Stapled ß-Hairpin Antimicrobial Peptides with Improved Stability and Activity against Drug-Resistant Gram-Negative Bacteria.

Different stapling techniques have been used recently to address the subpar performance of antimicrobial peptides (AMPs) in clinical trials with ample focus on α-helical AMPs. In comparison, a systematic evaluation of such strategies on ß-hairpin AMPs is lacking. Herein, we report the design, synthesis, and evaluation of a library of all-hydrocarbon-stapled ß-hairpin AMPs with variation in key parameters intended as potent therapeutics against drug-resistant pathogens. We observed an interesting interplay between the activity, stability, and structural strength. Single-stapled peptides with a 6-carbon staple at peptide termini such as 5(c6) displayed the most potent activity against colistin-resistant clinical isolates. Using imaging techniques, we observed translocation of 5(c6) across bacterial membranes without causing extensive damage. Overall, we have engineered novel all-hydrocarbon-stapled ß-hairpin AMPs with structural and functional proficiency that can effectively combat resistant pathogens, with findings from this study a point of reference for future interests in developing novel ß-hairpin AMPs.

Multifunctional Antibacterial Nanonets Attenuate Inflammatory Responses through Selective Trapping of Endotoxins and Pro-Inflammatory Cytokines.

Extracellular lipopolysaccharide (LPS) released from bacteria cells can enter the bloodstream and cause septic complications with excessive host inflammatory responses. Target-specific strategies to inactivate inflammation mediators have largely failed to improve the prognosis of septic patients in clinical trials. By utilizing their high density of positive charges, de novo designed peptide nanonets are shown to selectively entrap the negatively charged LPS and pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). This in turn enables the nanonets to suppress LPS-induced cytokine production by murine macrophage cell line and rescue the antimicrobial activity of the last-resort antibiotic, colistin, from LPS binding. Using an acute lung injury model in mice, it is demonstrated that intratracheal administration of the fibrillating peptides is effective at lowering local release of TNF-α and IL-6. Together with previously shown ability to simultaneously trap and kill pathogenic bacteria, the peptide nanonets display remarkable potential as a holistic, multifunctional anti-infective, and anti-septic biomaterial.

Addressing antimicrobial resistance with the IDentif.AI platform: Rapidly optimizing clinically actionable combination therapy regimens against nontuberculous mycobacteria.

Background: Current standard of care (SOC) regimens against nontuberculous mycobacteria (NTM) usually result in unsatisfactory therapeutic responses, primarily due to multi-drug resistance and antibiotic susceptibility-guided therapies. In the midst of rising incidences in NTM infections, strategies to develop NTM-specific treatments have been explored and validated. Methods: To provide an alternative approach to address NTM-specific treatment, IDentif.AI was harnessed to rapidly optimize and design effective combination therapy regimens against Mycobacterium abscessus (M. abscessus), the highly resistant and rapid growth species of NTM. IDentif.AI interrogated the drug interaction space from a pool of 6 antibiotics, and pinpointed multiple clinically actionable drug combinations. IDentif.AI-pinpointed actionable combinations were experimentally validated and their interactions were assessed using Bliss independence model and diagonal measurement of n-way drug interactions. Results: Notably, IDentfi.AI-designed 3- and 4-drug combinations demonstrated greater %Inhibition efficacy than the SOC regimens. The platform also pinpointed two unique drug interactions (Levofloxacin (LVX)/Rifabutin (RFB) and LVX/Meropenem (MEM)) that may serve as the backbone of potential 3- and 4-drug combinations like LVX/MEM/RFB, which exhibited 58.33±4.99 %Inhibition efficacy against M. abscessus. Further analysis of LVX/RFB via Bliss independence model pointed to dose-dependent synergistic interactions in clinically actionable concentrations. Conclusions: IDentif.AI-designed combinations may provide alternative regimen options to current SOC combinations that are often administered with Amikacin, which has been known to induce ototoxicity in patients. Furthermore, IDentif.AI pinpointed 2-drug interactions may also serve as the backbone for the development of other effective 3- and 4-drug combination therapies. The findings in this study suggest that this platform may contribute to NTM-specific drug development.

Sugar-Assisted Cryopreservation of Stem Cell-Laden Gellan Gum-Collagen Interpenetrating Network Hydrogels.

