higher BSR thresholds to a challenge 3 days following pump removal. However, stimulation thresholds for this group failed to reach statistical significance on both days (i.e., Days 3 and 10) following pump removal. Results showed that animals continuously infused with cocaine develop robust tolerance to cocaine's threshold-lowering effect during the 14-day infusion period. Tolerance to cocaine's threshold-lowering effect was short-lived and dissipated soon after minipump removal. L-NAME treatment failed to significantly alter the development of tolerance to cocaine's threshold-lowering suggesting that NO does not have a primary role in the development of cocaine tolerance.

Serum resistance, gallium nitrate tolerance and extrapulmonary dissemination are linked to heme consumption in a bacteremic strain of Acinetobacter baumannii.

Bacteremia caused by Acinetobacter baumannii is a highly lethal complication of hospital-acquired pneumonia. In the present study, we investigated the serum resistance, gallium nitrate tolerance and heme consumption of A. baumannii strain LAC-4 which was recently reported to display high virulence in a mouse pneumonia model with extrapulmonary dissemination leading to fatal bacteremia. This strain showed enhanced growth in mouse and fetal bovine serum that was independent of complement and was not observed with regular growth media. The LAC-4 strain was found to possess a high tolerance to gallium nitrate (GaN), whereas serum synergized with GaN in inhibiting A. baumannii strain ATCC 17978. We found that LAC-4 contains a heme oxygenase gene and expresses a highly efficient heme consumption system. This system can be fully blocked in vitro and in vivo by gallium protoporphyrin IX (GaPPIX). Inhibition of heme consumption by GaPPIX completely abrogated the growth advantage of LAC-4 in serum as well as its tolerance to GaN. More importantly, GaPPIX treatment of mice intranasally infected with LAC-4 prevented extrapulmonary dissemination and death. Thus, we propose that heme provides an additional source of iron for LAC-4 to bypass iron restriction caused by serum transferrin, lactoferrin or free gallium salts. Heme consumption systems in A. baumannii may constitute major virulence factors for lethal bacteremic isolates.

Modulation of toxin production by the flagellar regulon in Clostridium difficile.

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile.

In vitro and in vivo biological activities of iron chelators and gallium nitrate against Acinetobacter baumannii.

We investigated the ability of compounds interfering with iron metabolism to inhibit the growth of Acinetobacter baumannii. Iron restriction with transferrin or 2,2-bipyridyl significantly inhibited A. baumannii growth in vitro. Gallium nitrate alone was moderately effective at reducing A. baumannii growth but became bacteriostatic in the presence of serum or transferrin. More importantly, gallium nitrate treatment reduced lung bacterial burdens in mice. The use of gallium-based therapies shows promise for the control of multidrug-resistant A. baumannii.

Role of macrophages in early host resistance to respiratory Acinetobacter baumannii infection.

Acinetobacter baumannii is an emerging bacterial pathogen that causes nosocomial pneumonia and other infections. Although it is recognized as an increasing threat to immunocompromised patients, the mechanism of host defense against A. baumannii infection remains poorly understood. In this study, we examined the potential role of macrophages in host defense against A. baumannii infection using in vitro macrophage culture and the mouse model of intranasal (i.n.) infection. Large numbers of A. baumannii were taken up by alveolar macrophages in vivo as early as 4 h after i.n. inoculation. By 24 h, the infection induced significant recruitment and activation (enhanced expression of CD80, CD86 and MHC-II) of macrophages into bronchoalveolar spaces. In vitro cell culture studies showed that A. baumannii were phagocytosed by J774A.1 (J774) macrophage-like cells within 10 minutes of co-incubation, and this uptake was microfilament- and microtubule-dependent. Moreover, the viability of phagocytosed bacteria dropped significantly between 24 and 48 h after co-incubation. Infection of J774 cells by A. baumannii resulted in the production of large amounts of proinflammatory cytokines and chemokines, and moderate amounts of nitric oxide (NO). Prior treatment of J774 cells with NO inhibitors significantly suppressed their bactericidal efficacy (P<0.05). Most importantly, in vivo depletion of alveolar macrophages significantly enhanced the susceptibility of mice to i.n. A. baumannii challenge (P<0.01). These results indicate that macrophages may play an important role in early host defense against A. baumannii infection through the efficient phagocytosis and killing of A. baumannii to limit initial pathogen replication and the secretion of proinflammatory cytokines and chemokines for the rapid recruitment of other innate immune cells such as neutrophils.

