Acinetobacter baumanniiis a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence inA.baumanniiremain uncertain. By comparing genomes among a panel ofA.baumanniistrains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact genegtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenicgtr6mutants, we confirmed thatgtr6-disrupted strains were protected from phagocytosisin vitroand displayed higher bacterial burden and lethalityin vivo.Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illnessin vivo. We found that the CR3 receptor mediated phagocytosis ofgtr6+, but notgtr6-, strains in a complement-dependent manner. Furthermore, hypovirulentgtr6+strains demonstrated increased virulencein vivowhen CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multipleA.baumanniistrains. Thus, capsular structure can determine virulence amongA.baumanniistrains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Extremely drug-resistant (XDR) Acinetobacter baumannii is one of the most commonly encountered, highly resistant pathogens requiring novel therapeutic interventions. We developed C8, a monoclonal antibody (mAb), by immunizing mice with sublethal inocula of a hypervirulent XDR clinical isolate. C8 targets capsular carbohydrate on the bacterial surface, enhancing opsonophagocytosis. Treating with a single dose of C8 as low as 0.5 μg/mouse (0.0167 mg/kg) markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia models of XDR A. baumannii infection. C8 was also synergistic with colistin, substantially improving survival compared to monotherapy. Treatment with C8 significantly reduced blood bacterial density, cytokine production (tumor necrosis factor α, interleukin [IL] 6, IL-1β, and IL-10), and sepsis biomarkers. Serial in vitro passaging of A. baumannii in the presence of C8 did not cause loss of mAb binding to the bacteria, but did result in emergence of less-virulent mutants that were more susceptible to macrophage uptake. Finally, we developed a highly humanized variant of C8 that retains opsonophagocytic activity in murine and human macrophages and rescued mice from lethal infection. We describe a promising and novel mAb as therapy for lethal, XDR A. baumannii infections, and demonstrate that it synergistically improves outcomes in combination with antibiotics.

For more than a century, diabetic patients have been considered immunosuppressed due to defects in phagocytosis and microbial killing. We confirmed that diabetic mice were hypersusceptible to bacteremia caused by Gram-negative bacteria (GNB), dying at inocula nonlethal to nondiabetic mice. Contrary to the pervasive paradigm that diabetes impedes phagocytic function, the bacterial burden was no greater in diabetic mice despite excess mortality. However, diabetic mice did exhibit dramatically increased levels of proinflammatory cytokines in response to GNB infections, and immunosuppressing these cytokines with dexamethasone restored their resistance to infection, both of which are consistent with excess inflammation. Furthermore, disruption of the receptor for advanced glycation end products (RAGE), which is stimulated by heightened levels of AGEs in diabetic hosts, protected diabetic but not nondiabetic mice from GNB infection. Thus, rather than immunosuppression, diabetes drives lethal hyperinflammation in response to GNB by signaling through RAGE. As such, interventions to improve the outcomes from GNB infections should seek to suppress the immune response in diabetic hosts.IMPORTANCEPhysicians and scientists have subscribed to the dogma that diabetes predisposes the host to worse outcomes from infections because it suppresses the immune system. This understanding was based largely onex vivostudies of blood from patients and animals with diabetes. However, we have found that the opposite is true and worse outcomes from infection are caused by overstimulation of the immune system in response to bacteria. This overreaction occurs by simultaneous ligation of two host receptors: TLR4 and RAGE. Both signal via a common downstream messenger, MyD88, triggering hyperinflammation. In summary, contrary to hundred-year-old postulations about immune suppression in diabetic hosts, we find that diabetes instead predisposes to more severe infections because of additional inflammatory output through dual activation of MyD88 by not only TLR4 but also RAGE. It is the activation of RAGE during GNB infections in those with diabetes that accounts for their heightened susceptibility to infection compared to nondiabetic hosts.

Extremely drug-resistant (XDR) Acinetobacter baumannii is a notorious and frequently encountered pathogen demanding novel therapeutic interventions. An initial monoclonal antibody (MAb), C8, raised against A. baumannii capsule, proved a highly effective treatment against a minority of clinical isolates.

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 µL of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.