c Carbapenem-resistant bacteria represent a significant treatment challenge due to the lack of active antimicrobials available. MK-7655 is a novel ␤-lactamase inhibitor under clinical development. We investigated the combined killing activity of imipenem and MK-7655 against four imipenem-resistant bacterial strains, using a mathematical model previously evaluated in our laboratory. Time-kill studies (TKS) were conducted with imipenem and MK-7655 against a KPC-2-producing Klebsiella pneumoniae isolate (KP6339) as well as 3 Pseudomonas aeruginosa isolates (PA24226, PA24227, and PA24228) with OprD porin deletions and overexpression of AmpC. TKS were performed using 25 clinically achievable concentration combinations in a 5-by-5 array. Bacterial burden at 24 h was determined in triplicate by quantitative culture and mathematically modeled using a three-dimensional response surface. Mathematical model assessments were evaluated experimentally using clinically relevant dosing regimens of imipenem, with or without MK-7655, in a hollow-fiber infection model (HFIM). The combination of imipenem and MK-7655 was synergistic for all strains. Interaction indices were as follows: for KP6339, 0.50 (95% confidence interval [CI], 0.42 to 0.58); for PA24226, 0.60 (95% CI, 0.58 to 0.62); for PA24227, 0.70 (95% CI, 0.66 to 0.74); and for PA24228, 0.55 (95% CI, 0.49 to 0.61). In the HFIM, imipenem plus MK-7655 considerably reduced the bacterial burden at 24 h, while failure with imipenem alone was seen against all isolates. Sustained suppression of bacterial growth at 72 h was achieved with simulated doses of 500 mg imipenem plus 500 mg MK-7655 in 2 (KP6339 and PA24227) strains, and it was achieved in an additional strain (PA24228) when the imipenem dose was increased to 1,000 mg. Additional studies are being conducted to determine the optimal dose and combinations to be used in clinical investigations.

The development of β-lactam resistance during therapy could be suppressed by an optimized dosing exposure. Validation of the proposed target in a well-designed clinical study is warranted.

Antimicrobial resistance among Acinetobacter baumannii is increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates of A. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine ␤-naphthylamide (PA␤N). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 g/ml). Time-kill studies were performed over 24 h using approximately 10 6 CFU/ml of each strain with clinically relevant minocycline concentrations (2 g/ml and 8 g/ml), with and without polymyxin B (0.5 g/ml). The in vivo efficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (>4؋) was observed in the presence of PA␤N. The intracellular concentration and in vitro bactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocyclinesusceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.A cinetobacter baumannii is a common nosocomial pathogen globally which has been implicated as an etiologic agent in ventilator-associated pneumonia, skin and skin structure infections (traumatic battlefield and other wounds), urinary tract infections, meningitis, and bacteremia (1). Over the past decades, infections due to increasingly resistant strains of Acinetobacter have emerged (2, 3). This is particularly alarming because there are very limited therapeutic options for these infections (4, 5). Multidrug-resistant strains of A. baumannii have been reported to adversely affect patient outcomes. Studies have found significantly higher rates of hospital mortality in patients infected with multidrug-resistant strains than in patients with susceptible strains (6, 7).Current treatment strategies for multidrug-resistant A. baumannii include combination therapy with tigecycline, minocycline, carbapenems, polymyxins, and even daptomycin (8-11). However, the rationale of using certain agents together in a combination is not well established. In this study, the combination of minocycline and polymyxin B was studied based on a mechanistically plausible approach. Resistance to the tetracycline class in A. baumannii is commonly mediated through the upregulation of efflux pumps loca...

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2-to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (>64؋ MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop.

