Travis B. Nielsen

Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges

SUMMARY Acinetobacter is a complex genus, and historically, there has been confusion about the existence of multiple species. The species commonly cause nosocomial infections, predominantly aspiration pneumonia and catheter-associated bacteremia, but can also cause soft tissue and urinary tract infections. Community-acquired infections by Acinetobacter spp. are increasingly reported. Transmission of Acinetobacter and subsequent disease is facilitated by the organisms environmental tenacity, resistance to desiccation, and evasion of host immunity. The virulence properties demonstrated by Acinetobacter spp. primarily stem from evasion of rapid clearance by the innate immune system, effectively enabling high bacterial density that triggers lipopolysaccharide (LPS)–Toll-like receptor 4 (TLR4)-mediated sepsis. Capsular polysaccharide is a critical virulence factor that enables immune evasion, while LPS triggers septic shock. However, the primary driver of clinical outcome is antibiotic resistance. Administration of initially effective therapy is key to improving survival, reducing 30-day mortality threefold. Regrettably, due to the high frequency of this organism having an extreme drug resistance (XDR) phenotype, early initiation of effective therapy is a major clinical challenge. Given its high rate of antibiotic resistance and abysmal outcomes (up to 70% mortality rate from infections caused by XDR strains in some case series), new preventative and therapeutic options for Acinetobacter spp. are desperately needed.

Host Fate Is Rapidly Determined by Innate Effector-Microbial Interactions During Acinetobacter baumannii Bacteremia

BACKGROUND Acinetobacter baumannii is one of the most antibiotic-resistant pathogens. Defining mechanisms driving pathogenesis is critical to enable new therapeutic approaches. METHODS We studied virulence differences across a diverse panel of A. baumannii clinical isolates during murine bacteremia to elucidate host-microbe interactions that drive outcome. RESULTS We identified hypervirulent strains that were lethal at low intravenous inocula and achieved very high early, and persistent, blood bacterial densities. Virulent strains were nonlethal at low inocula but lethal at 2.5-fold higher inocula. Finally, relatively avirulent (hypovirulent) strains were nonlethal at 20-fold higher inocula and were efficiently cleared by early time points. In vivo virulence correlated with in vitro resistance to complement and macrophage uptake. Depletion of complement, macrophages, and neutrophils each independently increased bacterial density of the hypovirulent strain but insufficiently to change lethality. However, disruption of all 3 effector mechanisms enabled early bacterial densities similar to hypervirulent strains, rendering infection 100% fatal. CONCLUSIONS The lethality of A. baumannii strains depends on distinct stages. Strains resistant to early innate effectors are able to establish very high early bacterial blood density, and subsequent sustained bacteremia leads to Toll-like receptor 4-mediated hyperinflammation and lethality. These results have important implications for translational efforts to develop therapies that modulate host-microbe interactions.

Transferrin Iron Starvation Therapy for Lethal Bacterial and Fungal Infections

New strategies to treat antibiotic-resistant infections are urgently needed. We serendipitously discovered that stem cell conditioned media possessed broad antimicrobial properties. Biochemical, functional, and genetic assays confirmed that the antimicrobial effect was mediated by supra-physiological concentrations of transferrin. Human transferrin inhibited growth of gram-positive (Staphylococcus aureus), gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens by sequestering iron and disrupting membrane potential. Serial passage in subtherapeutic transferrin concentrations resulted in no emergence of resistance. Infected mice treated with intravenous human transferrin had improved survival and reduced microbial burden. Finally, adjunctive transferrin reduced the emergence of rifampin-resistant mutants of S. aureus in infected mice treated with rifampin. Transferrin is a promising, novel antimicrobial agent that merits clinical investigation. These results provide proof of principle that bacterial infections can be treated in vivo by attacking host targets (ie, trace metal availability) rather than microbial targets.

Monoclonal Antibody Protects Against Acinetobacter baumannii Infection by Enhancing Bacterial Clearance and Evading Sepsis

Background Extremely drug-resistant (XDR) Acinetobacter baumannii is one of the most commonly encountered, highly resistant pathogens requiring novel therapeutic interventions. Methods We developed C8, a monoclonal antibody (mAb), by immunizing mice with sublethal inocula of a hypervirulent XDR clinical isolate. Results 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. Conclusions 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.

Ly6G-mediated depletion of neutrophils is dependent on macrophages.

Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.

Cryopreservation of virulent Acinetobacter baumannii to reduce variability of in vivo studies.

BackgroundMicrobiological assays require accurate and reproducible preparation of bacterial inocula. Inocula prepared on different days by different individuals can vary significantly from experiment to experiment. This variance is particularly problematic for Gram-negative bacterial infections, for which threshold effects can result in marked variations in host outcome with minor differences in inocula.ResultsWe compared the accuracy of traditional methods versus using frozen stocks for preparing Acinetobacter baumannii inocula for infection in mice. Standard inoculum preparation resulted in substantial variability, both with respect to the actual inocula achieved compared to the targeted inocula, and with respect to the in vivo outcome resulting from similar inocula. Cryopreservation of the bacteria resulted in no significant decrement in growth of the bacteria. Furthermore, preparation of multiple infectious inocula from a frozen stock significantly improved the accuracy of the achieved inocula, and resulted in more reproducible in vivo outcomes from infection. Frozen stocks reduced inter-experiment variability associated with inoculum preparation, displayed no significant loss of growth capacity, and maintained virulence, increasing the reliability of infection.ConclusionsFrozen stocks require considerably less time to prepare and enhance reproducibility of in vivo experimental results when infecting with A. baumannii.

Diabetes Exacerbates Infection via Hyperinflammation by Signaling through TLR4 and RAGE

ABSTRACT 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. IMPORTANCE Physicians 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 on ex vivo studies 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. IMPORTANCE Physicians 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 on ex vivo studies 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.

Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1.

Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics. ABSTRACT Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

Evaluation of serotypes 5 and 8 capsular polysaccharides in protection against Staphylococcus aureus in murine models of infection

ABSTRACT Staphylococcus aureus is the leading cause of nosocomial and community-acquired infections, including soft tissue and skin infections and bacteremia. However, efforts to develop an effective vaccine against S. aureus infections have not been successful. We evaluated serotypes 5 and 8 capsule polysaccharides (CP) CRM197 conjugates as vaccine candidates in murine models of bacteremia, lethal sepsis, and skin infection. The conjugate vaccines elicited a good antibody response, and active immunization of CP5-CRM or CP8-CRM conjugates protected against staphylococcal bacteremia. In the skin infection model, CP8-CRM but not CP5-CRM protected against dermonecrosis, and CP8-CRM immunization significantly decreased the bacterial burden in the lesion. However, neither CP5-CRM nor CP8-CRM protected against mortality in the lethal sepsis model. The results indicate the capsular vaccines elicit protection against some, but not all, aspects of staphylococcal infection.

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

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.