Tissue engineering involves the transplantation of stem cell-laden hydrogels as synthetic constructs to replace damaged tissues. However, their time-consuming fabrication procedures are hurdles to widespread application in clinics. Fortunately, similar to cell banking, synthetic tissues could be cryopreserved for subsequent central distribution. Here, we report the use of trehalose and gellan gum as biomacromolecules to form a cryopreservable yet directly implantable hydrogel system for adipose-derived stem cell (ADSC) delivery. Through a modified cell encapsulation method and a preincubation step, adequate cryoprotection was afforded at 0.75 M trehalose to the encapsulated ADSCs. At this concentration, trehalose demonstrated lower propensity to induce apoptosis than 10% DMSO, the current gold standard cryoprotectant. Moreover, when cultured along with trehalose after thawing, the encapsulated ADSCs retained their stem cell-like phenotype and osteogenic differentiation capacity. Taken together, this study demonstrates the feasibility of an "off-the-shelf" biomacromolecule-based synthetic tissue to be applied in widespread tissue engineering applications.

Rationalisation of Antifungal Properties of α-Helical Pore-Forming Peptide, Mastoparan B.

The high mortality associated with invasive fungal infections, narrow spectrum of available antifungals, and increasing evolution of antifungal resistance necessitate the development of alternative therapies. Host defense peptides are regarded as the first line of defense against microbial invasion in both vertebrates and invertebrates. In this work, we investigated the effectiveness of four naturally occurring pore-forming antimicrobial peptides (melittin, magainin 2, cecropin A, and mastoparan B) against a panel of clinically relevant pathogens, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida glabrata. We present data on the antifungal activities of the four pore-forming peptides, assessed with descriptive statistics, and their cytocompatibility with cultured human cells. Among the four peptides, mastoparan B (MB) displayed potent antifungal activity, whereas cecropin A was the least potent. We show that MB susceptibility of phylogenetically distant non-candida albicans can vary and be described by different intrinsic physicochemical parameters of pore-forming α-helical peptides. These findings have potential therapeutic implications for the design and development of safe antifungal peptide-based drugs.

Novel tetrameric cell penetrating antimicrobial peptoids effective against mycobacteria and drug-resistant Staphylococcus aureus.

BACKGROUND: Antimicrobial peptides (AMPs) are short, cationic, amphipathic molecules that have gained tremendous popularity as alternatives to traditional antibiotics due to their lower propensity to develop bacterial resistance. However, the clinical developability of AMPs remains impeded due to shortcomings such as proteolytic instability and poor penetration leading to low bioavailability. AIMS: To improve the access of AMPs to cells and subsequent bacteria killing, we evaluated the cell-penetrating and antimicrobial properties of three novel libraries of synthetic peptoids using Minimum Inhibitory Concentration, killing efficacy and membrane permeabilization assays against mycobacteria and Staphylococcus aureus. In addition, we investigated cell selectivity using mammalian cells to assess peptoid toxicity. RESULTS: We showed that short tetrameric Rhodamine B-labeled peptoids composed of a balance of aromatic and lipophilic residues have potent selective antimicrobial activity against a range of microorganisms. The most potent candidates were active against drug-resistant S. aureus isolates as well as mycobacterial strains, with cell penetrating capabilities reported in HeLa and RAW 264.7 macrophage cells. CONCLUSIONS: These data suggest that peptoids with novel dual functionalities may potentially be an interesting class of therapeutics and/or molecular delivery agents for anti-infective purposes.

Wirelessly operated bioelectronic sutures for the monitoring of deep surgical wounds.

Monitoring surgical wounds post-operatively is necessary to prevent infection, dehiscence and other complications. However, the monitoring of deep surgical sites is typically limited to indirect observations or to costly radiological investigations that often fail to detect complications before they become severe. Bioelectronic sensors could provide accurate and continuous monitoring from within the body, but the form factors of existing devices are not amenable to integration with sensitive wound tissues and to wireless data transmission. Here we show that multifilament surgical sutures functionalized with a conductive polymer and incorporating pledgets with capacitive sensors operated via radiofrequency identification can be used to monitor physicochemical states of deep surgical sites. We show in live pigs that the sutures can monitor wound integrity, gastric leakage and tissue micromotions, and in rodents that the healing outcomes are equivalent to those of medical-grade sutures. Battery-free wirelessly operated bioelectronic sutures may facilitate post-surgical monitoring in a wide range of interventions.

