Development of Modernized Acinetobacter baumannii Susceptibility Test Interpretive Criteria for Recommended Antimicrobial Agents Using Pharmacometric Approaches.

Acinetobacter baumannii-Acinetobacter calcoaceticus complex (referred to herein as A. baumannii) treatment guidelines contain numerous older antimicrobial agents with susceptibility test interpretive criteria (STIC, also known as susceptibility breakpoints) set using only epidemiological data. We utilized a combination of in vitro surveillance data, preclinical murine thigh and lung infection models, population pharmacokinetics, simulation, and pharmacokinetic/pharmacodynamic (PK/PD) target attainment analyses to evaluate A. baumannii STIC for four commonly recommended antimicrobials from different classes (amikacin, ceftazidime, ciprofloxacin, and minocycline). Antimicrobial in vitro surveillance data were based on 1,647 clinical A. baumannii isolates obtained from 109 centers in the United States and Europe. Among these isolates, 5 were selected for evaluation in murine infection models based on fitness and MIC variability. PK and dose-ranging studies were conducted using neutropenic murine thigh and lung infection models The MIC ranges for the 5 isolates evaluated were as follows: amikacin, 2 to 32 µg/mL; ceftazidime, 4 to 16 µg/mL; ciprofloxacin, 0.12 to 2 µg/mL; minocycline, 0.25 to 4 µg/mL. All organisms grew ≥1.5 log10 CFU in both models in untreated controls. Plasma and epithelial lining fluid (ELF) pharmacokinetics for all drugs were determined in mice using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. For each isolate, 5 dose levels of each drug were tested individually in the thigh and lung infection model. The inoculum ranged from 7.9 to 8.4 and 6.8 to 7.7 log10 CFU/mL for the lung and thigh models, respectively. PK/PD targets associated with net bacterial stasis and 1- and 2-log10 CFU reductions from baseline were identified for each organism/infection model using Hill-type models. Population pharmacokinetic models for each agent were identified from the literature. Using demographic variables for simulated patients with hospital-acquired or ventilator-associated bacterial pneumonia or urinary tract infections (including acute pyelonephritis) who were administered maximal dosing regimens of each agent, estimates of protein binding, and ELF penetration ratios based on data from the literature, free-drug plasma and total-drug concentration-time profiles were generated, and PK/PD indices by MIC were calculated. Percent probabilities of attaining median and randomly assigned PK/PD targets associated with the above-described endpoints were determined. Recommended susceptible breakpoints for each agent were those representing the highest MIC at which the percent probabilities of achieving PK/PD targets associated with a 1-log10 CFU reduction from baseline approached or were ≥90%. The following susceptible breakpoints for A. baumannii were identified: amikacin, ≤8 µg/mL for pneumonia; ceftazidime, ≤32 and ≤8 µg/mL for pneumonia; ciprofloxacin, ≤1 µg/mL; and minocycline, ≤0.5/≤1 µg/mL which correspond to the standard and high minocycline dosing regimens of 200 mg per day and 200 mg every 12 h, respectively. Implementation of appropriate STIC will help clinicians optimally use the above-described agents and improve the likelihood of successful patient outcomes.

Conserved Role for Biofilm Matrix Polysaccharides in Candida auris Drug Resistance.

Candida auris has emerged as an outbreak pathogen associated with high mortality. Biofilm formation and linked drug resistance are common among Candida species. Drug sequestration by the biofilm matrix accounts for much of the antifungal tolerance. In this study, we examine the biofilm matrix composition and function for a diverse set of C. auris isolates. We show that matrix sequesters nearly 70% of the available triazole antifungal. Like the biofilms formed by other Candida spp., we find that the matrix of C. auris is rich in mannan-glucan polysaccharides and demonstrate that their hydrolysis reduces drug tolerance. This biofilm matrix resistance mechanism appears conserved among Candida species, including C. aurisIMPORTANCECandida auris is an emerging fungal threat linked to poor patient outcomes. The factors responsible for this apparent increase in pathogenicity remain largely unknown. Biofilm formation has been suggested as an important factor for persistence of this organism in patients and the environment. Our findings reveal one mechanism utilized by C. auris to evade the effect of triazole antifungal therapy during biofilm growth. The conservation of the protective biofilm matrix among Candida spp. suggests that is a promising pan-fungal Candida biofilm drug target.

