Crossing the Chasm: How to Approach Translational Pharmacokinetic-Pharmacodynamic Modeling of Phage Dosing.

Effectively treating multidrug-resistant bacterial infections remains challenging due to the limited drug development pipeline and a scarcity of novel agents effective against these highly resistant pathogens. Bacteriophages (phages) are a potential addition to the antimicrobial treatment arsenal. Though, phages are currently being tested in clinical trials for antibiotic-resistant infections, phages lack a fundamental understanding of optimal dosing in humans. Rationally designed preclinical studies using in vitro and in vivo infection models, allow us to assess clinically relevant phage +/- antibiotic exposure (pharmacokinetics), the resulting treatment impact on the infecting pathogen (pharmacodynamics) and host immune response (immunodynamics). A mechanistic modeling framework allows us to integrate this knowledge gained from preclinical studies to develop predictive models. We reviewed recently published mathematical models based on in vitro and/or in vivo data that evaluate the effects of varying bacterial or phage densities, phage characteristics (burst size, adsorption rate), phage pharmacokinetics, phage-antibiotic combinations and host immune responses. In our review, we analyzed study designs and the data used to inform the development of these mechanistic models. Insights gained from model-based simulations were reviewed as they help identify crucial phage parameters for determining effective phage dosing. These efforts contribute to bridging the gap between phage therapy research and its clinical translation.

Model-based learn and confirm: designing effective treatment regimens against multidrug resistant Gram-negative pathogens.

Over the last decade, there has been a growing appreciation for the use of in vitro and in vivo infection models to generate robust and informative nonclinical PK/PD data to accelerate the clinical translation of treatment regimens. The objective of this study was to develop a model-based "learn and confirm" approach to help with the design of combination regimens using in vitro infection models to optimise the clinical utility of existing antibiotics. Static concentration time-kill studies were used to evaluate the PD activity of polymyxin B (PMB) and meropenem against two carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates; BAA2146 (PMB-susceptible) and BRKP67 (PMB-resistant). A mechanism-based model (MBM) was developed to quantify the joint activity of PMB and meropenem. In silico simulations were used to predict the time-course of bacterial killing using clinically-relevant PK exposure profiles. The predictive accuracy of the model was further evaluated by validating the model predictions using a one-compartment PK/PD in vitro dynamic infection model (IVDIM). The MBM captured the reduction in bacterial burden and regrowth well in both the BAA2146 and BRKP67 isolate (R2 = 0.900 and 0.940, respectively). The bacterial killing and regrowth predicted by the MBM were consistent with observations in the IVDIM: sustained activity against BAA2146 and complete regrowth of the BRKP67 isolate. Differences observed in PD activity suggest that additional dose optimisation might be beneficial in PMB-resistant isolates. The model-based approach presented here demonstrates the utility of the MBM as a translational tool from static to dynamic in vitro systems to effectively perform model-informed drug optimisation.

High-level nitrofurantoin resistance in a clinical isolate of Klebsiella pneumoniae: a comparative genomics and metabolomics analysis.

