Synergistic Activity of Colistin and Rifampin Combination against Multidrug-Resistant Acinetobacter baumannii in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Combination therapy may be required for multidrug-resistant (MDR) Acinetobacter baumannii. This study systematically investigated bacterial killing and emergence of colistin resistance with colistin and rifampin combinations against MDR A. baumannii. Studies were conducted over 72 h in an in vitro pharmacokinetic (PK)/pharmacodynamic (PD) model at inocula of ∼10⁶ and ∼10⁸ CFU/ml using two MDR clinical isolates of A. baumannii, FADDI-AB030 (colistin susceptible) and FADDI-AB156 (colistin resistant). Three combination regimens achieving clinically relevant concentrations (constant colistin concentration of 0.5, 2, or 5 mg/liter and a rifampin maximum concentration [C_max] of 5 mg/liter every 24 hours; half-life, 3 h) were investigated. Microbiological response was measured by serial bacterial counts. Population analysis profiles assessed emergence of colistin resistance. Against both isolates, combinations resulted in substantially greater killing at the low inoculum; combinations containing 2 and 5 mg/liter colistin increased killing at the high inoculum. Combinations were additive or synergistic at 6, 24, 48, and 72 h with all colistin concentrations against FADDI-AB030 and FADDI-AB156 in, respectively, 8 and 11 of 12 cases (i.e., all 3 combinations) at the 10⁶-CFU/ml inoculum and 8 and 7 of 8 cases with the 2- and 5-mg/liter colistin regimens at the 10⁸-CFU/ml inoculum. For FADDI-AB156, killing by the combination was ∼2.5 to 7.5 and ∼2.5 to 5 log₁₀ CFU/ml greater at the low inoculum (all colistin concentrations) and high inoculum (2 and 5 mg/liter colistin), respectively. Emergence of colistin-resistant subpopulations was completely suppressed in the colistin-susceptible isolate with all combinations at both inocula. Our study provides important information for optimizing colistin-rifampin combinations against colistin-susceptible and -resistant MDR A. baumannii.     

Pharmacokinetic/Pharmacodynamic Investigation of Colistin against Pseudomonas aeruginosa Using an In Vitro Model

Colistin plays a key role in treatment of serious infections by Pseudomonas aeruginosa. The aims of this study were to (i) identify the pharmacokinetic/pharmacodynamic (PK/PD) index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC_max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT_>MIC]) that best predicts colistin efficacy and (ii) determine the values for the predictive PK/PD index required to achieve various magnitudes of killing effect. Studies were conducted in a one-compartment in vitro PK/PD model for 24 h using P. aeruginosa ATCC 27853, PAO1, and the multidrug-resistant mucoid clinical isolate 19056 muc. Six intermittent dosing intervals, with a range of ƒC_max colistin concentrations, and two continuous infusion regimens were examined. PK/PD indices varied from 0.06 to 18 for targeted ƒC_max/MIC, 0.36 to 312 for ƒAUC/MIC, and 0 to 100% for ƒT_>MIC. A Hill-type model was fit to killing effect data, which were expressed as the log₁₀ ratio of the area under the CFU/ml curve for treated regimens versus control. With ƒC_max values equal to or above the MIC, rapid killing was observed following the first dose; substantial regrowth occurred by 24 h with most regimens. The overall killing effect was best correlated with ƒAUC/MIC (R² = 0.931) compared to ƒC_max/MIC (R² = 0.868) and ƒT_>MIC (R² = 0.785). The magnitudes of ƒAUC/MIC required for 1- and 2-log₁₀ reductions in the area under the CFU/ml curve relative to growth control were 22.6 and 30.4, 27.1 and 35.7, and 5.04 and 6.81 for ATCC 27853, PAO1, and 19056 muc, respectively. The PK/PD targets identified will assist in designing optimal dosing strategies for colistin.Globally there is a growing threat from the emergence of multidrug-resistant (MDR) microorganisms (38), especially among a number of important Gram-negative bacterial pathogens (16, 29, 38). Colistin (polymyxin E) still retains significant activity against many of these MDR Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, which often leaves it as the only therapeutic option available (19, 26). With very few new chemical entities against Gram-negative infections in the drug development pipeline (29, 30, 38), particularly against P. aeruginosa (38), the use of colistin, a once-neglected antibiotic, has increased dramatically over the last 5 years (11, 26).Colistin is available commercially as colistin sulfate (hereafter referred to as colistin) and sodium colistin methanesulfonate (CMS), which is administered parenterally. CMS is an inactive prodrug of colistin (3) and, after parenteral administration, colistin is formed in vivo (21, 27, 33). Despite its newfound importance in therapy, there is a dearth of information on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of colistin, a situation of significant concern given that resistance to colistin is beginning to emerge (1, 15, 18, 26, 28). Thus, the aims of the present study were to utilize an in vitro PK/PD model to (i) identify the PK/PD index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC_max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT_>MIC]) that best predicts colistin efficacy and (ii) determine the magnitude of the predictive PK/PD index required to achieve various magnitudes of killing effect.(Parts of the present study were presented at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy [ICAAC], Washington, DC, 25 to 28 October 2008, and at the Second American Conference on Pharmacometrics, Mashantucket, CT, 4 to 7 October 2009.)     