The increasing prevalence of multidrug-resistant Gram-negative infections has led to the resurgence of systemic polymyxin B, but little is known about its pharmacokinetics. The objective of this study was to characterize the pharmacokinetics and renal disposition of polymyxin B. Eight female Sprague-Dawley rats (weight, 225 to 250 g) were administered a single intravenous polymyxin B dose (4 mg/kg of body weight). Serial serum samples were collected and assayed for major polymyxin B components using a validated ultraperformance liquid chromatography-tandem mass spectrometry method. The best-fit pharmacokinetic parameters of each component were derived and compared using one-way analysis of variance. Cumulative urine was also collected daily for 48 h and assayed for polymyxin B. Kidney drug concentrations were measured at 6 h (n ‫؍‬ 3) and 48 h (n ‫؍‬ 3) after the same dose. Additionally, three rats were administered 2 doses of intravenous polymyxin B (4 mg/kg) 7 days apart. Serial serum samples were collected pre-and post-renal insufficiency (induced by uranyl nitrate) and assayed for polymyxin B. The pharmacokinetic parameters of the major components did not appear to be significantly different (P > 0.05). Less than 1% of the dose was recovered unchanged in urine collected over 48 h following administration. Therapeutic drug concentrations persisted in kidney tissue at 48 h. The post-renal insufficiency to pre-renal insufficiency ratio of the area under the serum concentration-time curve from time zero to infinity was 1.33 ؎ 0.04. Polymyxin B components appear to have similar pharmacokinetics. Polymyxin B preferentially persists in kidneys, which suggests a selective uptake process in renal cells. A mechanism(s) other than renal excretion could be involved in polymyxin B elimination, and dosing adjustment in renal insufficiency may not be necessary.

Despite dose-limiting nephrotoxicity concerns, polymyxin B has resurged as the treatment of last resort for multidrug-resistant Gram-negative bacterial infections. However, the pharmacokinetic, pharmacodynamic, and nephrotoxic properties of polymyxin B still are not thoroughly understood. The objective of this study was to provide additional insights into the overall biodistribution and disposition of polymyxin B in an animal model. Sprague-Dawley rats were dosed with intravenous polymyxin B (3 mg/kg of body weight). Drug concentrations in the serum, urine, bile, and tissue (brain, heart, lungs, liver, spleen, kidneys, and skeletal muscle) samples over time were assayed by a validated methodology. Among all the organs evaluated, polymyxin B distribution was highest in the kidneys. The mean renal tissue/serum polymyxin B concentration ratios were 7.45 (95% confidence interval [CI], 4.63 to 10.27) at 3 h and 19.62 (95% CI, 5.02 to 34.22) at 6 h postdose. Intrarenal drug distribution was examined by immunostaining. Using a ratiometric analysis, proximal tubular cells showed the highest accumulation of polymyxin B (Mander's overlap coefficient, 0.998) among all cell types evaluated. Less than 5% of the administered dose was recovered in urine over 48 h, but all 4 major polymyxin B components were detected in the bile over 4 h. These findings corroborate previous results that polymyxin B is highly accumulated in the kidneys, but the elimination likely is via a nonrenal route. Biliary excretion could be one of the routes of polymyxin B elimination, and this should be further explored. The elucidation of mechanism(s) of drug uptake in proximal tubular cells is ongoing.T he emergence of multidrug-resistant bacterial infections has become a medical crisis worldwide (1, 2). Infections caused by Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter spp., are extremely challenging to treat (3-6). These infections also are associated with high rates of mortality and morbidity (7,8). Moreover, there are few new antibacterial agents available in the clinical drug development pipeline for these life-threatening infections. Consequently, this has led to the revival of old antibiotics, such as the polymyxins, as the treatment of last resort for infections caused by multidrugresistant Gram-negative pathogens (9-13).Polymyxins (primarily polymyxin B and polymyxin E [colistin]) are cyclic polypeptide antibiotics isolated from Bacillus polymyxa (14). Commercially available polymyxin B is a mixture of several related analogs, primarily polymyxin B1, B2, and B3 and isoleucine B1 (15, 16). Polymyxin B first became available for clinical use in the 1950s, but its clinical use has been limited largely due to its nephrotoxic potential.Despite being available for clinical use for more than 50 years, there is still a paucity of published reports correlating the pharmacokinetics of polymyxin B with its toxicity profile. Furthermore, we lack a thorough understanding of the biodistribution pattern, cel...