Design and immunological properties of Helicobacter pylori glycoconjugates based on a truncated lipopolysaccharide lacking Lewis antigen and comprising an α-1,6-glucan chain.

To investigate the vaccine potential of H. pylori lipopolysaccharide (LPS), truncated LPS of H. pylori strain 26695 HP0826::Kan lacking O-chain polysaccharide and comprising an extended α-1,6-linked glucan chain was conjugated to tetanus toxoid (TT) or bovine serum albumin (BSA). Two approaches were used for delipidation or partial delipidation of H. pylori LPS: (1) mild hydrolysis resulting in delipidated LPS (dLPS) and (2) treatment with anhydrous hydrazine resulting in removal of O-linked fatty acids (LPS-OH). Both LPS-OH and dLPS were covalently linked through a 2-keto-3-deoxy-octulosonic acid (Kdo) residue to a diamino group-containing spacer, followed by conjugation to thiolated TT or BSA to give conjugates LPS-OH-TT, dLPS-BSA and dLPS-TT, respectively. The LPS-OH-TT, dLPS-BSA and dLPS-TT conjugates were immunogenic in both rabbits and mice, inducing strong and specific IgG responses against homologous and heterologous strains of H. pylori. Moreover, the rabbit post-immune sera showed cross-reactivity against clinical isolates of H. pylori in a whole-cell indirect ELISA, which was further confirmed by indirect immunofluorescent microscopy. A tenfold stronger IgG immune response to the immunizing antigen was generated in mice and rabbits that received dLPS-containing conjugate. The post-immune sera of rabbits immunized with LPS-OH-TT, dLPS-BSA or dLPS-TT displayed significant bactericidal activity against mutant and wild-type α-1,6-glucan-expressing strains and selected clinical isolates of H. pylori. Finally, partial protection against H. pylori challenge was demonstrated in mice vaccinated with dLPS-TT conjugate adjuvanted with cholera toxin. In summary, this study shows that glycoconjugates based on delipidated or partially delipidated LPS from H. pylori 26695 HP0826::Kan mutant induce broadly cross-reactive functional antibodies in immunized animals and should be considered for further vaccine development and testing.

Simultaneous analysis of cardiolipin and lipid A from Helicobacter pylori by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cardiolipin (CL) is an anionic tetraacylphospholipid found in mammalian tissues, inner membrane of mitochondria and in the cytoplasmic membrane of Gram-positive and -negative bacteria. Lipid A is the principal structural component responsible for the range of biological activities of lipopolysaccharides. Here we report a MALDI-MS-based method for the sensitive simultaneous analysis of CL and lipid A from Helicobacter pylori cells. The sensitivity was demonstrated by the analysis of CL and lipid A from a single bacterial colony of in vitro grown H. pylori strain NCTC 11637 (ATCC 43504). We then characterized the CL and lipid A structures in H. pylori cells grown under three different conditions, on agar-horse blood plates, in liquid culture and ex vivo. The results revealed the presence of high amounts of myristic (C14:0) and 19-carbon cyclopropane (C19:0cyc) fatty acids. Alterations in CL structure were observed in H. pylori cells cultivated on plates as compared with the bacteria grown in broth culture. Furthermore, significant changes in lipid A acylation pattern were detected in H. pylori cells during formation of coccoids. In contrast, structural analysis of CL from ex vivo H. pylori cells recovered from the stomachs of infected Mongolian gerbils demonstrated only minor changes in acyl chain combination. This is the first report of simultaneous analysis of CL and lipid A from ex vivo cells of H. pylori.

Acinetobacter baumannii infection inhibits airway eosinophilia and lung pathology in a mouse model of allergic asthma.

Allergic asthma is a dysregulation of the immune system which leads to the development of Th2 responses to innocuous antigens (allergens). Some infections and microbial components can re-direct the immune response toward the Th1 response, or induce regulatory T cells to suppress the Th2 response, thereby inhibiting the development of allergic asthma. Since Acinetobacter baumannii infection can modulate lung cellular and cytokine responses, we studied the effect of A. baumannii in modulating airway eosinophilia in a mouse model of allergic asthma. Ovalbumin (OVA)-sensitized mice were treated with live A. baumannii or phosphate buffered saline (PBS), then intranasally challenged with OVA. Compared to PBS, A. baumannii treatment significantly reduced pulmonary Th2 cytokine and chemokine responses to OVA challenge. More importantly, the airway inflammation in A. baumannii-treated mice was strongly suppressed, as seen by the significant reduction of the proportion and the total number of eosinophils in the bronchoalveolar lavage fluid. In addition, A. baumannii-treated mice diminished lung mucus overproduction and pathology. However, A. baumannii treatment did not significantly alter systemic immune responses to OVA. Serum OVA-specific IgE, IgG1 and IgG2a levels were comparable between A. baumannii- and PBS-treated mice, and tracheobronchial lymph node cells from both treatment groups produced similar levels of Th1 and Th2 cytokines in response to in vitro OVA stimulation. Moreover, it appears that TLR-4 and IFN-γ were not directly involved in the A. baumannii-induced suppression of airway eosinophilia. Our results suggest that A. baumannii inhibits allergic airway inflammation by direct suppression of local pulmonary Th2 cytokine responses to the allergen.