Single-Dose Pharmacodynamics of Amphotericin B against Aspergillus Species in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MICs by using a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of Aspergillus fumigatus, A. terreus, and A. flavus with the same Clinical and Laboratory Standards Institute modal MICs of 1 mg/liter were exposed to amphotericin B concentrations following the plasma concentration-time profile after single-bolus administration with C_max values of 0.6, 1.2, 2.4, and 4.8 mg/liter. Fungal growth was monitored for up to 72 h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B with a C_max of ≥2.4 mg/liter. At the lower C_max values 0.6 and 1.2 mg/liter, significant growth delays of 34 and 52 h were observed, respectively (P < 0.001). For A. flavus, there was no complete inhibition but a progressive growth delay of 1 to 50 h at an amphotericin B C_max of 0.6 to 4.8 mg/liter (P < 0.001). For A. terreus, the growth delay was modest (up to 8 h) at all C_maxs (P < 0.05). The C_max (95% confidence interval) associated with 50% activity for A. fumigatus was 0.60 (0.49 to 0.72) mg/liter, which was significantly lower than for A. flavus 3.06 (2.46 to 3.80) mg/liter and for A. terreus 7.90 (5.20 to 12.29) mg/liter (P < 0.001). A differential in vitro activity of amphotericin B was found among Aspergillus species despite the same MIC in the order A. fumigatus > A. flavus > A. terreus in the in vitro PK/PD model, possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerted against these species.     

In Vitro Pharmacokinetic/Pharmacodynamic Modeling of Voriconazole Activity against Aspergillus Species in a New In Vitro Dynamic Model

The pharmacodynamics (PD) of voriconazole activity against Aspergillus spp. were studied using a new in vitro dynamic model simulating voriconazole human pharmacokinetics (PK), and the PK-PD data were bridged with human drug exposure to assess the percent target (near-maximum activity) attainment of different voriconazole dosages. Three Aspergillus clinical isolates (1 A. fumigatus, 1 A. flavus, and 1 A. terreus isolate) with CLSI MICs of 0.5 mg/liter were tested in an in vitro model simulating voriconazole PK in human plasma with C(max) values of 7, 3.5, and 1.75 mg/liter and a t(1/2) of 6 h. The area under the galactomannan index-time curve (AUC(GI)) was used as the PD parameter. In vitro PK-PD data were bridged with population human PK of voriconazole exposure, and the percent target attainment was calculated. The in vitro PK-PD relationship of fAUC(0-24)-AUC(GI) followed a sigmoid pattern (global R(2) = 0.97), with near-maximum activities (10% fungal growth) observed at an fAUC(0-24) (95% confidence interval [CI]) of 18.9 (14.4 to 23.1) mg · h/liter against A. fumigatus, 26.6 (21.1 to 32.9) mg · h/liter against A. flavus, and 36.2 (27.8 to 45.7) mg · h/liter against A. terreus (F test; P < 0.0001). Target attainment for 3, 4, and 5 mg/kg-of-body-weight voriconazole dosages was 24% (11 to 45%), 80% (32 to 97%), and 93% (86 to 97%) for A. fumigatus, 12% (5 to 26%), 63% (17 to 93%), and 86% (73 to 94%) for A. flavus, and 4% (2 to 11%), 36% (6 to 83%), and 68% (47 to 83%) for A. terreus. Based on the in vitro exposure-effect relationships, a standard dosage of voriconazole may be adequate for most patients with A. fumigatus but not A. flavus and A. terreus infections, for which a higher drug exposure may be required. This could be achieved using a higher voriconazole dosage, thus highlighting the usefulness of therapeutic drug monitoring in patients receiving a standard dosage.     

In Vitro Pharmacokinetic and Pharmacodynamic Evaluation of S-013420 against Haemophilus influenzae and Streptococcus pneumoniae

