Improved characterization of aminoglycoside penetration into human lung epithelial lining fluid via population pharmacokinetics.

Aminoglycosides are important treatment options for serious lung infections, but modeling analyses to quantify their human lung epithelial lining fluid (ELF) penetration are lacking. We estimated the extent and rate of penetration for five aminoglycosides via population pharmacokinetics from eight published studies. The area under the curve in ELF vs plasma ranged from 50% to 100% and equilibration half-lives from 0.61 to 5.80 h, indicating extensive system hysteresis. Aminoglycoside ELF peak concentrations were blunted, but overall exposures were moderately high.

Population pharmacokinetics and humanized dosage regimens matching the peak, area, trough, and range of amikacin plasma concentrations in immune-competent murine bloodstream and lung infection models.

Amikacin is an FDA-approved aminoglycoside antibiotic that is commonly used. However, validated dosage regimens that achieve clinically relevant exposure profiles in mice are lacking. We aimed to design and validate humanized dosage regimens for amikacin in immune-competent murine bloodstream and lung infection models of Acinetobacter baumannii. Plasma and lung epithelial lining fluid (ELF) concentrations after single subcutaneous doses of 1.37, 13.7, and 137 mg/kg of body weight were simultaneously modeled via population pharmacokinetics. Then, humanized amikacin dosage regimens in mice were designed and prospectively validated to match the peak, area, trough, and range of plasma concentration profiles in critically ill patients (clinical dose: 25-30 mg/kg of body weight). The pharmacokinetics of amikacin were linear, with a clearance of 9.93 mL/h in both infection models after a single dose. However, the volume of distribution differed between models, resulting in an elimination half-life of 48 min for the bloodstream and 36 min for the lung model. The drug exposure in ELF was 72.7% compared to that in plasma. After multiple q6h dosing, clearance decreased by ~80% from the first (7.35 mL/h) to the last two dosing intervals (~1.50 mL/h) in the bloodstream model. Likewise, clearance decreased by 41% from 7.44 to 4.39 mL/h in the lung model. The humanized dosage regimens were 117 mg/kg of body weight/day in mice [administered in four fractions 6 h apart (q6h): 61.9%, 18.6%, 11.3%, and 8.21% of total dose] for the bloodstream and 96.7 mg/kg of body weight/day (given q6h as 65.1%, 16.9%, 10.5%, and 7.41%) for the lung model. These validated humanized dosage regimens and population pharmacokinetic models support translational studies with clinically relevant amikacin exposure profiles.

Development of a Bispecific Antibody Targeting Clinical Isolates of Acinetobacter baumannii.

BACKGROUND: We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a bispecific mAb (bsAb) could improve avidity and efficacy while maximizing strain coverage in one molecule. METHODS: Humanized mAb 65 was cloned into a single-chain variable fragment and attached to humanized mAb C8, combining their paratopes into a single bsAb (C73). We tested bsAb C73's strain coverage, binding affinity, ex vivo opsonic activity, and in vivo efficacy compared to each mAb alone and combined. RESULTS: The bsAb demonstrated strain coverage, binding affinity, opsonization, and in vivo efficacy superior to either original mAb alone or combined. CONCLUSIONS: A humanized bsAb targeting distinct A. baumannii capsule moieties enabled potent and effective coverage of disparate A. baumannii clinical isolates. The bsAb enhances feasibility of development by minimizing the number of components of a promising novel therapeutic for these difficult-to-treat infections.

Antimicrobial Susceptibility Testing Performed in RPMI 1640 Reveals Azithromycin Efficacy against Carbapenem-Resistant Acinetobacter baumannii and Predicts In Vivo Outcomes in Galleria mellonella.

Antimicrobial susceptibility testing (AST) in RPMI 1640, a more physiologically relevant culture medium, revealed that a substantial proportion of carbapenem-resistant Acinetobacter baumannii isolates were susceptible to azithromycin, a macrolide antibiotic not currently considered effective against A. baumannii. Experiments using Galleria mellonella validated these in vitro data. Our finding that RPMI 1640's predictive accuracy for in vivo outcomes is superior to that of Mueller-Hinton II broth also supports the use of more physiologically relevant AST culturing conditions.

Therapeutic, Humanized Monoclonal Antibody Exhibits Broad Binding and Protective Efficacy against Acinetobacter baumannii.

Acinetobacter baumannii is an extremely drug-resistant pathogen necessitating the development of new therapies. We seek to generate a cocktail of monoclonal antibodies (MAbs) that can target the full diversity of A. baumannii isolates. We have newly identified the antibody MAb5. Here, we demonstrate that MAb5 has broad binding against U.S. (n = 300) and international (n = 250) isolates (72.24% and 28.76%, respectively), likely targets O-antigen capsular carbohydrates, and exhibits protective efficacy in vivo.