Nitrofurantoin is a commonly used chemotherapeutic agent in the treatment of uncomplicated urinary tract infections caused by the problematic multidrug resistant Gram-negative pathogen Klebsiella pneumoniae. The present study aims to elucidate the mechanism of nitrofurantoin action and high-level resistance in K. pneumoniae using whole-genome sequencing (WGS), qPCR analysis, mutation structural modeling and untargeted metabolomic analysis. WGS profiling of evolved highly resistant mutants (nitrofurantoin minimum inhibitory concentrations > 256 mg/L) revealed modified expression of several genes related to membrane transport (porin ompK36 and efflux pump regulator oqxR) and nitroreductase activity (ribC and nfsB, involved in nitrofurantoin reduction). Untargeted metabolomics analysis of total metabolites extracted at 1 and 4 h post-nitrofurantoin treatment revealed that exposure to the drug caused a delayed effect on the metabolome which was most pronounced after 4 h. Pathway enrichment analysis illustrated that several complex interrelated metabolic pathways related to nitrofurantoin bacterial killing (aminoacyl-tRNA biosynthesis, purine metabolism, central carbohydrate metabolism, and pantothenate and CoA biosynthesis) and the development of nitrofurantoin resistance (riboflavin metabolism) were significantly perturbed. This study highlights for the first time the key role of efflux pump regulator oqxR in nitrofurantoin resistance and reveals global metabolome perturbations in response to nitrofurantoin, in K. pneumoniae.IMPORTANCEA quest for novel antibiotics and revitalizing older ones (such as nitrofurantoin) for treatment of difficult-to-treat Gram-negative bacterial infections has become increasingly popular. The precise antibacterial activity of nitrofurantoin is still not fully understood. Furthermore, although the prevalence of nitrofurantoin resistance remains low currently, the drug's fast-growing consumption worldwide highlights the need to comprehend the emerging resistance mechanisms. Here, we used multidisciplinary techniques to discern the exact mechanism of nitrofurantoin action and high-level resistance in Klebsiella pneumoniae, a common cause of urinary tract infections for which nitrofurantoin is the recommended treatment. We found that the expression of multiple genes related to membrane transport (including active efflux and passive diffusion of drug molecules) and nitroreductase activity was modified in nitrofurantoin-resistant strains, including oqxR, the transcriptional regulator of the oqxAB efflux pump. Furthermore, complex interconnected metabolic pathways that potentially govern the nitrofurantoin-killing mechanisms (e.g., aminoacyl-tRNA biosynthesis) and nitrofurantoin resistance (riboflavin metabolism) were significantly inhibited following nitrofurantoin treatment. Our study could help inform the improvement of nitrofuran derivatives, the development of new pharmacophores, or drug combinations to support the resurgence of nitrofurantoin in the management of multidrug resistant K. pneumouniae infection.

Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility.

Infections caused by Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or ß-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or ß-lactam resistance. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes' expression was reduced. One of the mutations common to both heteroresistant strains was mapped within ppiA, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller-Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for ß-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.

Proof-of-concept for incorporating mechanistic insights from multi-omics analyses of polymyxin B in combination with chloramphenicol against Klebsiella pneumoniae.

Carbapenemase-resistant Klebsiella pneumoniae (KP) resistant to multiple antibiotic classes necessitates optimized combination therapy. Our objective is to build a workflow leveraging omics and bacterial count data to identify antibiotic mechanisms that can be used to design and optimize combination regimens. For pharmacodynamic (PD) analysis, previously published static time-kill studies (J Antimicrob Chemother 70, 2015, 2589) were used with polymyxin B (PMB) and chloramphenicol (CHL) mono and combination therapy against three KP clinical isolates over 24 h. A mechanism-based model (MBM) was developed using time-kill data in S-ADAPT describing PMB-CHL PD activity against each isolate. Previously published results of PMB (1 mg/L continuous infusion) and CHL (Cmax : 8 mg/L; bolus q6h) mono and combination regimens were evaluated using an in vitro one-compartment dynamic infection model against a KP clinical isolate (108 CFU/ml inoculum) over 24 h to obtain bacterial samples for multi-omics analyses. The differentially expressed genes and metabolites in these bacterial samples served as input to develop a partial least squares regression (PLSR) in R that links PD responses with the multi-omics responses via a multi-omics pathway analysis. PMB efficacy was increased when combined with CHL, and the MBM described the observed PD well for all strains. The PLSR consisted of 29 omics inputs and predicted MBM PD response (R2  = 0.946). Our analysis found that CHL downregulated metabolites and genes pertinent to lipid A, hence limiting the emergence of PMB resistance. Our workflow linked insights from analysis of multi-omics data with MBM to identify biological mechanisms explaining observed PD activity in combination therapy.

Transcriptomic Mapping of Neurotoxicity Pathways in the Rat Brain in Response to Intraventricular Polymyxin B.

Intraventricular or intrathecal administration of polymyxins are increasingly used to treat multidrug-resistant (MDR) Gram-negative bacteria caused infections in the central nervous system (CNS). However, our limited knowledge of the mechanisms underpinning polymyxin-induced neurotoxicity significantly hinders the development of safe and efficacious polymyxin dosing regimens. To this end, we conducted transcriptomic analyses of the rat brain and spinal cord 1 h following intracerebroventricular administration of polymyxin B into rat lateral ventricle at a clinically relevant dose (0.5 mg/kg). Following the treatment, 66 differentially expressed genes (DEGs) were identified in the brain transcriptome while none for the spinal cord (FDR ≤ 0.05, fold-change ≥ 1.5). DEGs were enriched in signaling pathways associated with hormones and neurotransmitters, including dopamine and (nor)epinephrine. Notably, the expression levels of Slc6a3 and Gabra6 were decreased by 20-fold and 4.3-fold, respectively, likely resulting in major perturbations of dopamine and γ-aminobutyric acid signaling in the brain. Mass spectrometry imaging of brain sections revealed a distinct pattern of polymyxin B distribution with the majority accumulating in the injection-side lateral ventricle and subsequently into third and fourth ventricles. Polymyxin B was not detectable in the left lateral ventricle or brain tissue. Electrophysiological measurements on primary cultured rat neurons revealed a large inward current and significant membrane leakage following polymyxin B treatment. Our work demonstrates, for the first time, the key CNS signaling pathways associated with polymyxin neurotoxicity. This mechanistic insight combined with pharmacokinetic/pharmacodynamic dosing strategies will help guide the design of safe and effective intraventricular/intrathecal polymyxin treatment regimens for CNS infections caused by MDR Gram-negative pathogens.