The pharmacokinetic (PK)/pharmacodynamic (PD) parameters and the antibacterial activity of S-013420, a novel bicyclolide, against Haemophilus influenzae and Streptococcus pneumoniae, including macrolide-resistant isolates, were investigated using an in vitro PD model. Various time-concentration curves were artificially constructed by modifying the PK data obtained in phase I studies. The activity against H. influenzae was evaluated using two parameters, that is, the area above the killing curve (AAC) and the viable cell reduction at 24 h. The relationships between the antibacterial activity of S-013420 and the three PK/PD parameters were investigated by fitting the data to the sigmoid maximum effective concentration model. The square of the correlation coefficient (R²) values for AAC versus the area under the concentration-time curve from 0 to 24 h (AUC_0-24)/MIC, the peak concentration (C_max)/MIC, and the cumulative percentage of a 24-h period that the drug concentration exceeded the MIC under steady-state PK conditions (%T_MIC) were 0.92, 0.87, and 0.49, respectively. The R² values for viable cell reduction at 24 h versus AUC_0-24/MIC, C_max/MIC, and %T_MIC were 0.93, 0.61, and 0.56, respectively. These results demonstrated that AUC_0-24/MIC is the most significant parameter for evaluation of the antibacterial activity of S-013420. The values of AUC_0-24/MIC required for maximum and static efficacy were 10.8 and 9.63, respectively, for H. influenzae and 16.3 to 22.3 and 4.66 to 9.01, respectively, for S. pneumoniae. This analysis is considered useful for determining the AUC value at the infection site, which would be required for efficacy in clinical use.In antimicrobial chemotherapy, the pharmacokinetic (PK)/pharmacodynamic (PD) concept has recently been used during drug development to determine susceptibility breakpoints, predict clinical efficacy, and optimize the antimicrobial regimen (1, 4, 13). Generally, antimicrobials can be categorized according to the dominant parameter of three PK/PD parameters correlated with antibacterial activity. These three parameters (1) are the area under the concentration-time curve from 0 to 24 h (AUC_0-24)/MIC, the peak concentration (C_max)/MIC, and the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state PK conditions (%T_MIC). Nonclinical PK/PD characterizations of antimicrobials using in vitro PD models or in vivo infection models have been reported (2, 5, 26, 29). In general, AUC_0-24/MIC is the dominant PK/PD parameter for macrolides, ketolides, and quinolones, while C_max/MIC is the main parameter for quinolones and aminoglycosides and %T_MIC is that for β-lactams (1, 4). Overall, nonclinical PK/PD data have agreed well with data obtained for infected patients, and a PK/PD evaluation would be helpful for cutting costs, addressing ethical issues, and shortening the duration of clinical trials (1, 4).Community-acquired respiratory tract infections (RTIs), such as community-acquired pneumonia, acute exacerbations of chronic bronchitis, sinusitis, and acute otitis media, are among the most common infectious diseases for which oral antibiotics are prescribed in primary clinical settings and are mainly caused by Streptococcus pneumoniae and Haemophilus influenzae (6). The macrolide antimicrobials, including clarithromycin and azithromycin, are often used for the treatment of community-acquired RTIs. However, the decreased susceptibility of S. pneumoniae to macrolide agents is a key problem worldwide. Recent reports have suggested that pneumococcal macrolide resistance rates were about 35% in some regions (7, 14, 23). In S. pneumoniae, two main mechanisms of macrolide resistance were reported, i.e., active efflux due to mef genes and ribosomal modification mediated by erm genes (11). S-013420 (EDP-420, EP-013420) is a novel bicyclolide (bridged bicyclic macrolide) that is characterized by a 6,11-O-bridged structure discovered by Enanta Pharmaceuticals, Inc. (Fig. ​(Fig.1),1), and that shows a broad spectrum of activity against a variety of the RTI pathogens, including erythromycin-resistant streptococci. (12a, 28).Open in a separate windowFIG. 1.Chemical structure of S-013420.The purpose of the present study was to investigate the pharmacodynamic relationship between the activity of S-013420 against H. influenzae and S. pneumoniae, including macrolide-resistant isolates, and the well-established PK/PD parameters AUC_0-24/MIC, C_max/MIC, and %T_MIC by using an in vitro PD model.     

Antibiotic Activity against Naive and Induced Streptococcus pneumoniae Biofilms in an In Vitro Pharmacodynamic Model

Biofilms play a role in the pathogenicity of pneumococcal infections. A pharmacodynamic in vitro model of biofilm was developed that allows characterization of the activity of antibiotics against viability and biomass by using in parallel capsulated (ATCC 49619) and noncapsulated (R6) reference strains. Naive biofilms were obtained by incubating fresh planktonic cultures for 2 to 11 days in 96-well polystyrene plates. Induced biofilms were obtained using planktonic bacteria collected from the supernatant of 6-day-old naive biofilms. Biomass production was more rapid and intense in the induced model, but the levels were similar for both strains. Full concentration responses fitting sigmoidal regressions allowed calculation of maximal efficacies and relative potencies of drugs. All antibiotics tested (amoxicillin, clarithromycin, solithromycin, levofloxacin, and moxifloxacin) were more effective against young naive biofilms than against old or induced biofilms, except macrolides/ketolides, which were as effective at reducing viability in 2-day-old naive biofilms and in 11-day-old induced biofilms of R6. Macrolides/ketolides, however, were less potent than fluoroquinolones against R6 (approximately 5- to 20-fold-higher concentrations needed to reduction viability of 20%). However, at concentrations obtainable in epithelial lining fluid, the viabilities of mature or induced biofilms were reduced 15 to 45% (amoxicillin), 17 to 44% (macrolides/ketolides), and 12 to 64% (fluoroquinolones), and biomasses were reduced 5 to 45% (amoxicillin), 5 to 60% (macrolides/ketolides), and 10 to 76% (fluoroquinolones), with solithromycin and moxifloxacin being the most effective and the most potent agents (due to lower MICs) in their respective classes. This study allowed the ranking of antibiotics with respect to their potential effectiveness in biofilm-related infections, underlining the need to search for still more effective options.     

In Vitro Pharmacodynamics of Various Antibiotics in Combination against Extensively Drug-Resistant Klebsiella pneumoniae