In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors, ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus.

ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10(7.4) to 10(8) CFU/thigh at the start of therapy with ACT-387042 and 10(6.7) to 10(8.3) CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R(2), 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors.

Endemic fungal infections in solid organ and hematopoietic cell transplant recipients enrolled in the Transplant-Associated Infection Surveillance Network (TRANSNET).

BACKGROUND: Invasive fungal infections are a major cause of morbidity and mortality among solid organ transplant (SOT) and hematopoietic cell transplant (HCT) recipients, but few data have been reported on the epidemiology of endemic fungal infections in these populations. METHODS: Fifteen institutions belonging to the Transplant-Associated Infection Surveillance Network prospectively enrolled SOT and HCT recipients with histoplasmosis, blastomycosis, or coccidioidomycosis occurring between March 2001 and March 2006. RESULTS: A total of 70 patients (64 SOT recipients and 6 HCT recipients) had infection with an endemic mycosis, including 52 with histoplasmosis, 9 with blastomycosis, and 9 with coccidioidomycosis. The 12-month cumulative incidence rate among SOT recipients for histoplasmosis was 0.102%. Occurrence of infection was bimodal; 28 (40%) infections occurred in the first 6 months post transplantation, and 24 (34%) occurred between 2 and 11 years post transplantation. Three patients were documented to have acquired infection from the donor organ. Seven SOT recipients with histoplasmosis and 3 with coccidioidomycosis died (16%); no HCT recipient died. CONCLUSIONS: This 5-year multicenter prospective surveillance study found that endemic mycoses occur uncommonly in SOT and HCT recipients, and that the period at risk extends for years after transplantation.

In vivo activities of ceftolozane, a new cephalosporin, with and without tazobactam against Pseudomonas aeruginosa and Enterobacteriaceae, including strains with extended-spectrum ß-lactamases, in the thighs of neutropenic mice.

Ceftolozane is a new cephalosporin with potent activity against Pseudomonas aeruginosa and Enterobacteriaceae. A neutropenic murine thigh infection model was used to determine which pharmacokinetic/pharmacodynamic index and magnitude drives the efficacy of ceftolozane with Gram-negative bacilli, to compare the rates of in vivo killing of P. aeruginosa by ceftolozane and ceftazidime, and to determine the impact of different ratios of ceftolozane plus tazobactam on Enterobacteriaceae containing extended-spectrum ß-lactamases (ESBLs). Neutropenic mice had 10(6.2-7.1) CFU/thigh when treated with ceftolozane for 24 h with (i) various doses (3.12 to 1,600 mg/kg) and dosage intervals (3, 6, 12, and 24 h) against two Enterobacteriaceae strains, (ii) 0.39 to 800 mg/kg every 6 h for four Enterobacteriaceae and four P. aeruginosa strains, and (iii) 400 or 800 mg/kg with 2:1. 4:1, and 8:1 ratios of tazobactam against five Enterobacteriaceae strains with ESBLs. The pharmacokinetics of ceftolozane at 25, 100, and 400 mg/kg were linear with peak/dose values of 1.0 to 1.4 and half-lives of 12 to 14 min. T>MIC was the primary index driving efficacy. For stasis (1 log kill), T>MIC was 26.3% ± 2.1% (31.6% ± 1.6%) for wild-type Enterobacteriaceae, 31.1% ± 4.9% (34.8% ± 4.4%) for Enterobacteriaceae with ESBLs, and 24.0% ± 3.3% (31.5% ± 3.9%) for P. aeruginosa. At 200 mg/kg every 3 h, the rate of in vivo killing of P. aeruginosa was faster with ceftolozane than with ceftazidime (-0.34 to -0.41 log10 CFU/thigh/h versus -0.21 to -0.24 log10 CFU/thigh/h). The 2:1 ratio of ceftolozane with tazobactam was the most potent combination studied. The T>MIC required for ceftolozane is less than with other cephalosporins and may be due to more rapid killing.