Untargeted metabolomics to evaluate polymyxin B toxicodynamics following direct intracerebroventricular administration into the rat brain.

There is a dearth of studies focused on understanding pharmacokinetics, pharmacodynamics and toxicodynamics of polymyxins following direct administration to the central nervous system (CNS). In this study, for the first time, untargeted metabolomics were employed to ascertain the perturbations of brain metabolism in the rat cerebral cortex following direct brain injection of 0.75 mg/kg polymyxin B (1 and 4 h) through the right lateral ventricle. In the right cortex metabolome, ICV polymyxin B induced a greater perturbation at 1 h compared to negligible effect at 4 h. Pathway enrichment analysis showed that sphingolipid, arginine, and histidine metabolism, together with aminoacyl-tRNA biosynthesis were significantly affected in the right cortex metabolome. Furthermore, intracerebroventricular (ICV) polymyxin B dysregulated the two arms (CDP-choline and CDP-ethanolamine) of the Kennedy pathway that governs the de novo biosynthesis of neuronal phospholipids. Importantly, the key intermediates of metabolic pathways that maintain cellular redox balance (e.g., glutathione metabolism) and mitochondrial function (e.g., electron transport chain) were markedly depleted. The abundance of key metabolites (e.g., N-acetyl-l-glutamate) associated with diverse CNS disorders (e.g., neurodegenerative disease) were also significantly perturbed. The biological significance of these metabolic perturbations on the CNS includes impaired oxidant-antioxidant balance, impaired neuronal lipid homeostasis and mitochondrial dysfunction. Furthermore, ICV polymyxin B caused a significant alteration in the abundance of several metabolic biomarkers associated with cerebral ischemia, oxidative stress as well as certain neurological disorders. These findings may facilitate the development of new pharmacokinetic/pharmacodynamic strategies to attenuate polymyxins ICV related CNS toxicities and stimulate the discovery of safer next-generation polymyxin-like lipopeptide antibiotics.

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection.

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary to perform reproducible burn research. The present protocol describes a simple and reproducible method to create ~20% total body surface area (TBSA) full-thickness burns in rats. Here, a 22.89 cm2 (5.4 cm diameter) copper rod heated at 97 °C in a water bath was applied to the rat skin surface to induce the burn injury. A copper rod with a high thermal conductivity was able to dissipate the heat deeper in the skin tissue to create a full-thickness burn. Histology analysis shows attenuated epidermis with coagulative damage to the full-thickness extent of the dermis and the subcutaneous tissue. Additionally, this model is representative of the clinical situations observed in hospitalized burn patients following burn injury such as immune dysregulation and bacterial infections. The model can recapitulate the systemic bacterial infection by both Gram-positive and Gram-negative bacteria. In conclusion, this paper presents an easy-to-learn and robust rat burn model that mimics the clinical situations, including immune dysregulation and bacterial infections, which is of considerable utility for the development of new topical antibiotic drugs for burn wound and infections.

A Systems-Based Analysis of Mono- and Combination Therapy for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections.