Extensively drug-resistant (XDR) Klebsiella pneumoniae is an emerging pathogen in Singapore. With limited therapeutic options available, combination antibiotics may be the only viable option. In this study, we aimed to elucidate effective antibiotic combinations against XDR K. pneumoniae isolates. Six NDM-1-producing and two OXA-181-producing K. pneumoniae strains were exposed to 12 antibiotics alone and in combination via time-kill studies. A hollow-fiber infection model (HFIM) with pharmacokinetic validation was used to simulate clinically relevant tigecycline-plus-meropenem dosing regimens against 2 XDR K. pneumoniae isolates over 240 h. The emergence of resistance against tigecycline was quantified using drug-free and selective (tigecycline at 3× the MIC) media. The in vitro growth rates were determined and serial passages on drug-free and selective media were carried out on resistant isolates obtained at 240 h. Both the polymyxin B and tigecycline MICs ranged from 1 to 4 mg/liter. In single time-kill studies, all antibiotics alone demonstrated regrowth at 24 h, except for polymyxin B against 2 isolates. Tigecycline plus meropenem was found to be bactericidal in 50% of the isolates. For the isolates that produced OXA-181-like carbapenemases, none of the 55 tested antibiotic combinations was bactericidal. Against 2 isolates in the HFIM, tigecycline plus meropenem achieved a >90% reduction in bacterial burden for 96 h before regrowth was observed until 10⁹ CFU/ml at 240 h. Phenotypically stable and resistant isolates, which were recovered from tigecycline-supplemented plates post-HFIM studies, had lower growth rates than those of their respective parent isolates, possibly implying a substantial biofitness deficit in this population. We found that tigecycline plus meropenem may be a potential antibiotic combination for XDR K. pneumoniae infections, but its efficacy was strain specific.     

In Vitro Activity of Fosfomycin against blaKPC-Containing Klebsiella pneumoniae Isolates,Including Those Nonsusceptible to Tigecycline and/or Colistin

In vitro activity of fosfomycin was evaluated against 68 bla_KPC-possessing Klebsiella pneumoniae (KpKPC) isolates, including 23 tigecycline- and/or colistin-nonsusceptible strains. By agar dilution, 93% of the overall KpKPC were susceptible (MIC_50/90 of 16/64 μg/ml, respectively). The subgroup of 23 tigecycline- and/or colistin-nonsusceptible strains showed susceptibility rates of 87% (MIC_50/90 of 32/128 μg/ml, respectively). Notably, 5 out of 6 extremely drug-resistant (tigecycline and colistin nonsusceptible) KpKPC were susceptible to fosfomycin. Compared to agar dilution, disk diffusion was more accurate than Etest.The worldwide spread of bla_KPC-possessing Klebsiella pneumoniae (KpKPC) isolates represents a serious threat to our health care systems (20). KpKPC isolates are responsible for hospital outbreaks in the United States, Israel, and Greece with mortality rates of approximately 35% (14, 18, 23, 26).KpKPC isolates are nonsusceptible (NS) in vitro to all β-lactams, including β-lactam/β-lactamase inhibitor combinations and carbapenems, quinolones, and frequently, aminoglycosides (10, 22). Thus, our therapeutic options against infections due to KpKPC are often limited to tigecycline and colistin. Unfortunately, an increasing number of colistin- and/or tigecycline-NS KpKPC isolates have been observed recently, primarily in the New York City area (1, 15). KpKPC isolates that are both colistin and tigecycline NS are defined as “extremely drug-resistant” (XDR) strains because all available standard antibiotics are ineffective in vitro (22). The spread of these XDR K. pneumoniae isolates may have devastating effects on patient outcomes (7).As a result of this urgency, new therapeutic strategies against KpKPC isolates need to be rapidly devised and implemented. Thus far, there are a few novel compounds in development that promise to be active (9, 11). However, these new drugs are currently in clinical trials and it will take a few years to see if their promise holds true in the clinic.Fosfomycin is an “old” antimicrobial that inhibits the first step of peptidoglycan synthesis and shows potent bactericidal action against many Gram-negative and Gram-positive pathogens (17). Fosfomycin tromethamine, an oral formulation, is approved in the United States and other countries for the treatment of uncomplicated urinary tract infections (UTIs) caused by Escherichia coli or Enterococcus faecalis (21, 27). In some European countries and Japan, fosfomycin disodium is also available for parenteral use. The drug shows little toxicity, and very high peak levels can be achieved in serum and urine (12, 13). Interestingly, fosfomycin rapidly penetrates tissues (12, 24), a property that is highly desirable in the treatment of serious infections. Unfortunately, resistance develops rapidly when fosfomycin is used as monotherapy (12, 19). Since fosfomycin shows synergistic action with other antimicrobials (2), it is used in combination to treat a wide range of infections, including pneumonia and septicemia. Overall, cure rates of >80% are observed (12, 13). Surprisingly, data regarding in vitro and in vivo activity of fosfomycin against KpKPC isolates are lacking.In the present work, we analyzed the in vitro activity of fosfomycin against a collection of 68 KpKPC clinical isolates. Forty-two isolates were previously characterized strains collected in the Eastern United States (8, 10, 11), whereas the remaining 26 were recently (January to July 2009) isolated in institutions located in New York City (n = 17) and Cleveland, OH (n = 9).MICs for tigecycline and colistin were obtained using the Etest method (AB bioMérieux) on Mueller-Hinton agar (MHA; BBL, Becton Dickinson). The results for tigecycline were interpreted according to the U.S. FDA criteria, whereas those for colistin were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria established for organisms that are not members of the Enterobacteriaceae (i.e., susceptibility of ≤2 μg/ml for both antimicrobials) (5).Susceptibility to fosfomycin was determined using three methods: agar dilution (AD), disk diffusion (DD), and Etest. AD was performed using fosfomycin disodium salt (Sigma-Aldrich Co.) on MHA containing 25 μg/ml of glucose-6-phosphate (G6P; Roche Diagnostics), employing a Steers replicator that delivered 10⁴ CFU/10-μl spot (4, 5). Disk diffusion was carried out on MHA with disks (BBL, Becton Dickinson) containing 200 μg of fosfomycin and 50 μg of G6P (5). Etest was performed on MHA containing G6P, following the manufacturer''s instructions. Since CLSI criteria to evaluate fosfomycin susceptibility in K. pneumoniae are not available, results were interpreted according to guidelines approved for Escherichia coli in UTIs (i.e., susceptible at MICs of ≤64 μg/ml or with zones of ≥16 mm) (5); these breakpoints have been used by authors of similar studies (6, 16, 17, 25). ATCC strains Escherichia coli 25922 and Pseudomonas aeruginosa 27853 were used as controls for all experiments.Among the 68 KpKPC isolates analyzed, 23 were tigecycline and/or colistin NS (i.e., 5 tigecycline NS, 12 colistin NS, and 6 XDR). In Fig. ​Fig.1,1, we present the susceptibility results for fosfomycin obtained with the AD, Etest, and DD. By AD, an overall susceptibility of 92.6% (MIC₅₀ and MIC₉₀ of 16 and 64 μg/ml, respectively) was found. The subgroup of tigecycline- and/or colistin-NS isolates showed susceptibility rates of 87.0% (MIC₅₀ and MIC₉₀ of 32 and 128 μg/ml, respectively). Notably, fosfomycin was active in vitro against five of the six XDR KpKPC isolates (i.e., two with MICs of 32 μg/ml, three with MICs of 64 μg/ml, and one with a MIC of 256 μg/ml). By Etest and DD, overall susceptibility rates of approximately 60% were recorded (Fig. ​(Fig.11).Open in a separate windowFIG. 1.MIC distributions for fosfomycin were obtained using agar diffusion and Etest (MICs were adjusted up to the next highest doubling concentration, e.g., from 48 to 64 μg/ml). Inhibition zone (IZ) diameter distribution was obtained with disk diffusion. Results were interpreted according to CLSI criteria for E. coli (5). The number of KpKPC isolates that had each result is presented above the bars. Dashed line, susceptible cutoff; solid line, resistant cutoff; S, susceptible; I, intermediate; R, resistant.The results of Etest and DD were compared with those obtained from the AD, used as the reference method, to establish their ability to characterize fosfomycin susceptibility. Essential agreement (EA), categorical agreement (CA), minor errors (MiE), major errors (MaE), and very major errors (VME) were calculated (see definitions in Table ​Table1).1). CLSI recommends that <10% MiE, <3% MaE, and <1.5% VME should be obtained to approve the performance of susceptibility tests (3).