The scarcity of new antibiotics against drug-resistant bacteria has led to the development of inhibitors targeting specific resistance mechanisms, which aim to restore the effectiveness of existing agents. However, there are few guidelines for the optimal dosing of inhibitors. Extending the utility of mathematical modeling, which has been used as a decision support tool for antibiotic dosing regimen design, we developed a novel mathematical modeling framework to guide optimal dosing strategies for a beta-lactamase inhibitor. To illustrate our approach, MK-7655 was used in combination with imipenem against a clinical isolate of Klebsiella pneumoniae known to produce KPC-2. A theoretical concept capturing fluctuating susceptibility over time was used to define a novel pharmacodynamic index (time above instantaneous MIC [T>MIC i ]). The MK-7655 concentration-dependent MIC reduction was characterized by using a modified sigmoid maximum effect (E max )-type model. Various dosing regimens of MK-7655 were simulated to achieve escalating T>MIC i values in the presence of a clinical dose of imipenem (500 mg every 6 h). The effectiveness of these dosing exposures was subsequently validated by using a hollow-fiber infection model (HFIM). An apparent trend in the bacterial response was observed in the HFIM with increasing T>MIC i values. In addition, different dosing regimens of MK-7655 achieving a similar T>MIC i (69%) resulted in comparable bacterial killing over 48 h. The proposed framework was reasonable in predicting the in vitro activity of a novel beta-lactamase inhibitor, and its utility warrants further investigations.

The rapid increase in the prevalence of antibiotic-resistant pathogens is a global problem that has challenged our ability to treat serious infections. Currently, clinical decisions on treatment are often based on in vitro susceptibility data. The role of the immune system in combating bacterial infections is unequivocal, but it is not well captured quantitatively. In this study, the impact of neutrophils on bacterial clearance was quantitatively assessed in a murine pneumonia model. In vitro time-growth studies were performed to determine the growth rate constants of Acinetobacter baumannii ATCC BAA 747 and Pseudomonas aeruginosa PAO1. The absolute neutrophil count in mice resulting from different cyclophosphamide preparatory regimens was determined. The dynamic change of bacterial (A. baumannii BAA 747) burden in mice with graded immunosuppression over 24 h was captured by a mathematical model. The fit to the data was satisfactory (r 2 ‫؍‬ 0.945). The best-fit maximal kill rate (K k ) of the bacterial population by neutrophils was 1.743 h ؊1 , the number of neutrophils necessary for 50% maximal killing was 190.8/l, and the maximal population size was 1.8 ؋ 10 9 CFU/g, respectively. Using these model parameter estimates, the model predictions were subsequently validated by the bacterial burden change of P. aeruginosa PAO1 at 24 h. A simple mathematical model was proposed to quantify the contribution of neutrophils to bacterial clearance and predict the bacterial growth/ suppression in animals. Our results provide a novel framework to link in vitro and in vivo information and may be used to improve clinical treatment of bacterial infections.The rapid increase in the prevalence of antibiotic-resistant pathogens is a global problem that has challenged our ability to treat serious infections. To prevent further spread of multidrug resistance and returning to the preantibiotic era, it is imperative that the current approach to treatment be improved. Presently, clinical decisions on treatment are often based on in vitro susceptibility data (MICs), which only provide information about antibacterial activity at a single time point and potentially miss information on the changes in bacterial population dynamics over time. Drug selections solely based on MIC values may not always correlate to patient outcomes (11). One major reason for the discrepancy may be due to in vitro investigations neglecting the presence of the immune system in patients, which is well known to be significant.The immune system (especially neutrophils) plays an important role in combating bacterial infections. This is best exemplified by patients with neutropenia or after transplantation, who have significantly higher mortality than patients without neutropenia (8, 10, 13). However, the effect of the immune system is not well accounted for when data obtained from in vitro investigations are used to provide guidance for treatment. The overall antimicrobial effect in a patient is the sum of the antibacterial activity from drug therapy and the effect ...