Exploration of the Pharmacokinetic-Pharmacodynamic Relationships for Fosfomycin Efficacy Using an In Vitro Infection Model

Fosfomycin, a phosphonic class antibiotic with a broad spectrum of antibacterial activity, has been used outside the United States since the early 1970s for the treatment of a variety of infections. In the United States, an oral (tromethamine salt) formulation is used for uncomplicated urinary tract infections. Recently, there has been interest in the use of an intravenous solution (ZTI-01) for the treatment of a broad range of infections associated with multidrug-resistant bacteria. In this era of multidrug-resistant bacteria with few treatment options, it is critical to understand the pharmacokinetic-pharmacodynamic (PK-PD) determinants for fosfomycin efficacy. Since such data are limited, a one-compartment in vitro infection model was used to determine the PK-PD index associated with efficacy and the magnitude of this measure necessary for various levels of effect. One challenge isolate (Escherichia coli ATCC 25922, for which the fosfomycin agar MIC is 0.5 mg/liter and the broth microdilution MIC is 1 mg/liter) was evaluated in the dose fractionation studies, and two additional clinical E. coli isolates were evaluated in the dose-ranging studies. Mutation frequency studies indicated the presence of an inherently fosfomycin resistant E. coli subpopulation (agar MIC = 32 to 64 mg/liter) within the standard starting inoculum of a susceptibility test. Due to the presence of this resistant subpopulation, we identified the percentage of the dosing interval that drug concentrations were above the inherent resistance inhibitory concentration found at baseline to be the PK-PD index associated with efficacy (r² = 0.777). The magnitudes of this PK-PD index associated with net bacterial stasis and 1- and 2-log₁₀ CFU/ml reductions from baseline at 24 h were 11.9, 20.9, and 32.8, respectively. These data provide useful information for modernizing and optimizing ZTI-01 dosing regimens for further study.     

Activities of Fosfomycin,Tigecycline, Colistin,and Gentamicin against Extended-Spectrum-β-Lactamase-Producing Escherichia coli in a Foreign-Body Infection Model

Limited antimicrobial agents are available for the treatment of implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli. We compared the activities of fosfomycin, tigecycline, colistin, and gentamicin (alone and in combination) against a CTX-M15-producing strain of Escherichia coli (Bj HDE-1) in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration in logarithmic phase (MBC_log) and stationary phase (MBC_stat) were 0.12, 0.12, and 8 μg/ml for fosfomycin, 0.25, 32, and 32 μg/ml for tigecycline, 0.25, 0.5, and 2 μg/ml for colistin, and 2, 8, and 16 μg/ml for gentamicin, respectively. In time-kill studies, colistin showed concentration-dependent activity, but regrowth occurred after 24 h. Fosfomycin demonstrated rapid bactericidal activity at the MIC, and no regrowth occurred. Synergistic activity between fosfomycin and colistin in vitro was observed, with no detectable bacterial counts after 6 h. In animal studies, fosfomycin reduced planktonic counts by 4 log₁₀ CFU/ml, whereas in combination with colistin, tigecycline, or gentamicin, it reduced counts by >6 log₁₀ CFU/ml. Fosfomycin was the only single agent which was able to eradicate E. coli biofilms (cure rate, 17% of implanted, infected cages). In combination, colistin plus tigecycline (50%) and fosfomycin plus gentamicin (42%) cured significantly more infected cages than colistin plus gentamicin (33%) or fosfomycin plus tigecycline (25%) (P < 0.05). The combination of fosfomycin plus colistin showed the highest cure rate (67%), which was significantly better than that of fosfomycin alone (P < 0.05). In conclusion, the combination of fosfomycin plus colistin is a promising treatment option for implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli.     

Evaluation of fosfomycin alone and in combination with ceftriaxone or vancomycin in an experimental model of meningitis caused by two strains of cephalosporin-resistant Streptococcus pneumoniae