c-di-GMP protects against intranasal Acinetobacter baumannii infection in mice by chemokine induction and enhanced neutrophil recruitment.

Acinetobacter baumannii has emerged as a major cause of both community-associated and nosocomial infections worldwide. A. baumannii rapidly develops resistance to multiple antibiotics; as a result, infections by this pathogen have become increasingly difficult to treat. In this study, we evaluated the effect of 3',5'-cyclic diguanylic acid (c-di-GMP), a bacterial second messenger and immunomodulator, in the host defense against A. baumannii infection in a mouse model of intranasal infection. Our results showed that 50 µg of c-di-GMP administered 18 h prior to infection provided the best protection against intranasal infection with A. baumannii. Mechanistically, administration of c-di-GMP induced the production of chemokines KC, MCP-1, MIP-1α, MIP-2 and RANTES, and enhanced neutrophil recruitment in the lung. Moreover, depletion of neutrophils abolished the protective role of c-di-GMP. Taken together, our data suggest that c-di-GMP confers resistance against intranasal A. baumannii infection in mice through a neutrophil-dependent mechanism and that c-di-GMP should be further explored as an immunomodulator for the treatment of A. baumannii infection.

Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice.

Francisella tularensis subspecies (subsp.) tularensis is a CDC Category A biological warfare agent and inhalation of as few as 15 bacilli can initiate severe disease. Relatively little is known about the cellular and molecular mechanisms of host defense against respiratory infection with subsp. tularensis. In this study, we examined the role of neutrophils and NADPH phagocyte oxidase in host resistance to pulmonary infection in a mouse intranasal infection model. We found that despite neutrophil recruitment to the lungs and increased concentrations of neutrophil-chemotactic chemokines (KC, MIP-2 and RANTES) in the bronchoalveolar lavage fluid following intranasal inoculation of the pathogen, neither depletion of neutrophils nor enhancement of their recruitment into the lungs had any impact on bacterial burdens or survival rate/time. Nevertheless, mice deficient in NADPH phagocyte oxidase (gp91(phox⁻/⁻)) did exhibit higher tissue and blood bacterial burdens and succumbed to infection one day earlier than wild-type C57BL/6 mice. These results imply that although neutrophils are not a major effector cell in defense against subsp. tularensis infection, NADPH phagocyte oxidase does play a marginal role.

The potential of 3',5'-cyclic diguanylic acid (c-di-GMP) as an effective vaccine adjuvant.

3', 5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial intracellular signaling molecule that plays a crucial role in the regulation of bacterial motility, adhesion, cell-to-cell communication, exopolysaccharide synthesis, biofilm formation and virulence. The recent finding that c-di-GMP can act as a danger signal on eukaryotic cells has prompted the study of the immunostimulatory and immunomodulatory properties of c-di-GMP in an effort to determine whether c-di-GMP might be further developed as a potential vaccine adjuvant. In this review, we discussed the recent in vitro and in vivo studies of the immunostimulatory properties of c-di-GMP and the progress that has been made in the preclinical development of c-di-GMP as a potential vaccine adjuvant for systemic and mucosal vaccination.

3',5'-Cyclic diguanylic acid elicits mucosal immunity against bacterial infection.

3',5'-Cyclic diguanylic acid (cdiGMP) is emerging as a universal bacterial second messenger in regulating bacterial growth on surfaces. It has been recently shown that cdiGMP stimulates innate immunity and enhances antigen-specific humoral and cellular immune responses. We herein report that intranasal (i.n.) administration with cdiGMP induces an acute but transient inflammatory response and activation of dendritic cells in the lungs. Moreover, i.n. immunization of mice with pneumococcal surface adhesion A (PsaA) in conjunction with cdiGMP elicited strong antigen-specific serum immunoglobulin G (IgG) and secretory IgA antibody responses at multiple mucosal surfaces. More importantly, the immunized mice showed significantly reduced nasopharyngeal Streptococcus pneumoniae colonization. These results, for the first time, provide direct evidence for the induction of protection against mucosal bacterial infections by cdiGMP as an adjuvant.