Antimicrobial resistance is a global threat. As "proof-of-concept," we employed a system-based approach to identify patient, bacterial, and drug variables contributing to mortality in patients with carbapenem-resistant Klebsiella pneumoniae (CRKp) bloodstream infections exposed to colistin (COL) and ceftazidime-avibactam (CAZ/AVI) as mono- or combination therapies. Patients (n = 49) and CRKp isolates (n = 22) were part of the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE-1), a multicenter, observational, prospective study of patients with carbapenem-resistant Enterobacterales (CRE) conducted between 2011 and 2016. Pharmacodynamic activity of mono- and combination drug concentrations was evaluated over 24 h using in vitro static time-kill assays. Bacterial growth and killing dynamics were estimated with a mechanism-based model. Random Forest was used to rank variables important for predicting 30-day mortality. Isolates exposed to COL+CAZ/AVI had enhanced early bacterial killing compared to CAZ/AVI alone and fewer incidences of regrowth compared to COL and CAZ/AVI. The mean coefficient of determination (R2) for the observed versus predicted bacterial counts was 0.86 (range: 0.75 - 0.95). Bacterial subpopulation susceptibilities and drug mechanistic synergy were essential to describe bacterial killing and growth dynamics. The combination of clinical (hypotension), bacterial (IncR plasmid, aadA2, and sul3) and drug (KC50) variables were most predictive of 30-day mortality. This proof-of-concept study combined clinical, bacterial, and drug variables in a unified model to evaluate clinical outcomes.

Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen causing nosocomial infections. K. pneumoniae rapidly acquires antibiotic resistance and is known as a reservoir for resistance genes. Polymyxins remain effective as a last-line therapy against infections caused by multidrug-resistant (MDR) K. pneumoniae; however, resistance to polymyxins emerges rapidly with monotherapy. Synergistic combinations of polymyxins with FDA-approved non-antibiotics are a novel approach to preserve its efficacy whilst minimising the emergence of polymyxin resistance in K. pneumoniae. This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the anti-fungal caspofungin against K. pneumoniae. The combination of polymyxin B and caspofungin showed marked synergistic antibacterial killing activity in checkerboard broth microdilution and static time-kill assays at clinically relevant concentrations at early (0.5 and 1 h) and later (4 h) time points. The potential bacterial killing mechanism of the combination was studied against K. pneumoniae FADDI-KP001 using metabolomics and transcriptomics studies at 0.5, 1 and 4 h. The key pathways involved in the synergistic killing action of the combination were cell wall assembly (peptidoglycan and lipopolysaccharide biosynthesis), central carbon metabolism (glycolysis, pentose phosphate pathway and tricarboxylic acid cycle) and fatty acid biosynthesis. Moreover, the combination inhibited the most common bacterial virulence pathway (phosphotransferase system) as well as the multi-resistant efflux mechanisms, including ATP-binding cassette (ABC) transporter pathway. Overall, this study sheds light on the possibility of a polymyxin-caspofungin combination for the treatment of infections caused by K. pneumoniae and may help repurpose FDA-approved caspofungin against MDR K. pneumoniae infections.

Pharmacodynamic and immunomodulatory effects of polymyxin B in combination with fosfomycin against KPC-2-producing Klebsiella pneumoniae.

Determining the role of the immune response in preventing antimicrobial resistance and optimising antibiotic regimens against carbapenemase-producing Klebsiella pneumoniae is a research gap that exists and needs to be further explored. The objective of this study was to determine the pharmacodynamic and immunomodulatory effects of fosfomycin alone and in combination with polymyxin B against KPC-2-producing K. pneumoniae clinical isolates. Six K. pneumoniae isolates were selected (polymyxin B MIC, 0.5-64 mg/L; fosfomycin MIC, 16-128 mg/L) to evaluate the pharmacodynamics of monotherapy and combination therapies in static time-kill studies. A mechanism-based model was used to characterise the joint activity of polymyxin B and fosfomycin. A549 human airway epithelial cells were infected with four isolates to evaluate the immunomodulatory effects of treatment. Our mechanism-based model indicated greater bacterial killing efficacy of fosfomycin with polymyxin B compared with monotherapy. In combination, polymyxin B was assumed to exert an outer membrane effect that resulted in an increase in the ability of fosfomycin to reach its target site. The mechanism-based model described the data well across all six strains, with R2 values ranging from 0.705-0.935. Combination therapy reduced K. pneumoniae-induced IL-6 and IL-8 but not TNFα expression. The reduction in cytokine expression was greater with polymyxin B than fosfomycin alone; combination therapy showed significantly greater reduction compared to either monotherapy. Our findings suggest that further research is needed to better understand immune-mediated killing in order to identify a strategy which harnesses the power of the immune response against these hard-to-treat bacteria.

Evaluation Strategies for Triple-Drug Combinations against Carbapenemase-Producing Klebsiella Pneumoniae in an In Vitro Hollow-Fiber Infection Model.