TABLE 1.

Agreement of the Etest and disk diffusion methods with agar dilution in testing susceptibility to fosfomycin

In Vitro Synergy of Colistin Combinations against Colistin-Resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae Isolates

Colistin resistance, although uncommon, is increasingly being reported among Gram-negative clinical pathogens, and an understanding of its impact on the activity of antimicrobials is now evolving. We evaluated the potential for synergy of colistin plus trimethoprim, trimethoprim-sulfamethoxazole (1/19 ratio), or vancomycin against 12 isolates of Acinetobacter baumannii (n = 4), Pseudomonas aeruginosa (n = 4), and Klebsiella pneumoniae (n = 4). The strains included six multidrug-resistant clinical isolates, K. pneumoniae ATCC 700603, A. baumannii ATCC 19606, P. aeruginosa ATCC 27853, and their colistin-resistant derivatives (KPm1, ABm1, and PAm1, respectively). Antimicrobial susceptibilities were assessed by broth microdilution and population analysis profiles. The potential for synergy of colistin combinations was evaluated using a checkerboard assay, as well as static time-kill experiments at 0.5× and 0.25× MIC. The MIC ranges of vancomycin, trimethoprim, and trimethoprim-sulfamethoxazole (1/19) were ≥128, 4 to ≥128, and 2/38 to >128/2,432 μg/ml, respectively. Colistin resistance demonstrated little impact on vancomycin, trimethoprim, or trimethoprim-sulfamethoxazole MIC values. Isolates with subpopulations heterogeneously resistant to colistin were observed to various degrees in all tested isolates. In time-kill assays, all tested combinations were synergistic against KPm1 at 0.25× MIC and 0.5× MIC and ABm1 and PAm1 at 0.5× MIC. In contrast, none of the tested combinations demonstrated synergy against any colistin-susceptible P. aeruginosa isolates and clinical strains of K. pneumoniae isolates. Only colistin plus trimethoprim or trimethoprim-sulfamethoxazole was synergistic and bactericidal at 0.5× MIC against K. pneumoniae ATCC 700603. Colistin resistance seems to promote the in vitro activity of unconventional colistin combinations. Additional experiments are warranted to understand the clinical significance of these observations.     