Role of matrix ß-1,3 glucan in antifungal resistance of non-albicans Candida biofilms.

Candida biofilm infections pose an increasing threat in the health care setting due to the drug resistance associated with this lifestyle. Several mechanisms underlie the resistance phenomenon. In Candida albicans, one mechanism involves drug impedance by the biofilm matrix linked to ß-1,3 glucan. Here, we show this is important for other Candida spp. We identified ß-1,3 glucan in the matrix, found that the matrix sequesters antifungal drug, and enhanced antifungal susceptibility with matrix ß-1,3 glucan hydrolysis.

Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant.

An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.

In vivo pharmacodynamics of new lipopeptide MX-2401.

MX-2401 is a novel lipopeptide (amphomycin analog) with a broad-spectrum bactericidal activity against Gram-positive organisms. We used murine thigh and lung infection models in neutropenic and normal mice to characterize the in vivo pharmacokinetic/pharmacodynamic (PK/PD) activities of MX-2401. The compound (2.5 to 40 mg/kg of body weight) demonstrated linear PK characterized by an area under the concentration-time curve (AUC) of 228 to 3,265 µg·h/ml and half-lives of 5.7 to 8.8 h. MICs ranged from 0.25 to 2 µg/ml. The in vivo postantibiotic effect was prolonged (8.5 h with Staphylococcus aureus and 10.3 to 12.3 with Streptococcus pneumoniae). MX-2401 exhibited dose-dependent in vivo activity against various strains of S. pneumoniae and S. aureus; penicillin and macrolide resistance in the pneumococci and methicillin resistance in the staphylococci had no impact on the antimicrobial activity of the drug. To determine which PK/PD index correlated best with MX-2401 activity, dose fractionation studies over a 72-hour period were performed. The maximum concentration of drug in serum divided by the MIC (C(max)/MIC) correlated best with the efficacy for both S. aureus and S. pneumoniae. Static doses required free-drug C(max)/MIC values of 0.683 to 1.06. Free-drug 72-h AUC/MIC values for the static dose were in the range of 7.49 to 32.3 and were less than expected. The drug showed modest enhancement in activity in the presence of white blood cells (1.7- to 3.4-fold). The potency of the drug in the lung was only marginally lower than in the thigh (1.3- to 1.9-fold). Based on its PK/PD profile, MX-2401 appears to be a promising new lipopeptide agent for treatment of infections by Gram-positive bacteria, including those induced by antibiotic-resistant pathogens.

Statins are associated with improved outcomes of bloodstream infection in solid-organ transplant recipients.

Among recipients of intra-abdominal solid-organ transplants, bloodstream infections (BSIs) are a major cause of mortality. We undertook a retrospective cohort study of recipients of kidney, pancreas, and/or liver transplants with BSIs at a single center over an 11-year period. Multivariate analysis using logistic regression was used to determine independent predictors of 15-day mortality and clinical cure, with a focus on the use of statins. Three hundred and eleven recipients of solid-organ transplants had 604 episodes of BSI. Forty-four (14%) died within 15 days of BSI. Sixteen percent did not achieve clinical cure. In the multivariate model, each one point increase in the APACHE score was associated with a 1.09-fold increased risk of death (95% confidence interval [CI] 1.00-1.18, P = 0.03). The lack of appropriate antibiotic therapy was associated with a four-fold higher risk of death within 15 days (odds ratio [OR] 4.65, 95% CI 1.46-14.78, P = 0.009). Statin use was protective (OR 0.18, 95% CI 0.04-0.78). Patients with high APACHE scores, nosocomial rather than community source of BSI, lack of appropriate antibiotic therapy, and mental status changes were less likely to achieve clinical cure of their BSIs. In conclusion, appropriate antibiotic therapy and statin use are associated with lower risk of mortality from BSIs in this patient population.