Mounting antimicrobial resistance to carbapenemase-producing Klebsiella pneumoniae (CPKP) highlights the need to optimize currently available treatment options. The objective of this study was to explore alternative dosing strategies that limit the emergence of resistance to preserve the utility of last-line antibiotics by: (i) evaluating the pharmacodynamic (PD) killing activity of simulated humanized exposures to monotherapy and two-drug and three-drug combinations against CPKP bacterial isolates with different resistance mechanisms; and (ii) optimizing polymyxin B (PMB) exposure simulated in the three-drug combination regimens to maximize the killing activity. Two CPKP clinical isolates (BAA2146 (NDM-1) and BRKP76 (KPC-2)) were evaluated over 168 hours using a hollow-fiber infection model simulating clinically relevant PMB, fosfomycin, and meropenem dosing regimens. PMB-based three-drug combinations were further optimized by varying the initial exposure (0-24 hours) or maintenance dose received over the duration of treatment. The area under the bacterial load-versus-time curve (AUCFU) was used to determine PD activity. Overall reductions in PMB exposure ranged from 2 to 84%. BAA2146 and BRKP76 had median (range) AUCFUs of 11.0 (10.6-11.6) log10  CFU hour/mL and 7.08 (7.04-11.9) log10 CFU hour/mL, respectively. The PMB "front loaded" 2.5 mg/kg/day + 0.5 mg/kg maintenance dose in combination with meropenem and fosfomycin was a promising regimen against BRKP76, with an overall reduction in PMB exposure of 56% while still eradicating the bacteria. Tailored triple-combination therapy allows for the optimization of dose and treatment duration of last-line agents like PMB to achieve adequate drug exposure and appropriate PD activity while minimizing the emergence of resistance.

The unusual glycine-rich C terminus of the Acinetobacter baumannii RNA chaperone Hfq plays an important role in bacterial physiology.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that causes soft tissue infections in patients who spend a long time in intensive care units. This recalcitrant bacterium is very well known for developing rapid drug resistance, which is a combined outcome of its natural competence and mobile genetic elements. Successful efforts to treat these infections would be aided by additional information on the physiology of A. baumannii Toward that end, we recently reported on a small RNA (sRNA), AbsR25, in this bacterium that regulates the genes of several efflux pumps. Because sRNAs often require the RNA chaperone Hfq for assistance in binding to their cognate mRNA targets, we identified and characterized this protein in A. baumannii The homolog in A. baumannii is a large protein with an extended C terminus unlike Hfqs in other Gram-negative pathogens. The extension has a compositional bias toward glycine and, to a lower extent, phenylalanine and glutamine, suggestive of an intrinsically disordered region. We studied the importance of this glycine-rich tail using truncated versions of Hfq in biophysical assays and complementation of an hfq deletion mutant, finding that the tail was necessary for high-affinity RNA binding. Further tests implicate Hfq in important cellular processes of A. baumannii like metabolism, drug resistance, stress tolerance, and virulence. Our findings underline the importance of the glycine-rich C terminus in RNA binding, ribo-regulation, and auto-regulation of Hfq, demonstrating this hitherto overlooked protein motif to be an indispensable part of the A. baumannii Hfq.

Comparison of the composition and in vitro activity of polymyxin B products.

A number of companies manufacture polymyxin B using United States Pharmacopeia (USP) metrics, rather than chemical composition, to report biological activity. Given that polymyxin B contains several different components, it is unknown whether pharmacokinetic and pharmacodynamic variability exists between the different brands and whether USP metrics capture this variability. Here we investigated the composition of polymyxin B obtained from four manufacturers (Sigma-Aldrich, AK Scientific, USP and MP Biomedicals) and evaluated their rate and extent of killing against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae using in vitro static time-kill experiments. Ultraviolet (UV) fingerprinting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed similarities and differences between component distributions. The significant differences between products, based on UV fingerprinting and LC-MS/MS, did not translate into pharmacodynamic differences at the three concentrations evaluated. The aggregate polymyxin B concentration, rather than that of the individual components, influences overall bacterial killing.

Evaluation of Activity and Emergence of Resistance of Polymyxin B and ZTI-01 (Fosfomycin for Injection) against KPC-Producing Klebsiella pneumoniae.

