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.

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.

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.

A protein-free vaccine stimulates innate immunity and protects against nosocomial pathogens.

Traditional vaccines are difficult to deploy against the diverse antimicrobial-resistant, nosocomial pathogens that cause health care-associated infections. We developed a protein-free vaccine composed of aluminum hydroxide, monophosphoryl lipid A, and fungal mannan that improved survival and reduced bacterial burden of mice with invasive blood or lung infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-expressing Escherichia coli, and carbapenem-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The vaccine also conferred protection against the fungi Rhizopus delemar and Candida albicans. Efficacy was apparent by 24 hours and lasted for up to 28 days after a single vaccine dose, with a second dose restoring efficacy. The vaccine acted through stimulation of the innate, rather than the adaptive, immune system, as demonstrated by efficacy in the absence of lymphocytes that were abrogated by macrophage depletion. A role for macrophages was further supported by the finding that vaccination induced macrophage epigenetic alterations that modulated phagocytosis and the inflammatory response to infection. Together, these data show that this protein-free vaccine is a promising strategy to prevent deadly antimicrobial-resistant health care-associated infections.

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.

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.

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.

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.

Acacia Fiber Protects the Gut from Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Colonization Enabled by Antibiotics.

Novel approaches to combating antibiotic resistance are needed given the ever-continuing rise of antibiotic resistance and the scarce discovery of new antibiotics. Little is known about the colonization dynamics and the role of intrinsic plant-food characteristics in this process. We sought to determine whether plant fiber could alter colonization dynamics by antibiotic-resistant bacteria in the gut. We determined that ingestion of antibiotics in mice markedly enhanced gut colonization by a pathogenic extended-spectrum beta-lactamase-producing Escherichia coli strain of human origin, E. coli JJ1886 (ST131-H30Rx). Furthermore, ingestion of soluble acacia fiber before and after antibiotic exposure significantly reduced pathogenic E. coli colonization. 16S rRNA analysis and ex vivo cocultures demonstrated that fiber protected the microbiome by serving as a prebiotic, which induced native gut E. coli to inhibit pathogenic E. coli via colicin M. Fiber may be a useful prebiotic with which to administer antibiotics to protect human and livestock gut microbiomes against colonization from antibiotic-resistant, pathogenic bacteria. IMPORTANCE A One Health-based strategy-the concept that human health and animal health are interconnected with the environment-is necessary to determine the drivers of antibiotic resistance from food to the clinic. Moreover, humans can ingest antibiotic-resistant bacteria on food and asymptomatically, or "silently," carry such bacteria in the gut long before they develop an opportunistic extraintestinal infection. Here, we determined that fiber-rich foods, in particular acacia fiber, may be a new, promising, and inexpensive prebiotic to administer with antibiotics to protect the mammalian (i.e., human and livestock) gut against such colonization by antibiotic-resistant, pathogenic bacteria.

In Vitro Activity of Rifabutin and Rifampin against Antibiotic-Resistant Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

We recently reported that the antimicrobial activity of rifabutin against Acinetobacter baumannii is best modeled by the use of RPMI for in vitro susceptibility testing. Here, we define the effects of medium on the susceptibility and frequency of resistance emergence in a panel of A. baumannii, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa clinical isolates. Only A. baumannii was hypersusceptible to rifabutin in vitro and in vivo using a Galleria mellonella infection model. In vitro, the frequency of resistance emergence was greater when the bacteria were selected on RPMI versus tryptic soy agar (TSA) or Mueller-Hinton II (MHII) agar plates. However, the frequency of resistance emergence was lower in vivo than in the RPMI in vitro condition. IMPORTANCE Rifabutin has been recently described as a potential adjunctive therapy for antibiotic-resistant A. baumannii infections due to hypersensitivity in iron-depleted media, which may more closely mimic an in vivo environment. Here, we report that this hyperactivity is specific for A. baumannii, rather than being a general effect for other pathogens.

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.

Environmental Spread of Antibiotic Resistance.