In vivo pharmacodynamics of ceftobiprole against multiple bacterial pathogens in murine thigh and lung infection models.

Ceftobiprole medocaril is the parenteral prodrug of ceftobiprole, a novel pyrrolidinone broad-spectrum cephalosporin with in vitro and in vivo bactericidal activities against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae (PRSP). We have used murine thigh and lung infection models in neutropenic and normal mice to characterize the in vivo pharmacokinetic (PK)-pharmacodynamic (PD) activities of ceftobiprole against multiple strains of S. aureus (including MRSA), S. pneumoniae (including PRSP), and gram-negative bacilli. Serum levels of ceftobiprole following the administration of multiple doses were determined by a microbiological assay. In vivo bactericidal activities and postantibiotic effects (PAEs) of ceftobiprole against MRSA and PRSP strains were determined from serial CFU/thigh values following single doses of ceftobiprole (40 and 160 mg/kg of body weight). Dose fractionation studies were used to determine which PK-PD index correlated best with activity. Magnitudes of the PK-PD indices were calculated from MICs and PK parameters. A sigmoid dose-response model was used to estimate the dose (mg/kg/24 h) required to achieve a static and 2-log(10) kill effects over 24 h. PK results showed area under the concentration-time curve/dose values of 1.8 to 2.8 and half-lives of 0.29 to 0.51 h. MICs ranged from 0.015 to 2 microg/ml. Ceftobiprole demonstrated time-dependent killing; its in vivo PAEs varied from 3.8 h to 4.8 h for MRSA and from 0 to 0.8 h for PRSP. The time above MIC (T > MIC) correlated best with efficacy for both MRSA and PRSP. The T > MIC values required for the static doses were significantly longer (P < 0.001) for Enterobacteriaceae (36 to 45%) than for S. aureus (14 to 28%) and S. pneumoniae (15 to 22%). The drug showed activities in the lung model similar to those in the thigh model. The presence of neutrophils significantly enhanced the activity of ceftobiprole against S. pneumoniae but only slightly against Klebsiella pneumoniae. Based on its PD profile, ceftobiprole is a promising new beta-lactam agent with activity against gram-negative and gram-positive organisms including MRSA and PRSP.

In vivo pharmacodynamic target investigation for micafungin against Candida albicans and C. glabrata in a neutropenic murine candidiasis model.

Previous studies using in vivo candidiasis models have demonstrated that the concentration-associated pharmacodynamic indices, the maximum concentration of a drug in serum/MIC and 24-h area under the curve (AUC)/MIC, are associated with echinocandin treatment efficacy. The current investigations used a neutropenic murine model of disseminated Candida albicans and C. glabrata infection to identify the 24-h AUC/MIC index target associated with a stasis and killing endpoint for the echinocandin, micafungin. The kinetics after intraperitoneal micafungin dosing were determined in neutropenic infected mice. Peak levels and AUC values were linear over the 16-fold dose range studied. The serum drug elimination half-life ranged from 7.5 to 16 h. Treatment studies were conducted with 4 C. albicans and 10 C. glabrata isolates with micafungin MICs varying from 0.008 to 0.25 microg/ml to determine whether similar 24-h AUC/MIC ratios were associated with efficacy. The free drug AUC/MICs associated with stasis and killing (1-log) endpoints were near 10 and 20, respectively. The micafungin exposures associated with efficacy were similar for the two Candida species. Furthermore, the free drug micafungin exposures required to produce stasis and killing endpoints were similar to those recently reported for another echinocandin, anidulafungin, against the identical Candida isolates in this model.

Blastomycosis in solid organ transplant recipients.