Manipulating turn residues on de novo designed ß-hairpin peptides for selectivity against drug-resistant bacteria.

Synthetic ß-hairpin antimicrobial peptides (AMPs) offer a useful source for the development of novel antimicrobial agents. ß-hairpin peptides generally consist of two side strands bridged by a reverse turn. In literature, most studies focused on the modifications of the side strands to manipulate the stability and activity of ß-hairpin peptides, and much less is known about the impact of the turn region. By designing a series of de novo ß-hairpin peptides with identical side strands but varied turns, we demonstrated that mutations of only 2 to 4 amino acids at the turn region could impart a wide range of antimicrobial profiles among synthetic ß-hairpin AMPs. BTT2-4 and BTT6 displayed selective potency against Gram-negative bacteria, with minimum inhibitory concentrations (MICs) of 4-8 µM. In contrast, BTT1 exhibited broad-spectrum activity, with MICs of 4-8 µM against both Gram-positive and Gram-negative strains. Additionally, BTT1 was potent against methicillin-resistant Staphylococcus aureus (MRSA) and colistin-resistant Enterobacterales. The antimicrobial potency of BTT1 persisted after 14 days of serial passage. Mechanistic studies revealed that interactions between lipopolysaccharide (LPS) and the peptides were critical to their membranolytic activity against the bacterial inner membrane. Aside from folding stability, we observed that a degree of conformational flexibility was required for disruptive membrane interactions. STATEMENT OF SIGNIFICANCE: By examining the significance of the turn region of ß-hairpin peptides, we present valuable knowledge to the design toolkit of novel antimicrobial peptides as alternative therapeutics to overcome antibiotic resistance. Our de novo designed synthetic peptides displayed selective activity against Gram-negative bacteria and potent activity against clinically relevant antibiotic-resistant strains (e.g. colistin-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus). The bactericidal activity of our peptides was shown to be robust in the presence of proteolytic trypsin and saline, conditions that could suppress peptide activity. Our peptides were also determined to be non-cytotoxic against a human cell line.

Pristine Gellan Gum-Collagen Interpenetrating Network Hydrogels as Mechanically Enhanced Anti-inflammatory Biologic Wound Dressings for Burn Wound Therapy.

Gellan gum is a biologically inert natural polymer that is increasingly favored as a material-of-choice to form biorelevant hydrogels. However, as a burn wound dressing, native gellan gum hydrogels do not drive host's biology toward regeneration and are mechanically inadequate wound barriers. To overcome these issues, we fabricateda gellan gum-collagen full interpenetrating network (full-IPN) hydrogel that can house adipose-derived mesenchymal stem cells (ADSCs) and employ their multilineage differentiation potential and produce wound-healing paracrine factors to reduce inflammation and promote burn wound regeneration. Herein, a robust temperature-dependent simultaneous IPN (SIN) hydrogel fabrication process was demonstrated using applied rheology for the first time. Subsequently after fabrication, mechanical characterization assays showed that the IPN hydrogels were easy to handle without deforming and retained sufficient mass to effect ADSCs' anti-inflammation property in a simulated wound environment. The IPN hydrogels' increased stiffness proved conducive for mechanotransduced cell adhesion. Scanning electron microscopy revealed theIPN's porous network, which enabled encapsulated ADSCs to spread and proliferate, for up to 3 weeks of culture, further shown by cells' dynamic filopodia extension observed in 3D confocal images. Successful incorporation of ADSCs accorded the IPN hydrogels with biologic wound-dressing properties, which possess the ability to promote human dermal fibroblast migration and secrete an anti-inflammatory paracrine factor, TSG-6 protein, as demonstrated in the 2D scratch wound assay and ELISA, respectively. More importantly, upon application onto murine full thickness burn wounds, our biologic wound dressing enhanced early wound closure, reduced inflammation, and promoted complete skin regeneration. Altogether, our results highlight the successful mechanical and biological enhancement of the inert matrix of gellan gum. Through completely natural procedures, a highly applicable biologic wound dressing is introduced for cell-based full thickness burn wound therapy.