Evaluation of Ceftaroline Alone and in Combination against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Daptomycin and Vancomycin in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Annually, medical device infections are associated with >250,000 catheter-associated bloodstream infections (CLABSI), with up to 25% mortality. Staphylococcus aureus, a primary pathogen in these infections, is capable of biofilm production, allowing organism persistence in harsh environments, offering antimicrobial protection. With increases in S. aureus isolates with reduced susceptibility to current agents, ceftaroline (CPT) offers a therapeutic alternative. Therefore, we evaluated whether CPT would have a role against biofilm-producing methicillin-resistant S. aureus (MRSA), including those with decreased susceptibilities to alternative agents. In this study, we investigated CPT activity alone or combined with daptomycin (DAP) or rifampin (RIF) against 3 clinical biofilm-producing MRSA strains in an in vitro biofilm pharmacokinetic/pharmacodynamic (PK/PD) model. Simulated antimicrobial regimens were as follows: 600 mg of CPT every 8 h (q8h) (free maximum concentration of drug [fC_max], 17.04 mg/liter; elimination half-life [t_1/2], 2.66 h), 12 mg/kg of body weight/day of DAP (fC_max, 14.7 mg/liter; t_1/2, 8 h), and 450 mg of RIF q12h (fC_max, 3.5 mg/liter; t_1/2, 3.4 h), CPT plus DAP, and CPT plus RIF. Samples were obtained and plated to determine colony counts. Differences in log₁₀ CFU/cm² were evaluated by analysis of variance with Tukey''s post hoc test. The strains were CPT and vancomycin susceptible and DAP nonsusceptible (DNS). CPT displayed activity throughout the experiment. DAP demonstrated initial activity with regrowth at 24 h in all strains. RIF was comparable to the drug-free control, and little benefit was observed when combined with CPT. CPT plus DAP displayed potent activity, with an average log₁₀ CFU/cm² reduction of 3.33 ± 1.01 from baseline. CPT demonstrated activity against biofilm-producing DNS MRSA. CPT plus DAP displayed therapeutic enhancement over monotherapy, providing a potential option for difficult-to-treat medical device infections.     

Pharmacodynamics of the Antibacterial Effect of and Emergence of Resistance to Doripenem in Pseudomonas aeruginosa and Acinetobacter baumannii in an In Vitro Pharmacokinetic Model

An in vitro dilutional pharmacokinetic model of infection was used to study the pharmacodynamics of doripenem in terms of the ability to kill Pseudomonas aeruginosa or Acinetobacter baumannii and also changes in their population profiles. In dose-ranging studies, the cumulative percentages of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (T(MIC)s) required for doripenem to produce a 24-h bacteriostatic effect and a -2-log-unit reduction in viable count were 25% ± 11% and 35% ± 13%, respectively, for P. aeruginosa (MIC range, 0.24 to 3 mg/liter) and 20% ± 11% and 33% ± 12%, respectively, for Acinetobacter spp. (MIC range, 0.45 to 3.0 mg/liter). A T(MIC) of >40 to 50% produced a maximum response with both species at 24 h or 48 h of exposure. After 24 h of exposure to doripenem at a T(MIC) in the range of 12.5 to 37.5%, P. aeruginosa and A. baumannii population profiles revealed mutants able to grow on 4× MIC-containing medium; such changes were further amplified by 48 h of exposure. Dose-fractionation experiments targeting T(MIC)s of 12.5%, 25%, or 37.5% as six exposures, two exposures, or a single exposure over 48 h with a single strain of P. aeruginosa indicated that changes in population profiles were greatest with multiple exposures at T(MIC) targets of 12.5 or 25%. In contrast, multiple exposures at 37.5% T(MIC) most effectively suppressed total bacterial counts and changes in population profiles. Simulations of human doses of doripenem of 500 mg, 1,000 mg, 2,000 mg, and 3,000 mg every 8 h over 96 h showed marked initial killing up to 6 h but growback thereafter. Changes in population profiles occurred only in the regimen of 500 mg every 8 h against P. aeruginosa but occurred with all dose regimens for A. baumannii strains. A doripenem T(MIC) of ≥40 to 50% is maximally effective in killing P. aeruginosa or A. baumannii and suppressing changes in population profiles in short-term experiments for up to 48 h; however, a T(MIC) of 12.5 to 25% amplifies population changes, especially with exposures every 8 h. In longer-term experiments, up to 96 h, even doripenem doses of 4 to 6 times those used in human studies proved incapable of pathogen eradication and prevention of changes in population profiles. The association of a T(MIC) of 25 to 37.5% with changes in population profiles has implications in terms of future clinical breakpoint setting.     

Activity of Imipenem against Klebsiella pneumoniae Biofilms In Vitro and In Vivo

Encapsulated Klebsiella pneumoniae has emerged as one of the most clinically relevant and more frequently encountered opportunistic pathogens in combat wounds as the result of nosocomial infection. In this report, we show that imipenem displayed potent activity against established K. pneumoniae biofilms under both static and flow conditions in vitro. Using a rabbit ear model, we also demonstrated that imipenem was highly effective against preformed K. pneumoniae biofilms in wounds.     