Individual Components of Polymyxin B Modeled via Population Pharmacokinetics to Design Humanized Dosage Regimens for a Bloodstream and Lung Infection Model in Immune-Competent Mice.

Polymyxin B is a "last-line-of-defense" antibiotic approved in the 1960s. However, the population pharmacokinetics (PK) of its four main components has not been reported in infected mice. We aimed to determine the PK of polymyxin B1, B1-Ile, B2, and B3 in a murine bloodstream and lung infection model of Acinetobacter baumannii and develop humanized dosage regimens. A linear 1-compartment model, plus an epithelial lining fluid (ELF) compartment for the lung model, best described the PK. Clearance and volume of distribution were similar among the four components. The bioavailability fractions were 72.6% for polymyxin B1, 12.0% for B1-Ile, 11.5% for B2, and 3.81% for B3 for the lung model and were similar for the bloodstream model. While the volume of distribution was comparable between both models (17.3 mL for the lung and ~27 mL for the bloodstream model), clearance was considerably smaller for the lung (2.85 mL/h) compared to that of the bloodstream model (5.59 mL/h). The total drug exposure (AUC) in ELF was high due to the saturable binding of polymyxin B presumably to bacterial lipopolysaccharides. However, the modeled unbound AUC in ELF was ~16.7% compared to the total drug AUC in plasma. The long elimination half-life (~4 h) of polymyxin B enabled humanized dosage regimens with every 12 h dosing in mice. Daily doses that optimally matched the range of drug concentrations observed in patients were 21 mg/kg for the bloodstream and 13 mg/kg for the lung model. These dosage regimens and population PK models support translational studies for polymyxin B at clinically relevant drug exposures.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, 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 isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when 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 multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Monoclonal Antibody Requires Immunomodulation for Efficacy Against Acinetobacter baumannii Infection.

Monoclonal antibodies (mAbs) are gaining significant momentum as novel therapeutics for infections caused by antibiotic-resistant bacteria. We evaluated the mechanism by which antibacterial mAb therapy protects against Acinetobacter baumannii infections. Anticapsular mAb enhanced macrophage opsonophagocytosis and rescued mice from lethal infections by harnessing complement, macrophages, and neutrophils; however, the degree of bacterial burden did not correlate with survival. Furthermore, mAb therapy reduced proinflammatory (interleukin-1ß [IL-1ß], IL-6, tumor necrosis factor-α [TNF-α]) and anti-inflammatory (IL-10) cytokines, which correlated inversely with survival. Although disrupting IL-10 abrogated the survival advantage conferred by the mAb, IL-10-knockout mice treated with mAb could still survive if TNF-α production was suppressed directly (via anti-TNF-α neutralizing antibody) or indirectly (via macrophage depletion). Thus, even for a mAb that enhances microbial clearance via opsonophagocytosis, clinical efficacy required modulation of pro- and anti-inflammatory cytokines. These findings may inform future mAb development targeting bacteria that trigger the sepsis cascade.

Monoclonal Antibody Therapy against Acinetobacter baumannii.

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. To overcome this limitation, we broadened coverage by developing a second antibody for use in a combination regimen. We sought to develop an additional anti-A. baumannii MAb through hybridoma technology by immunizing mice with sublethal inocula of virulent, XDR clinical isolates not bound by MAb C8. We identified a new antibacterial MAb, 65, which bound to strains in a pattern distinct from and complementary to that of MAb C8. MAb 65 enhanced macrophage opsonophagocytosis of targeted strains and markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia murine models of A. baumannii infection. MAb 65 was also synergistic with colistin, substantially enhancing protection compared to monotherapy. Treatment with MAb 65 significantly reduced blood bacterial density, ameliorated cytokine production (interleukin-1ß [IL-1ß], IL-6, IL-10, and tumor necrosis factor), and sepsis biomarkers. We describe a novel MAb targeting A. baumannii that broadens immunotherapeutic strain coverage, is highly potent and effective, and synergistically improves outcomes in combination with antibiotics.

Synergistic Rifabutin and Colistin Reduce Emergence of Resistance When Treating Acinetobacter baumannii.

Recently, we reported rifabutin hyperactivity against Acinetobacter baumannii We sought to characterize potential interactions between rifabutin and colistin, the last-resort drug for carbapenem-resistant infections. Rifabutin and colistin were synergistic in vitro and in vivo, and low-dose colistin significantly suppressed emergence of resistance to rifabutin. Thus, this combination is a promising therapeutic option for highly resistant A. baumannii infections.