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression.

The global cell culture market is experiencing significant growth due to the rapid advancement in antibody-based and cell-based therapies. Both rely on the capacity of different living factories, namely prokaryotic and eukaryotic cells, plants or animals for reliable and mass production. The ability to improve production yield is of important concern. Among many strategies pursued, optimizing the complex nutritional requirements for cell growth and protein production has been frequently performed via culture media component titration and serum replacement. The addition of specific ingredients into culture media to modulate host cells' metabolism has also recently been explored. In this study, we examined the use of extracted bioactive components of the microalgae Chlorella vulgaris, termed chlorella growth factor (CGF), as a cell culture additive for serum replacement and protein expression induction. We first established a chemical fingerprint of CGF using ultraviolet-visible spectroscopy and liquid chromatography-mass spectrometry and evaluated its ability to enhance cell proliferation in mammalian host cells. CGF successfully promoted the growth of Chinese hamster ovary (CHO) and mesenchymal stem cells (MSC), in both 2D and 3D cell cultures under reduced serum conditions for up to 21 days. In addition, CGF preserved cell functions as evident by an increase in protein expression in CHO cells and the maintenance of stem cell phenotype in MSC. Taken together, our results suggest that CGF is a viable culture media additive and growth matrix component, with wide ranging applications in biotechnology and tissue engineering.

Nucleic acid peptide nanogels for the treatment of bacterial keratitis.

Cage-shaped nucleic acid nanocarriers are promising molecular scaffolds for the organization of polypeptides. However, there is an unmet need for facile loading strategies that truly emulate nature's host-guest systems to drive encapsulation of antimicrobial peptides (AMPs) without loss of biological activity. Herein, we develop DNA nanogels with rapid in situ loading of L12 peptide during the thermal annealing process. By leveraging the binding affinity of L12 to the polyanionic core, we successfully confine the AMPs within the DNA nanogel. We report that the thermostability of L12 in parallel with the high encapsulation efficiency, low toxicity and sustained drug release of the pre-loaded L12 nanogels can be translated into significant antimicrobial activity. Using an S. aureus model of infectious bacterial keratitis, we observe fast resolution of clinical symptoms and significant reduction of bacterial bioburden. Collectively, this study paves the way for the development of DNA nanocarriers for caging AMPs with immense significance to address the rise of resistance.

Silica Nanoparticles-A Versatile Tool for the Treatment of Bacterial Infections.

The rapid emergence of drug resistance continues to outpace the development of new antibiotics in the treatment of infectious diseases. Conventional therapy is currently limited by drug access issues such as low intracellular drug accumulations, drug efflux by efflux pumps and/or enzymatic degradation. To improve access, targeted delivery using nanocarriers could provide the quantum leap in intracellular drug transport and retention. Silica nanoparticles (SiNPs) with crucial advantages such as large surface area, ease-of-functionalization, and biocompatibility, are one of the most commonly used nanoparticles in drug delivery applications. A porous variant, called the mesoporous silica nanoparticles (MSN), also confers additional amenities such as tunable pore size and volume, leading to high drug loading capacity. In the context of bacterial infections, SiNPs and its variants can act as a powerful tool for the targeted delivery of antimicrobials, potentially reducing the impact of high drug dosage and its side effects. In this review, we will provide an overview of SiNPs synthesis, its structural proficiency which is critical in loading and conjugation of antimicrobials and its role in different antimicrobial applications with emphasis on intracellular drug targeting in anti-tuberculosis therapy, nitric oxide delivery, and metal nanocomposites. The role of SiNPs in antibiofilm coatings will also be covered in the context of nosocomial infections and surgical implants.

Biomimicry of microbial polysaccharide hydrogels for tissue engineering and regenerative medicine - A review.

