Kamilia Abdelraouf

Prevalence, Resistance Mechanisms, and Susceptibility of Multidrug-Resistant Bloodstream Isolates of Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is an important pathogen commonly implicated in nosocomial infections. The occurrence of multidrug-resistant (MDR) P. aeruginosa strains is increasing worldwide and limiting our therapeutic options. The MDR phenotype can be mediated by a variety of resistance mechanisms, and the corresponding relative biofitness is not well established. We examined the prevalence, resistance mechanisms, and susceptibility of MDR P. aeruginosa isolates (resistant to ≥3 classes of antipseudomonal agents [penicillins/cephalosporins, carbapenems, quinolones, and aminoglycosides]) obtained from a large, university-affiliated hospital. Among 235 nonrepeat bloodstream isolates screened between 2005 and 2007, 33 isolates (from 20 unique patients) were found to be MDR (crude prevalence rate, 14%). All isolates were resistant to carbapenems and quinolones, 91% were resistant to penicillins/cephalosporins, and 21% were resistant to the aminoglycosides. By using the first available isolate for each bacteremia episode (n = 18), 13 distinct clones were revealed by repetitive-element-based PCR. Western blotting revealed eight isolates (44%) to have MexB overexpression. Production of a carbapenemase (VIM-2) was found in one isolate, and mutations in gyrA (T83I) and parC (S87L) were commonly found. Growth rates of most MDR isolates were similar to that of the wild type, and two isolates (11%) were found to be hypermutable. All available isolates were susceptible to polymyxin B, and only one isolate was nonsusceptible to colistin (MIC, 3 mg/liter), but all isolates were nonsusceptible to doripenem (MIC, >2 mg/liter). Understanding and continuous monitoring of the prevalence and resistance mechanisms of MDR P. aeruginosa would enable us to formulate rational treatment strategies to combat nosocomial infections.

Characterization of Polymyxin B-Induced Nephrotoxicity: Implications for Dosing Regimen Design

ABSTRACT The increasing prevalence of multidrug-resistant Gram-negative infections has led to renewed interest in the use of systemic polymyxin B. However, the nephrotoxic properties of polymyxin B are still poorly understood. The objective of this study was to characterize nephrotoxicity associated with polymyxin B, with an emphasis on examining the impact of dosing frequencies on the onset of nephrotoxicity. Sprague-Dawley rats were divided into two groups and administered the same total daily dose of polymyxin B subcutaneously but with different dosing frequencies (either 20 mg/kg of body weight every 24 h [q24h] or 5 mg/kg q6h). Drug concentrations in renal tissue were compared between the two groups at 24 h. Kidney tissues were harvested at 48 h and compared histologically. Serum creatinine was measured daily for up to 10 days, and nephrotoxicity was defined as a significant elevation in serum creatinine (≥2× baseline). Kaplan-Meier analysis was used to compare the onset of nephrotoxicity. Polymyxin B-induced nephrotoxicity manifested as elevation in serum creatinine and acute tubular necrosis. Extensive injury of the proximal tubules was observed. The lesions were more severe and higher drug concentrations were achieved in the kidneys of the q6h dosing group. The q24h dosing group experienced a more gradual onset of nephrotoxicity, which could be attributed to the lower kidney tissue drug concentrations (48.5 ± 17.4 μg/g versus 92.1 ± 18.1 μg/g of polymyxin B1, P = 0.04). Preferential accumulation of polymyxin B in the kidneys suggests that uptake to renal cells is a nonpassive process and q24h dosing was less nephrotoxic than q6h dosing.

Pharmacokinetics and renal disposition of polymyxin B in an animal model.

ABSTRACT 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.

Effect of multidrug resistance-conferring mutations on the fitness and virulence of Pseudomonas aeruginosa

BACKGROUND Multidrug resistance has become a quandary in the treatment of bacterial infections. The effect of resistance mutations and the fitness cost on the pathogenicity of Pseudomonas aeruginosa is not well established. The objective of this study was to examine the impact of multidrug resistance on the fitness and virulence of P. aeruginosa. METHODS Fourteen P. aeruginosa strains with various resistance mechanisms were used. In vitro growth of these isolates was investigated in full-strength and 0.25-strength Mueller-Hinton broth (MHB). Exponential growth rates were estimated from serial bacterial burden over 24 h. In vitro growth of two multidrug-resistant strains (PAO1ΔmexRΔoprD and PA9019) was studied when each was grown in co-culture with wild-type strain PAO1. In vivo growth was compared between PAO1 and PAO1ΔmexRΔopD using a murine pneumonia model; virulence over 10 days was studied in six isolates. RESULTS Significant reduction in growth rate was observed in selected mutants (P < 0.01). PAO1 out-competed PAO1ΔmexRΔoprD and PA9019 in vitro, and in vivo growth of PAO1 was faster than PAO1ΔmexRΔoprD. Compared with PAO1, PAO1ΔmexR and PAO1ΔoprD showed a slight reduction in mortality rate; significantly lower mortality was seen in PAO1ΔmexRΔoprD (P < 0.01). However, virulence of PA9019 was not significantly different from that of PAO1. CONCLUSIONS Specific resistance mutations were associated with fitness cost in P. aeruginosa, and accumulation of such mutations was associated with a reduction in virulence. However, it was difficult to predict the impact in clinical isolates. Knowledge of multidrug resistance mechanisms and compensatory mutations would likely be helpful.