BACKGROUND: Blastomyces dermatitidis, the etiologic agent of blastomycosis, causes severe disease and substantial mortality in those immunocompromised by acquired immunodeficiency syndrome or malignancy. In solid organ transplant recipients, the epidemiology, clinical features, and outcomes have not been fully described. METHODS: We conducted a retrospective case-series at the University of Wisconsin Hospital and Clinics. Case patients were solid organ transplant recipients with blastomycosis. RESULTS: From 1986 to 2004, we identified 11 cases of post-transplant blastomycosis with 64% occurring between 2000 and 2004. Onset of infection occurred a median of 26 months post transplantation with near equal distribution before and after the first year of transplantation. Rejection did not precede any case of post-transplant blastomycosis. Opportunistic co-infections were common, occurring in 36% of patients. Pneumonia was the most common clinical presentation and was frequently complicated by acute respiratory distress syndrome (ARDS). Extrapulmonary disease predominantly involved the skin and spared the central nervous system. The overall mortality rate was 36%; however, this increased to 67% in those with ARDS. None of the surviving patients relapsed or received routine secondary antifungal prophylaxis. CONCLUSION: Blastomycosis is an uncommon infection following solid organ transplantation that is frequently complicated by ARDS, dissemination, and opportunistic co-infection. After cure, post-transplant blastomycosis may not require lifelong antifungal suppression.

Pharmacokinetics and pharmacodynamics of antibiotics in meningitis.

The penetration of antimicrobials into the CSF is dependent on lipid solubility, molecular size, capillary and choroid plexus efflux pumps, protein binding, and the degree of inflammation. Penicillins, certain cephalosporins, carbapenems, fluoroquinolones, vancomycin, and rifampin provide the highest ratios of CSF levels to the MBC for common infecting organisms. For beta-lactam antibiotics, it is the duration of time that CSF concentrations exceed the MBC that determines the rate of bactericidal activity. It appears that levels should exceed the MBC for more than 50% of the dosing interval. The peak/MBC and AUC/MBC ratios are important determinants of efficacy for aminoglycosides and fluoroquinolones. Once-daily dosing of aminoglycosides is as effective as multiple-daily dosing regimens in experimental meningitis, probably because of drug-induced prolonged persistent effects. Fluoroquinolones do not produce as prolonged persistent effects and are slightly less effective when administered once daily. Although steroid use can reduce the penetration and decrease the bactericidal activity of some antimicrobials, such as vancomycin, in experimental meningitis, the clinical impact of steroid use in human meningitis is still unclear.

Pharmacodynamics of fluoroquinolones in experimental models of endocarditis.

We calculated the magnitude of various serum pharmacodynamic parameters for fluoroquinolones in models of experimental endocarditis (EE) described in the literature. Nineteen publications contained data that allowed calculation of these parameters. Data were available for eight fluoroquinolones against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, methicillin-resistant Staphylococcus epidermidis, viridans streptococci, Enterobacter aerogenes, and Pseudomonas aeruginosa in rabbit or rat models. Enterococci were excluded because of poor bactericidal activity. A 24-hour area under the concentration curve (AUC)/minimal inhibitory concentration (MIC) ratio > or = 100, a peak level/MIC ratio > 8, and continuous levels above the time were associated with a significantly lower number of cfu per vegetation after 3-6 days of therapy. The 24-hour AUC/MIC exhibited the best linear correlation with cfu per vegetation after 3-6 days of therapy (r2 = 45%). The pharmacodynamic parameters predictive of efficacy for fluoroquinolones in the treatment of experimental endocarditis are similar to those for other infectious models.

Parenteral versus oral antibiotic therapy.

The past decade has seen the increased use of oral therapy for a variety of serious infections that were previously treated exclusively by parenteral therapy. A variety of clinical trials in patients with pneumonia, urinary tract infections, skin and soft tissue infections, osteomyelitis, and bacteremia have demonstrated equal efficacy between oral and parenteral therapy. Much of this success is due to the availability of new oral agents, such as the fluoroquinolones and cephalosporins, with enhanced activity against gram-negative bacilli and improved pharmacokinetics. Oral therapy with certain of these drugs provides the same therapeutic serum levels required for efficacy that are obtained with parenteral therapy, that is, a 24-hour AUC/MIC above 125 for fluoroquinolones and levels constantly above the MIC for cephalosporins and other beta-lactams. Oral therapy can also reduce the costs of antimicrobial therapy.