Evaluation of the Novel Combination of High-Dose Daptomycin plus Trimethoprim-Sulfamethoxazole against Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus Using an In Vitro Pharmacokinetic/Pharmacodynamic Model of Simulated Endocardial Vegetations

Daptomycin-nonsusceptible (DNS) Staphylococcus aureus is found in difficult-to-treat infections, and the optimal therapy is unknown. We investigated the activity of high-dose (HD) daptomycin plus trimethoprim-sulfamethoxazole de-escalated to HD daptomycin or trimethoprim-sulfamethoxazole against 4 clinical DNS methicillin-resistant S. aureus (MRSA) isolates in an in vitro pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations (10⁹ CFU/g). Simulated regimens included HD daptomycin at 10 mg/kg/day for 14 days, trimethoprim-sulfamethoxazole at 160/800 mg every 12 h for 14 days, HD daptomycin plus trimethoprim-sulfamethoxazole for 14 days, and the combination for 7 days de-escalated to HD daptomycin for 7 days and de-escalated to trimethoprim-sulfamethoxazole for 7 days. Differences in CFU/g (at 168 and 336 h) were evaluated by analysis of variance (ANOVA) with a Tukey''s post hoc test. Daptomycin MICs were 4 μg/ml (SA H9749-1, vancomycin-intermediate Staphylococcus aureus; R6212, heteroresistant vancomycin-intermediate Staphylococcus aureus) and 2 μg/ml (R5599 and R5563). Trimethoprim-sulfamethoxazole MICs were ≤0.06/1.19 μg/ml. HD daptomycin plus trimethoprim-sulfamethoxazole displayed rapid bactericidal activity against SA H9749-1 (at 7 h) and R6212 (at 6 h) and bactericidal activity against R5599 (at 72 h) and R5563 (at 36 h). A ≥8 log₁₀ CFU/g decrease was observed with HD daptomycin plus trimethoprim-sulfamethoxazole against all strains (at 48 to 144 h), which was maintained with de-escalation to HD daptomycin or trimethoprim-sulfamethoxazole at 336 h. The combination for 14 days and the combination for 7 days de-escalated to HD daptomycin or trimethoprim-sulfamethoxazole was significantly better than daptomycin monotherapy (P < 0.05) and trimethoprim-sulfamethoxazole monotherapy (P < 0.05) at 168 and 336 h. Combination therapy followed by de-escalation offers a novel bactericidal therapeutic alternative for high-inoculum, serious DNS MRSA infections.     

Ceftaroline plus Avibactam Demonstrates Bactericidal Activity against Pathogenic Anaerobic Bacteria in a One-Compartment In Vitro Pharmacokinetic/Pharmacodynamic Model

Anaerobic pathogens are often associated with polymicrobial infections, such as diabetic foot infections. Patients with these infections are often treated with broad-spectrum, multidrug therapies targeting resistant Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. The broad-spectrum, non-beta-lactam, beta-lactamase inhibitor avibactam has been combined with ceftaroline and may provide a single-product alternative for complicated polymicrobial infections. We compared the activity of ceftaroline-avibactam (CPA) to that of ertapenem (ERT) against common anaerobic pathogens in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. Simulations of doses of ceftaroline-fosamil at 600 mg every 8 h (q8h) (maximum free drug concentration [fC_max], 17.04 mg/liter, and half-life [t_1/2], 2.66 h) plus avibactam at 600 mg q8h (fC_max, 11.72 mg/liter, and t_1/2, 1.8 h) and of ertapenem at 1 g q24h (fC_max, 13 mg/liter, and t_1/2, 4 h) were evaluated against two strains of Bacteroides fragilis, one strain of Prevotella bivia, and one strain of Finegoldia magna in an anaerobic one-compartment in vitro PK/PD model over 72 h with a starting inoculum of ∼8 log₁₀ CFU/ml. Bactericidal activity was defined as a reduction of ≥3 log₁₀ CFU/ml from the starting inoculum. Both CPA and ERT were bactericidal against all four strains. CPA demonstrated improved activity against Bacteroides strains compared to that of ERT but had similar activity against Finegoldia magna and P. bivia, although modest regrowth was observed with CPA against P. bivia. No resistance emerged from any of the models. The pharmacokinetics achieved were 92 to 105% of the targets. CPA has potent in vitro activity against common anaerobic pathogens at clinically relevant drug exposures and may be a suitable single product for the management of complicated polymicrobial infections.     

Activity of Telavancin against Staphylococcus aureus Strains with Various Vancomycin Susceptibilities in an In Vitro Pharmacokinetic/Pharmacodynamic Model with Simulated Endocardial Vegetations