Pharmacokinetics of Polymyxin B in a Patient With Renal Insufficiency: A Case Report

TO THE EDITOR—The pharmacokinetics of polymyxin B, especially with regard to the renal insufficiency cohort, are extremely lacking despite the increasing clinical use of this agent [1]. We described the pharmacokinetics of polymyxin B in a patient with renal insufficiency who was not undergoing dialysis.

Predicting bacterial fitness cost associated with drug resistance

OBJECTIVES It has been proposed that antimicrobial resistance could be associated with a fitness cost in bacteria, which is often determined by competition experiments between isogenic strains (wild-type and mutant). However, this conventional approach is time consuming and labour intensive. An alternative method was developed to assess the fitness cost in drug-resistant bacteria. METHODS Time-growth studies were performed with approximately 1 × 10(5) cfu/mL of Acinetobacter baumannii or Pseudomonas aeruginosa at baseline. Serial samples were obtained to quantify the bacterial burden over 24 h. The growth rates (K(g)) of isogenic strains (antibiotic susceptible and resistant) were determined individually and used to predict their relative abundance in a co-culture over an extended period of time. The predicted difference between the two strains was subsequently validated by in vitro growth competition experiments. RESULTS The growth rates of A. baumannii were not significantly different in different strengths of growth medium. The difference in bacterial burden observed in competition studies was in general agreement with the predicted difference based on K(g) values, suggesting good predicting ability of the mathematical model. CONCLUSIONS The proposed mathematical model was found to be reasonable in characterizing bacterial growth and predicting the fitness cost of resistance. This simple method appears robust in the assessment of fitness cost associated with drug resistance and warrants further investigations.

Uptake of Polymyxin B into Renal Cells

ABSTRACT Polymyxin B is increasingly used as a treatment of last resort against multidrug-resistant Gram-negative infections. Using a mammalian kidney cell line, we demonstrated that polymyxin B uptake into proximal tubular epithelial cells was saturable and occurred primarily through the apical membrane, suggesting the involvement of transporters in the renal uptake of polymyxin B. Megalin might play a role in the uptake and accumulation of polymyxin B into renal cells.

Pharmacokinetics and efficacy of liposomal polymyxin B in a murine pneumonia model.

Polymyxin B (PB) is increasingly used as the last treatment for multidrug-resistant (MDR) Gram-negative bacterial infections. In this study, serum and epithelial lining fluid (ELF) pharmacokinetics and the efficacy of a PB liposomal formulation were investigated. Two groups of 24 Swiss Webster mice were intravenously administered PB liposomes or PB aqueous solution at ca. 3 mg/kg. Serum and ELF samples were collected for up to 6 h to quantify major PB components. Three groups of neutropenic mice (n = 6/group) were infected with a clinical MDR Pseudomonas aeruginosa strain followed by intravenous administration of PB liposomes or PB aqueous solution at 3 mg/kg every 6 h or sham (drug-free) liposomes every 6 h. Bacterial burden in animal lung tissues was quantified after 24 h of therapy and was compared using one-way ANOVA. Survival of infected animals over time (n = 10/group) was evaluated by Kaplan-Meier analysis and log-rank test. In the pharmacokinetic study, the AUC ratio in ELF between liposome and aqueous solution groups ranged from 4.6 to 11.1 for various major PB components. In the efficacy study, for strain PA 9019 a significantly lower bacterial burden was seen in the liposomal group (3.8 ± 0.7 vs. 7.9 ± 0.8 log(10)CFU/g in the aqueous solution group), which subsequently prolonged survival of infected animals. In this study, treatment with a PB liposomal formulation yielded higher drug penetration into pulmonary ELF, which resulted in superior efficacy. However, further investigations on the clinical utility of the PB liposomal formulation are warranted.

Quantitative Impact of Neutrophils on Bacterial Clearance in a Murine Pneumonia Model

ABSTRACT 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 (r2 = 0.945). The best-fit maximal kill rate (Kk) of the bacterial population by neutrophils was 1.743 h−1, the number of neutrophils necessary for 50% maximal killing was 190.8/μl, and the maximal population size was 1.8 × 109 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.

Pharmacokinetics of polymyxin B in an infant with multidrug-resistant Klebsiella pneumoniae bacteremia.

We report our experience with a 9-month-old infant treated with intravenous polymyxin B for multidrug-resistant Klebsiella pneumoniae bacteremia. Serial blood samples were obtained at steady state and serum drug concentrations were determined using a validated liquid chromatography-mass spectrometry method. The elimination half-lives of polymyxin B1 and isoleucine-polymyxin B1 were found to be 3.1 and 4.7 hours, respectively.