Hydrogels as artificial biomaterial scaffolds offer a much favoured 3D microenvironment for tissue engineering and regenerative medicine (TERM). Towards biomimicry of the native ECM, polysaccharides from Nature have been proposed as ideal surrogates given their biocompatibility. In particular, derivatives from microbial sources have emerged as economical and sustainable biomaterials due to their fast and high yielding production procedures. Despite these merits, microbial polysaccharides do not interact biologically with human tissues, a critical limitation hampering their translation into paradigmatic scaffolds for in vitro 3D cell culture. To overcome this, chemical and biological functionalization of polysaccharide scaffolds have been explored extensively. This review outlines the most recent strategies in the preparation of biofunctionalized gellan gum, xanthan gum and dextran hydrogels fabricated exclusively via material blending. Using inorganic or organic materials, we discuss the impact of these approaches on cell adhesion, proliferation and viability of anchorage-dependent cells for various TERM applications.'

Ultra-Short Antimicrobial Peptoids Show Propensity for Membrane Activity Against Multi-Drug Resistant Mycobacterium tuberculosis.

Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.

Developing inhaled protein therapeutics for lung diseases.

Biologic therapeutics such as protein/polypeptide drugs are conventionally administered systemically via intravenous injection for the treatment of diseases including lung diseases, although this approach leads to low target site accumulation and the potential risk for systemic side effects. In comparison, topical delivery of protein drugs to the lung via inhalation is deemed to be a more effective approach for lung diseases, as proteins would directly reach the target in the lung while exhibiting poor diffusion into the systemic circulation, leading to higher lung drug retention and efficacy while minimising toxicity to other organs. This review examines the important considerations and challenges in designing an inhaled protein therapeutics for local lung delivery: the choice of inhalation device, structural changes affecting drug deposition in diseased lungs, clearance mechanisms affecting an inhaled protein drug's lung accumulation, protein stability, and immunogenicity. Possible approaches to overcoming these issues will also be discussed.

Facile and efficient encapsulation of antimicrobial peptides via crosslinked DNA nanostructures and their application in wound therapy.

There is growing interest in the development of nucleic acid nanostructures as smart functional materials for applications in drug delivery. Inspired by the diverse physical interactions that exist in nature, crosslinked DNA nanostructures can serve as attractive affinity binding networks that interact with therapeutic cargos or living cells. Herein we report a strategy that addresses the challenges of topical oligopeptide therapy by exploiting high binding affinity between polyanionic DNA nanostructures and cationic antimicrobial peptides (AMPs) to fabricate hydrogels that release a model antimicrobial L12 peptide in response to pathogenic S. aureus infections. We further demonstrated controlled peptide release profiles via the DNA hydrogels that were biocompatible and delivered superior antimicrobial activity against nuclease-releasing susceptible and methicillin-resistant S. aureus infections. Single application of the L12-loaded DNA hydrogels on porcine explant S. aureus infections revealed potent efficacy after 24h. As a result of the capacity of the crosslinked DNA nanostructures to elicit a strong anti-inflammatory response, in vivo treatment of mice excision wounds translated into faster healing rates. Overall, the crosslinked DNA nanostructures reported in this study offer significant advantage as functional wound dressings and their future adaptation holds equally great promise for the delivery of cationic antimicrobials.

Correction to: Candidemia in a major regional tertiary referral hospital - epidemiology, practice patterns and outcomes.

[This corrects the article DOI: 10.1186/s13756-017-0184-1.].

Integrated pharmacokinetic-pharmacodynamic modeling to evaluate empiric carbapenem therapy in bloodstream infections.