We investigated the activity of telavancin, a novel lipoglycopeptide, alone and combined with gentamicin or rifampin (rifampicin) against strains of Staphylococcus aureus with various vancomycin susceptibilities. Strains tested included methicillin (meticillin)-resistant S. aureus (MRSA) 494, methicillin-sensitive S. aureus (MSSA) 1199, heteroresistant glycopeptide-intermediate S. aureus (hGISA) 1629, which was confirmed by a population analysis profile, and glycopeptide-intermediate S. aureus (GISA) NJ 992. Regimens of 10 mg/kg telavancin daily and 1 g vancomycin every 12 h were investigated alone and combined with 5 mg/kg gentamicin daily or 300 mg rifampin every 8 h in an in vitro model with simulated endocardial vegetations over 96 h. Telavancin demonstrated significantly greater killing than did vancomycin (P < 0.01) for all isolates except MRSA 494 (P = 0.07). Telavancin absolute reductions, in log₁₀ CFU/g, at 96 h were 2.8 ± 0.5 for MRSA 494, 2.8 ± 0.3 for MSSA 1199, 4.2 ± 0.2 for hGISA 1629, and 4.1 ± 0.3 for GISA NJ 992. Combinations of telavancin with gentamicin significantly enhanced killing compared to telavancin alone against all isolates (P < 0.001) except MRSA 494 (P = 0.176). This enhancement was most evident against hGISA 1629, where killing to the level of detection (2 log₁₀ CFU/g) was achieved at 48 h (P < 0.001). The addition of rifampin to telavancin resulted in significant (P < 0.001) enhancement of killing against only MSSA 1199. No changes in telavancin susceptibilities were observed. These results suggest that telavancin may have therapeutic potential, especially against strains with reduced susceptibility to vancomycin. Combination therapy, particularly with gentamicin, may improve bacterial killing against certain strains.The worldwide dissemination and poor treatment outcomes of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) present therapeutic difficulties for clinicians. Vancomycin has historically been the mainstay of therapy for serious infections caused by MRSA, but decades of selective pressure have led to evolutionary changes in S. aureus that diminish the utility of this agent (5, 17, 18, 34). Of note is the emergence of S. aureus with reduced susceptibility to glycopeptides, including glycopeptide-intermediate S. aureus (GISA) and heteroresistant GISA (hGISA) strains (16, 33). hGISA is of particular concern, because this organism is not detected by traditional MIC testing or automated methods used in clinical microbiology laboratories (17, 25). Due to these difficulties in detection, the true prevalence of hGISA is difficult to estimate but ranges from ∼2% to ∼11% for isolates from a variety of clinical sources (5, 13, 25, 28, 33). Our own study of hGISA at the Detroit Medical Center demonstrated 8.3% hGISA isolates from the time period 2003 to 2007 and also demonstrated that the number of MRSA strains displaying this phenotype may be increasing (33). This is problematic, as preliminary studies have found an association between infection with hGISA and poor treatment outcomes, including prolonged fever and bacteremia, increased length of hospital stay, and vancomycin treatment failure (5, 17, 18).Telavancin is a novel lipoglycopeptide antimicrobial with antibacterial activity against a broad range of gram-positive pathogens (7, 8, 14, 19, 20, 22, 23). It is structurally derived from vancomycin by the addition of a hydrophobic decylaminoethyl side chain on the vancosamine sugar and a hydrophilic phosphonomethylaminomethyl group on the 4′ position of amino acid 7 (21). This modification has resulted in a molecule with two mechanisms of action. Similar to vancomycin, telavancin inhibits late-stage peptidoglycan synthesis and cross-linking, but additionally, it targets the bacterial membrane causing disruption of membrane potential and increased permeability (15). The multifunctional mechanism of action of telavancin accounts for its activity against strains of S. aureus with reduced susceptibility to vancomycin, such as GISA and hGISA strains (2, 12, 23, 27).Telavancin has been studied in a variety of in vitro and in vivo models. In two separate rabbit models of endocarditis, telavancin was found to be effective against both MRSA and GISA strains, though statistically significant differences between telavancin and vancomycin were only observed against GISA strains (27, 29). Clinically, telavancin has been found to be noninferior to vancomycin for the treatment of complicated skin and soft tissue infections caused by gram-positive pathogens in a large prospective, double-blind, randomized evaluation of patients, including a large number infected with MRSA (MRSA isolated at baseline from 579 clinically evaluable patients) (35). The objective of this investigation was to evaluate the activity of telavancin versus that of vancomycin, both alone and combined with gentamicin or rifampin (rifampicin) against S. aureus strains harboring a spectrum of vancomycin susceptibilities in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations (SEVs). (A portion of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy [ICAAC]/46th Annual Meeting of the Infectious Diseases Society of America [IDSA], Washington, DC, 2008.)     

Pharmacodynamics of Trovafloxacin and Levofloxacin against Bacteroides fragilis in an In Vitro Pharmacodynamic Model

An in vitro pharmacodynamic investigation was conducted to explore whether the area under the concentration time curve from 0 to 24 h (AUC(0-24))/MIC ratio could predict fluoroquinolone performance against Bacteroides fragilis. An in vitro model was used to generate kill curves for trovafloxacin (TVA) and levofloxacin (LVX) at AUC(0-24)/MIC ratios of 1 to 406 against three strains of B. fragilis (ATCC 25285, ATCC 23745, and clinical isolate M97-117). TVA and LVX were bolused prior to the start of experiments to achieve the corresponding AUC(0-24)/MIC ratio. Experiments were performed in duplicate over 24 h and in an anaerobic environment. Analyses of antimicrobial performance were conducted by comparing the rates of bacterial kill (K) using nonlinear regression analysis with 95% confidence intervals. Statistical significance was defined as a lack of overlap in the 95% confidence limits generated from the slope of each kill curve. For both TVA and LVX, K was maximized once an AUC(0-24)/MIC ratio of > or =40 was achieved and was not further increased despite a 10-fold increase in AUC(0-24)/MIC from approximately 40 to 400 against all three strains of B. fragilis. No significant differences were found in K between AUC(0-24)/MIC ratios of approximately 40 to 200. In experiments where AUC(0-24)/MIC ratios that were > or = 5 and < or = 44 were conducted, 64% demonstrated regrowth at 24 h. Resistant strains were selected in 50% of those experiments, demonstrating regrowth, which resulted in increased MICs of two- to 16-fold for both TVA and LVX. Regrowth did not occur, nor were resistant strains selected in any studies with an AUC/MIC that was > 44. Our findings suggest that fluoroquinolones provide antibacterial effects against B. fragilis in a concentration-independent manner associated with an AUC(0-24)/MIC ratio of > or =40. Also, the potential for the selection of resistant strains of B. fragilis may increase with an AUC(0-24)/MIC ratio of < or =44.