FDA Public Workshop Summary: Advancing Animal Models for Antibacterial Drug Development.

The U.S. Food and Drug Administration (FDA) hosted a public workshop entitled "Advancing Animal Models for Antibacterial Drug Development" on 5 March 2020. The workshop mainly focused on models of pneumonia caused by Pseudomonas aeruginosa and Acinetobacter baumannii The program included discussions from academic investigators, industry, and U.S. government scientists. The potential use of mouse, rabbit, and pig models for antibacterial drug development was presented and discussed.

Apotransferrin in Combination with Ciprofloxacin Slows Bacterial Replication, Prevents Resistance Amplification, and Increases Antimicrobial Regimen Effect.

There has been renewed interest in combining traditional small-molecule antimicrobial agents with nontraditional therapies to potentiate antimicrobial effects. Apotransferrin, which decreases iron availability to microbes, is one such approach. We conducted a 48-h one-compartment in vitro infection model to explore the impact of apotransferrin on the bactericidal activity of ciprofloxacin. The challenge panel included four Klebsiella pneumoniae isolates with ciprofloxacin MIC values ranging from 0.08 to 32 mg/liter. Each challenge isolate was subjected to an ineffective ciprofloxacin monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio] ranging from 0.19 to 96.6) with and without apotransferrin. As expected, the no-treatment and apotransferrin control arms showed unaltered prototypical logarithmic bacterial growth. We identified relationships between exposure and change in bacterial density for ciprofloxacin alone (R2 = 0.64) and ciprofloxacin in combination with apotransferrin (R2 = 0.84). Addition of apotransferrin to ciprofloxacin enabled a remarkable reduction in bacterial density across a wide range of ciprofloxacin exposures. For instance, at a ciprofloxacin AUC/MIC ratio of 20, ciprofloxacin monotherapy resulted in nearly 2 log10 CFU increase in bacterial density, while the combination of apotransferrin and ciprofloxacin resulted in 2 log10 CFU reduction in bacterial density. Furthermore, addition of apotransferrin significantly reduced the emergence of ciprofloxacin-resistant subpopulations compared to monotherapy. These data demonstrate that decreasing the rate of bacterial replication with apotransferrin in combination with antimicrobial therapy represents an opportunity to increase the magnitude of the bactericidal effect and to suppress the growth rate of drug-resistant subpopulations.

Adjunctive transferrin to reduce the emergence of antibiotic resistance in Gram-negative bacteria.

BACKGROUND: New strategies are needed to slow the emergence of antibiotic resistance among bacterial pathogens. In particular, society is experiencing a crisis of antibiotic-resistant infections caused by Gram-negative bacterial pathogens and novel therapeutics are desperately needed to combat such diseases. Acquisition of iron from the host is a nearly universal requirement for microbial pathogens-including Gram-negative bacteria-to cause infection. We have previously reported that apo-transferrin (lacking iron) can inhibit the growth of Staphylococcus aureus in culture and diminish emergence of resistance to rifampicin. OBJECTIVES: To define the potential of apo-transferrin to inhibit in vitro growth of Klebsiella pneumoniae and Acinetobacter baumannii, key Gram-negative pathogens, and to reduce emergence of resistance to antibiotics. METHODS: The efficacy of apo-transferrin alone or in combination with meropenem or ciprofloxacin against K. pneumoniae and A. baumannii clinical isolates was tested by MIC assay, time-kill assay and assays for the selection of resistant mutants. RESULTS: We confirmed that apo-transferrin had detectable MICs for all strains tested of both pathogens. Apo-transferrin mediated an additive antimicrobial effect for both antibiotics against multiple strains in time-kill assays. Finally, adding apo-transferrin to ciprofloxacin or meropenem reduced the emergence of resistant mutants during 20 day serial passaging of both species. CONCLUSIONS: These results suggest that apo-transferrin may have promise to suppress the emergence of antibiotic-resistant mutants when treating infections caused by Gram-negative bacteria.

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

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 organism's 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.

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-1ß, 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.

Cathepsin K Contributes to Cavitation and Collagen Turnover in Pulmonary Tuberculosis.

Cavitation in tuberculosis enables highly efficient person-to-person aerosol transmission. We performed transcriptomics in the rabbit cavitary tuberculosis model. Among 17 318 transcripts, we identified 22 upregulated proteases. Five type I collagenases were overrepresented: cathepsin K (CTSK), mast cell chymase-1 (CMA1), matrix metalloproteinase 1 (MMP-1), MMP-13, and MMP-14. Studies of collagen turnover markers, specifically, collagen type I C-terminal propeptide (CICP), urinary deoxypyridinoline (DPD), and urinary helical peptide, revealed that cavitation in tuberculosis leads to both type I collagen destruction and synthesis and that proteases other than MMP-1, MMP-13, and MMP-14 are involved, suggesting a key role for CTSK. We confirmed the importance of CTSK upregulation in human lung specimens, using immunohistochemical analysis, which revealed perigranulomatous staining for CTSK, and we showed that CTSK levels were increased in the serum of patients with tuberculosis, compared with those in controls (3.3 vs 0.3 ng/mL; P = .005).