OBJECTIVES: Treatment for nosocomial bloodstream infections (BSI) caused by multidrug-resistant (MDR) Gram-negative bacteria (GNB) is challenging. Rising antimicrobial resistance, especially in extended spectrum beta-lactamase production, inadvertently increases empiric carbapenem consumption. Three antipseudomonal carbapenems (imipenem, meropenem [MER], and doripenem [DOR]) are available commercially against MDR GNB in Singapore. The study aims to determine the most optimal empiric carbapenem dosing regimens (CDR) and evaluate their cost-effectiveness for GNB-BSI in the face of increasing MDR GNB. METHODS: Carbapenem minimum inhibitory concentrations (MICs) were generated for non-repeat GNB-BSI obtained in 2013-2014 from two hospitals. Monte Carlo simulations were used to assess the cumulative fraction of response (CFR) of various CDRs using the percentage of time above MIC for 40% (%T > MIC of 40%) as the pharmacokinetic (PK)-pharmacodynamic (PD) parameter for efficacy. Carbapenem costs were based on patient antibiotic costs. Antibiotic cost-effectiveness was calculated as total daily drug cost/CFR. RESULTS: A total of 1,140 bloodstream isolates were collected. They comprised 116 Acinetobacter baumannii, 237 Pseudomonas aeruginosa, and 787 Enterobacteriaceae. All CDRs achieved ~40, ~80, and ≥90% CFRs against A. baumannii, P. aeruginosa, and Enterobacteriaceae, respectively. Against P. aeruginosa, MER 2 g every 8 h infused over 3 h and DOR 1 g every 8 h infused over 4 h achieved CFRs 84 and 81%, respectively. Against Enterobacteriaceae, the cost of MER 2 g every 8 h infused over 3 h was the lowest among the three carbapenems at $0.40/percentage of CFR. CONCLUSION: This study demonstrates the utility of PK-PD modeling to formulate the optimal selection of a cost-effective empiric CDR in antibiotics guidelines and formulary inclusion. The findings support the selection of high MER doses of prolonged infusions as empiric coverage for GNB-BSI in our institutions.

Antimicrobial and Anti-Biofilm Activities of Surface Engineered Polycationic Albumin Nanoparticles with Reduced Hemolytic Activity.

Protein-based polymeric polyelectrolytes are emerging as alternative synthetic nanoparticles owing to their biodegradability and biocompatibility. However, potential in vivo toxicity remains a significant challenge. Herein an array of protein polyelectrolytes generated from cationic human serum albumin (cHSA) and polyethylene glycol (PEG) are synthesized via synthetic customization as antimicrobials for the treatment of systemic infections. By varying PEG molecular weight and chain length, in vitro hemolytic activity can be fine-tuned without significantly affecting antimicrobial potency. The optimal hybrid material, PEG (2000)18 -cHSA, with potent antimicrobial character, low hemolytic activity, and in vitro biofilm disruptive properties is identified. Surface plasmon resonance (SPR) evaluation demonstrates significantly higher binding activity of the protein nanoparticles to bacteria cell wall components and microfluidic live-cell imaging indicates that the nanoparticles act through a membranolytic mechanism. Given their low susceptibility to drug resistance and potent activity against resistant bacteria strains, these findings establish the PEGylated albumin nanoparticles as a potent weaponry against drug resistance and biofilm-related infection.

Rapid Antibiotic Combination Testing for Carbapenem-Resistant Gram-Negative Bacteria within Six Hours Using ATP Bioluminescence.

To guide the timely selection of antibiotic combinations against carbapenem-resistant Gram-negative bacteria (CR-GNB), an in vitro test with a short turnaround time is essential. We developed an in vitro ATP bioluminescence assay to determine effective antibiotic combinations against CR-GNB within 6 h. We tested 42 clinical CR-GNB strains (14 Acinetobacter baumannii, 14 Pseudomonas aeruginosa, and 14 Klebsiella pneumoniae strains) against 74 single antibiotics and two-antibiotic combinations. Bacteria (approximately 5 log10 CFU/ml) were incubated with an antibiotic(s) at 35°C; ATP bioluminescence was measured at 6 h and 24 h; and the measurements were compared to viable counts at 24 h. Receiver operating characteristic (ROC) curves were used to determine the optimal luminescence thresholds (TRLU) for distinguishing between inhibitory and noninhibitory combinations. The areas under the 6-h and 24-h ROC curves were compared using the DeLong method. Prospective validation of the established thresholds was conducted using 18 additional CR-GNB. The predictive accuracy of TRLU for the 6-h ATP bioluminescence assay was 77.5% when all species were analyzed collectively. Predictive accuracies ranged from 73.7% to 82.7% when each species was analyzed individually. Upon comparison of the areas under the 6-h and 24-h ROC curves, the 6-h assay performed significantly better than the 24-h assay (P < 0.01). Predictive accuracy remained high upon prospective validation of the 6-h ATP assay (predictive accuracy, 79.8%; 95% confidence interval [CI], 77.6 to 81.9%), confirming the external validity of the assay. Our findings indicate that our 6-h ATP bioluminescence assay can provide guidance for prospective selection of antibiotic combinations against CR-GNB in a timely manner and may be useful in the management of CR-GNB infections.