ZTI-01 (fosfomycin for injection) is a broad-spectrum antibiotic with a novel mechanism of action and is currently under development in the United States for treatment of complicated urinary tract infections. Globally, fosfomycin and polymyxin B are increasingly being used to treat multidrug-resistant Gram-negative infections. The objectives were to evaluate the pharmacodynamic activity of polymyxin B and fosfomycin alone and in combination against KPC-producing Klebsiella pneumoniae and to assess the rate and extent of emergence of resistance to different antibiotic regimens. Two clinical isolates, BRKP26 (MIC of polymyxin B[MICPMB], 0.5 mg/liter; MIC of fosfomycin [MICFOF], 32 mg/liter) and BRKP67 (MICPMB, 8 mg/liter; MICFOF, 32 mg/liter) at an initial inoculum of 107 CFU/ml, were evaluated over 168 h in a hollow-fiber infection model simulating clinically relevant polymyxin B (2.5-mg/kg loading dose as a 2 h-infusion followed by 1.5-mg/kg dose every 12 h [q12h] as a 1-h infusion) and fosfomycin (6 g q6h as a 1-h or 3-h infusion) regimens alone and in combination. Population analysis profiles (PAPs) and MIC testing were performed to assess emergence of resistance. Polymyxin B or fosfomycin monotherapy was ineffective and selected for resistance by 24 h. Polymyxin B plus a fosfomycin 1-h infusion demonstrated sustained bactericidal activity by 4 h, with undetectable colony counts beyond 144 h. Polymyxin B plus a fosfomycin 3-h infusion demonstrated bactericidal activity at 4 h, followed by regrowth similar to that of the control by 144 h. PAPs revealed resistant subpopulations by 120 h. The combination of polymyxin B and a fosfomycin 1-h infusion is a promising treatment option for KPC-producing K. pneumoniae and suppresses the emergence of resistance. Further evaluation of novel dosing strategies is warranted to optimize therapy.

In Vitro Assessment of Combined Polymyxin B and Minocycline Therapy against Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae.

The multidrug resistance profiles of Klebsiella pneumoniae carbapenemase (KPC) producers have led to increased clinical polymyxin use. Combination therapy with polymyxins may improve treatment outcomes, but it is uncertain which combinations are most effective. Clinical successes with intravenous minocycline-based combination treatments have been reported for infections caused by carbapenemase-producing bacteria. The objective of this study was to evaluate the in vitro activity of polymyxin B and minocycline combination therapy against six KPC-2-producing K. pneumoniae isolates (minocycline MIC range, 2 to 32 mg/liter). Polymyxin B monotherapy (0.5, 1, 2, 4, and 16 mg/liter) resulted in a rapid reduction of up to 6 log in bactericidal activity followed by regrowth by 24 h. Minocycline monotherapy (1, 2, 4, 8, and 16 mg/liter) showed no reduction of activity of >1.34 log against all isolates, although concentrations of 8 and 16 mg/liter prolonged the time to regrowth. When the therapies were used in combination, rapid bactericidal activity was followed by slower regrowth, with synergy (60 of 120 combinations at 24 h, 19 of 120 combinations at 48 h) and additivity (43 of 120 combinations at 24 h, 44 of 120 combinations at 48 h) against all isolates. The extent of killing was greatest against the more susceptible polymyxin B isolates (MICs of ≤0.5 mg/liter) regardless of the minocycline MIC. The pharmacodynamic activity of combined polymyxin B-minocycline therapy against KPC-producing K. pneumoniae is dependent on polymyxin B susceptibility. Further in vitro and animal studies must be performed to fully evaluate the efficacy of this drug combination.

Polymyxin B in combination with meropenem against carbapenemase-producing Klebsiella pneumoniae: pharmacodynamics and morphological changes.

Combination therapy provides a useful therapeutic approach to overcome resistance until new antibiotics become available. In this study, the pharmacodynamics, including the morphological effects, of polymyxin B (PMB) and meropenem alone and in combination against KPC-producing Klebsiella pneumoniae clinical isolates was examined. Ten clinical isolates were obtained from patients undergoing treatment for mediastinitis. KPCs were identified and MICs were measured using microbroth dilution. Time-kill studies were conducted over 24 h with PMB (0.5-16 mg/L) and meropenem (20-120 mg/L) alone or in combination against an initial inoculum of ca. 106 CFU/mL. Scanning electron microscopy (SEM) was employed to analyse changes in bacterial morphology after treatment, and the log change method was used to quantify the pharmacodynamic effect. All isolates harboured the blaKPC-2 gene and were resistant to meropenem (MICs ≥8 mg/L). Clinically relevant PMB concentrations (0.5, 1.0 and 2.0 mg/L) in combination with meropenem were synergistic against all isolates except BRKP28 (polymyxin- and meropenem-resistant, both MICs >128 mg/L). All PMB and meropenem concentrations in combination were bactericidal against polymyxin-susceptible isolates with meropenem MICs ≤16 mg/L. SEM revealed extensive morphological changes following treatment with PMB in combination with meropenem compared with the changes observed with each individual agent. Additionally, morphological changes decreased with increasing resistance profiles of the isolate, i.e. increasing meropenem MIC. These antimicrobial effects may not only be a summation of the effects due to each antibiotic but also a result of differential action that likely inhibits protective mechanisms in bacteria.