Mycobacterium tuberculosis dysregulates MMP/TIMP balance to drive rapid cavitation and unrestrained bacterial proliferation.

Active tuberculosis (TB) often presents with advanced pulmonary disease, including irreversible lung damage and cavities. Cavitary pathology contributes to antibiotic failure, transmission, morbidity and mortality. Matrix metalloproteinases (MMPs), in particular MMP-1, are implicated in TB pathogenesis. We explored the mechanisms relating MMP/TIMP imbalance to cavity formation in a modified rabbit model of cavitary TB. Our model resulted in consistent progression of consolidation to human-like cavities (100% by day 28), with resultant bacillary burdens (>10(7) CFU/g) far greater than those found in matched granulomatous tissue (10(5) CFU/g). Using a novel, breath-hold computed tomography (CT) scanning and image analysis protocol, we showed that cavities developed rapidly from areas of densely consolidated tissue. Radiological change correlated with a decrease in functional lung tissue, as estimated by changes in lung density during controlled pulmonary expansion (R(2) = 0.6356, p < 0.0001). We demonstrated that the expression of interstitial collagenase (MMP-1) was specifically greater in cavitary compared to granulomatous lesions (p < 0.01), and that TIMP-3 significantly decreased at the cavity surface. Our findings demonstrated that an MMP-1/TIMP imbalance is associated with the progression of consolidated regions to cavities containing very high bacterial burdens. Our model provided mechanistic insight, correlating with human disease at the pathological, microbiological and molecular levels. It also provided a strategy to investigate therapeutics in the context of complex TB pathology. We used these findings to predict a MMP/TIMP balance in active TB and confirmed this in human plasma, revealing the potential of MMP/TIMP levels as key components of a diagnostic matrix aimed at distinguishing active from latent TB (PPV = 92.9%, 95% CI 66.1-99.8%, NPV = 85.6%; 95% CI 77.0-91.9%).

In vivo prediction of tuberculosis-associated cavity formation in rabbits.

The presence of cavitary lesions in patients with tuberculosis poses a significant clinical concern due to the risk of infectivity and the risk of antibiotic treatment failure. We describe 2 algorithms that use noninvasive positron emission tomography (PET) and computed tomography (CT) to predict the development of cavitary lesions in rabbits. Analysis of the PET region of interest predicted cavitary disease with 100% sensitivity and 76% specificity, and analysis of the CT region of interest predicted cavitary disease with 83.3% sensitivity and 76.9% specificity. Our results show that restricting our analysis to regions with high [(18)F]-fluorodeoxyglucose uptake provided the best combination of sensitivity and specificity.

Clinical assays rapidly predict bacterial susceptibility to monoclonal antibody therapy.

With antimicrobial resistance (AMR) emerging as a major threat to global health, monoclonal antibodies (MAbs) have become a promising means to combat difficult-to-treat AMR infections. Unfortunately, in contrast with standard antimicrobials, for which there are well-validated clinical laboratory methodologies to determine whether an infecting pathogen is susceptible or resistant to a specific antimicrobial drug, no assays have been described that can inform clinical investigators or clinicians regarding the clinical efficacy of a MAb against a specific pathogenic strain. Using Acinetobacter baumannii as a model organism, we established and validated 2 facile clinical susceptibility assays, which used flow cytometry and latex bead agglutination, to determine susceptibility (predicting in vivo efficacy) or resistance (predicting in vivo failure) of 1 newly established and 3 previously described anti-A. baumannii MAbs. These simple assays exhibited impressive sensitivity, specificity, and reproducibility, with clear susceptibility breakpoints that predicted the in vivo outcomes in our preclinical model with excellent fidelity. These MAb susceptibility assays have the potential to enable and facilitate clinical development and deployment of MAbs that generally target the surface of microbes.

Defining the minimum inhibitory concentration of 22 rifamycins in iron limited, physiologic medium against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates.

Recently, we reported rifabutin hyper-activity against Acinetobacter baumannii. We sought to characterize if any additional rifamycins (n = 22) would also display hyper-activity when tested in iron-limited media against A. baumannii, K. pneumoniae, and E. coli. MICs were determined against representative clinical isolates using the iron-limited media RPMI-1640. Only rifabutin was hyperactive against A. baumannii.