Antibiotic resistance represents a global health concern. Soil, water, livestock and plant foods are directly or indirectly exposed to antibiotics due to their agricultural use or contamination. This selective pressure has acted synergistically to bacterial competition in nature to breed antibiotic-resistant (AR) bacteria. Research over the past few decades has focused on the emergence of AR pathogens in food products that can cause disease outbreaks and the spread of antibiotic resistance genes (ARGs), but One Health approaches have lately expanded the focus to include commensal bacteria as ARG donors. Despite the attempts of national and international authorities of developed and developing countries to reduce the over-prescription of antibiotics to humans and the use of antibiotics as livestock growth promoters, the selective flow of antibiotic resistance transmission from the environment to the clinic (and vice-versa) is increasing. This review focuses on the mechanisms of ARG transmission and the hotspots of antibiotic contamination resulting in the subsequent emergence of ARGs. It follows the transmission of ARGs from farm to plant and animal food products and provides examples of the impact of ARG flow to clinical settings. Understudied and emerging antibiotic resistance selection determinants, such as heavy metal and biocide contamination, are also discussed here.

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.

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.

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.

Pharmacology, Dosing, and Side Effects of Rifabutin as a Possible Therapy for Antibiotic-Resistant Acinetobacter Infections.

Acinetobacter baumannii has among the highest rates of antibiotic resistance encountered in hospitals. New therapies are critically needed. We found that rifabutin has previously unrecognized hyperactivity against most strains of A. baumannii. Here we review the pharmacology and adverse effects of rifabutin to inform potential oral dosing strategies in patients with A. baumannii infections. Rifabutin demonstrates dose-dependent increases in blood levels up to 900 mg per day, but plateaus thereafter. Furthermore, rifabutin induces its own metabolism after prolonged dosing, lowering its blood levels. Pending future development of an intravenous formulation, a rifabutin oral dose of 900-1200 mg per day for 1 week is a rational choice for adjunctive therapy of A. baumannii infections. This dosage maximizes AUC24 to drive efficacy while simultaneously minimizing toxicity. Randomized controlled trials will be needed to definitively establish the safety and efficacy of rifabutin to treat A. baumannii infections.

The Bactericidal Activity and Spore Inhibition Effect of Manuka Honey against Clostridioides Difficile.

Clostridioides difficile colitis overgrowth occurs when the normal gut microbiome becomes disrupted, often due to antibiotics. Effective treatment remains elusive, due partly to the persistence of its spores in the gut. Natural substances like manuka honey offer an alternative antimicrobial mechanism of action to conventional antibiotics. We investigated the antibiotic activity of manuka honey against 20 C. difficile isolates. The minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBC) of manuka honeys of methylglyoxal (MGO) grades 30+, 100+, 250+, and 400+ were determined based on broth microdilution. Sporicidal activity was assessed in a range of honey concentrations by enumerating total viable cell and spore counts at 0-96 h after organism inoculation. The MICs of C. difficile ranged from 4% to >30% (w/v). MIC50 for the four MGO grades were similar at 10-14%. MBC results for the majority of isolates were distributed bimodally at MBC/MIC ratios ≤4 or MBC >30%. Growth kinetics in honey showed total viable cell counts remaining >105 colony-forming units (CFU)/mL at all time points, whereas spore counts remained within 1-log of baseline (102 CFU/mL) in honey but steadily increased in the drug-free control to >105 CFU/mL by 96 h. Manuka honey demonstrated variable inhibitory and bactericidal activity against C. difficile. MGO grade had no noticeable impact on overall MIC distributions or bactericidal activity. Although manuka honey could inhibit spore proliferation, it did not eradicate spores completely.

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.

A nutrient-limited screen unmasks rifabutin hyperactivity for extensively drug-resistant Acinetobacter baumannii.

Industry screens of large chemical libraries have traditionally relied on rich media to ensure rapid bacterial growth in high-throughput testing. We used eukaryotic, nutrient-limited growth media in a compound screen that unmasked a previously unknown hyperactivity of the old antibiotic, rifabutin (RBT), against highly resistant Acinetobacter baumannii. In nutrient-limited, but not rich, media, RBT was 200-fold more potent than rifampin. RBT was also substantially more effective in vivo. The mechanism of enhanced efficacy was a Trojan horse-like import of RBT, but not rifampin, through fhuE, only in nutrient-limited conditions. These results are of fundamental importance to efforts to discover antibacterial agents.