Polymyxin B in Combination with Rifampin and Meropenem against Polymyxin B-Resistant KPC-Producing Klebsiella pneumoniae.

Safe and effective therapies are urgently needed to treat polymyxin-resistant KPC-producing Klebsiella pneumoniae infections and suppress the emergence of resistance. We investigated the pharmacodynamics of polymyxin B, rifampin, and meropenem alone and as polymyxin B-based double and triple combinations against KPC-producing K. pneumoniae isolates. The rates and extents of killing with polymyxin B (1 to 128 mg/liter), rifampin (2 to 16 mg/liter), and meropenem (10 to 120 mg/liter) were evaluated against polymyxin B-susceptible (PBs) and polymyxin B-resistant (PBr) clinical isolates using 48-h static time-kill studies. Additionally, humanized triple-drug regimens of polymyxin B (concentration at steady state [Css] values of 0.5, 1, and 2 mg/liter), 600 mg rifampin every 12 or 8 h, and 1 or 2 g meropenem every 8 h dosed as an extended 3-h infusion were simulated over 48 h by using a one-compartment in vitro dynamic infection model. Serial bacterial counts were performed to quantify the pharmacodynamic effect. Population analysis profiles (PAPs) were used to assess the emergence of polymyxin B resistance. Monotherapy was ineffective against both isolates. Polymyxin B with rifampin demonstrated early bactericidal activity against the PBs isolate, followed by regrowth by 48 h. Bactericidal activity was sustained at all polymyxin B concentrations of ≥2 mg/liter in combination with meropenem. No two-drug combinations were effective against the PBr isolate, but all simulated triple-drug regimens showed early bactericidal activity against both strains by 8 h that was sustained over 48 h. PAPs did not reveal the emergence of resistant subpopulations. The triple-drug combination of polymyxin B, rifampin, and meropenem may be a viable consideration for the treatment of PBr KPC-producing K. pneumoniae infections. Further investigation is warranted to optimize triple-combination therapy.

Fosfomycin resistance in Acinetobacter baumannii is mediated by efflux through a major facilitator superfamily (MFS) transporter-AbaF.

OBJECTIVES: To decipher the function of A1S_1331, named AbaF (Acinetobacter baumannii Fosfomycin efflux), one of the primary targets of AbsR25, a small RNA of A. baumannii. METHODS: abaF was cloned in a multicopy plasmid and expressed from its native promoter in an efflux-deficient strain-Escherichia coli KAM32. Drug susceptibility, accumulation and efflux of ethidium bromide (EtBr) were determined in this strain. abaF was disrupted in A. baumannii using homologous recombination and its effect on drug susceptibility, biofilm formation and virulence was studied. Expression of abaF was followed by quantitative PCR in fosfomycin-challenged A. baumannii and fosfomycin-resistant mutants of A. baumannii. Expression of abaF in clinical strains of A. baumannii was determined by RT-PCR. RESULTS: Expression of abaF in E. coli KAM32 resulted in increased resistance to fosfomycin. Lower accumulation and higher efflux of EtBr from this strain confirmed the role of AbaF as an efflux pump. Disruption of abaF in A. baumannii caused an increase in fosfomycin susceptibility and a decrease in biofilm formation and virulence. The expression of abaF was higher in A. baumannii cells exposed to fosfomycin and in cells resistant to higher concentrations of fosfomycin. The clinically relevant strains of A. baumannii also tested positive for the expression of abaF. CONCLUSIONS: The results of this study suggest that efflux is an important mechanism of fosfomycin resistance and AbaF is involved in fosfomycin resistance in A. baumannii. AbaF also seems to play a role in biofilm formation and virulence of A. baumannii.