High susceptibility to respiratory Acinetobacter baumannii infection in A/J mice is associated with a delay in early pulmonary recruitment of neutrophils.

Acinetobacter baumannii is an important cause of both community-associated and nosocomial pneumonia, which have become increasingly difficult to treat because of the rapid development of resistance to multiple antibiotics. Despite its clinical importance, the pathogenesis of and host defense against respiratory A. baumannii infection remains largely unknown. To examine host factors that could contribute to the defense, we compared the susceptibilities of A/J and C57BL/6 mice to intranasal (i.n.) inoculation with A. baumannii. We found that A/J mice were significantly more susceptible to infection with higher mortality (P<0.05) and tissue bacterial burdens (P<0.01) as well as greater histopathology in the lung and spleen than C57BL/6 mice. More importantly, the high susceptibility of A/J mice was associated with a reduced local proinflammatory cytokine/chemokine (particularly IL-1beta, MIP-2 and TNF-alpha) responses and a significant delay and reduction in the early influx of neutrophils in the lung (P<0.05). Intranasal administration of neutrophil-inducing chemokine MIP-2 to A/J mice enhanced pulmonary neutrophil influx and partially restored host resistance to A. baumannii to a level comparable to the more resistant C57BL/6 mice. Our results imply that the early recruitment of neutrophils into the lung is critical for initiating an efficient host defense against respiratory A. baumannii infection.

Pentabody-mediated antigen delivery induces antigen-specific mucosal immune response.

An efficient immunization system is essential for the development of mucosal vaccine. Cholera toxin (CT) and Escherichia coli heat labile toxin (LT) are among the strongest adjuvants tested in experimental animals but their use in humans has been hindered by their toxicity. On the other hand, the role of their non-toxic B-subunits, CTB or LTB, in enhancing mucosal immune response is not clear. We propose here a novel strategy for the induction of mucosal immune responses. Single domain antibodies (sdAbs) against a model antigen bovine serum albumin (BSA) were raised from the antibody repertoire of a llama immunized with BSA, pentamerized by fusing the sdAbs to CTB, generating the so-called pentabodies. These pentabodies were used to deliver the antigen by mixing the two components and administering the mixture to mice intranasally. One construct was equivalent to CT in helping induce mucosal immune response. It was also found that this ability was probably due to its high affinity to BSA, providing some insight into the controversial role of CTB in mucosal immunization: at least for BSA, the model antigen BSA employed in this study, CTB has to be tightly linked to the antigen to have adjuvant/immune-enhancing effect.

6th Annual Vaccines: all things considered.

The 6th Annual Vaccines: All Things Considered conference was hosted by GTCbio. The purpose of the conference was to provide a forum for researchers from academia, industry and government to update one another about novel vaccine developments, regulatory perspectives, new technologies and government policies. The meeting was attended by approximately 80 delegates, many from European countries, and included two keynote addresses, 20 oral presentations and 15 posters presented over a 2-day period. As part of the conference's tradition, two posters were selected for oral presentation. This meeting report will highlight the main issues discussed.

Role of NADPH phagocyte oxidase in host defense against acute respiratory Acinetobacter baumannii infection in mice.

Acinetobacter baumannii is an emerging bacterial pathogen that rapidly develops multiple-drug resistance and is responsible for many nosocomial pulmonary infections. This study investigated the role of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (NOS2) in the host defense against respiratory infection with A. baumannii in mouse models of intranasal A. baumannii infection. gp91(phox-/-) mice showed higher susceptibility to A. baumannii infection than wild-type (WT) C57BL/6 mice, with significantly greater bacterial counts in their lungs (1,000-fold) (P < 0.005) and spleens (10-fold) (P < 0.05). Moreover, all of the gp91(phox-/-) mice succumbed to infection within 48 h. In contrast, only a moderate increase in bacterial burdens was detected in the lungs of NOS2(-/-) mice, and all NOS2(-/-) mice survived infection. Compared to WT mice, the pulmonary influx of inflammatory cells and serum and local inflammatory cytokine/chemokine responses were not obviously impaired at 4 h and were significantly higher at 24 h (P < 0.05) in gp91(phox-/-) mice, but NADPH-deficient neutrophils were unable to control bacterial replication and extrapulmonary dissemination. Thus, NADPH phagocyte oxidase appears to play a crucial role in the neutrophil-mediated host defense against A. baumannii.