OBJECTIVES: To study the in vitro and in vivo efficacy of fosfomycin, alone and in combination with ceftriaxone or vancomycin, against two strains of Streptococcus pneumoniae: HUB 2349 (fosfomycin and ceftriaxone, MICs 16 and 2 mg/L) and ATCC 51916 (MICs 4 and 32 mg/L). METHODS: Pharmacokinetics/pharmacodynamics data were collected from the study of eight infected animals after a single intravenous dose of 300 mg/kg of fosfomycin. Time-kill curves were plotted using CSF antibiotic concentrations achievable clinically. In the rabbit model, we studied the efficacy and effects on inflammation of treatment with fosfomycin 1200 mg/kg/day, ceftriaxone 100 mg/kg/day and vancomycin 30 mg/kg/day, over 26 h. RESULTS: Fosfomycin peak level in serum was 324.48 +/- 102.1 mg/L at 0.5 h; CSF penetration was 49.2%. Time-kill curves showed that fosfomycin was bactericidal against the ATCC 51916 strain and that the addition of fosfomycin to ceftriaxone or vancomycin was synergic against the HUB 2349 strain. Resistance to fosfomycin was detected both when fosfomycin was studied alone and in combination. In the rabbit model, fosfomycin showed bactericidal activity only against the ATCC 51916 strain. Combinations of fosfomycin with ceftriaxone or vancomycin were bactericidal against both strains; they improved efficacy and decreased CSF inflammatory parameters over monotherapies, without showing statistical differences in comparison with the combination of ceftriaxone and vancomycin. CONCLUSIONS: Fosfomycin in combination with ceftriaxone or vancomycin appeared to be effective for the treatment of experimental cephalosporin-resistant pneumococcal meningitis. These combinations are possible alternatives in cases of allergy or intolerance to first-line drugs or in rare meningitis caused by highly cephalosporin-resistant pneumococci.     

Synergistic activity of fosfomycin and chloramphenicol against vancomycin-resistant Enterococcus faecium (VREfm) isolates from bloodstream infections

Vancomycin-resistant Enterococcus faecium (VREfm) infections are increasing. Current anti-VREfm options (linezolid and daptomycin) are suboptimal. Fosfomycin maintains good efficacy against VREfm and chloramphenicol is active against ≥ 90% of VREfm. We tested chloramphenicol + fosfomycin (CAF+FOS) against 10 VREfm isolated from blood. MICs were 64 to 512 µg/mL for fosfomycin and 8 to 16 µg/mL for chloramphenicol. The combination decreased both MICs, with a synergic effect in 50% of the isolates and an additive effect in the remaining 50%. Time-kill assays performed on fractional inhibitory concentration index ≤ 0.5 strains confirmed the synergism. The antibiotic combination at ¼ of minimum inhibitory concentrations (MICs) caused a ≥ 2 log₁₀ reduction compared to the two antibiotics alone. Finally, we provided a proof of concept of the in vitro efficacy of CAF+FOS in G. mellonella. The survival of G. mellonella larvae treated with the combination was significantly higher. The activity of fosfomycin and chloramphenicol against VREfm increases when they are used in combination.     

Activity of Trovafloxacin (with or without Ampicillin-Sulbactam) against Enterococci in an In Vitro Dynamic Model of Infection

Antibiotic-resistant enterococci are being increasingly identified as causal agents of infection. Trovafloxacin is a new fluoronaphthyridone with enhanced activity against gram-positive cocci and variable activity reported against Enterococcus spp. Twenty-one strains of vancomycin-resistant Enterococcus faecium and two strains of Enterococcus faecalis (one vancomycin resistant) were studied at an initial inoculum of 10⁶ CFU/ml in time-kill assays with trovafloxacin (3 mg/liter), ampicillin-sulbactam (100/50 mg/liter), and the combination. Six strains of E. faecium (five vancomycin resistant) also were studied in an in vitro two-compartment dynamic model that mimics human pharmacokinetics with trovafloxacin simulated at 300 mg every 12 h (q12h), ampicillin-sulbactam at 2/1 g q6h, and the combination. Peripheral compartments were sampled q2h for 30 h for bacterial counts. Trovafloxacin MICs ranged from 0.5 to 32 mg/liter, and the nine strains of vancomycin-resistant E. faecium for which MICs were ≤2 mg/liter were more likely to show a reduction of 2 log units or more in viable counts in time-kill assays than were strains for which MICs were higher. Synergism with ampicillin-sulbactam was found for only one strain (trovafloxacin MIC, 16 mg/liter). Similar results were obtained in the pharmacokinetic model, with 2- to 4-log-unit reductions in viable bacteria for trovafloxacin-susceptible strains. Although no convincing evidence of synergism was found, ampicillin-sulbactam in combination minimized late bacterial regrowth of two trovafloxacin-susceptible strains. These data suggest that this high dose of trovafloxacin (with or without ampicillin-sulbactam) might be useful against strains of vancomycin-resistant E. faecium for which MICs were ≤2 mg/liter.     

High Activity of Fosfomycin and Rifampin against Methicillin-Resistant Staphylococcus aureus Biofilm In Vitro and in an Experimental Foreign-Body Infection Model

Increasing antimicrobial resistance reduces treatment options for implant-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the activity of fosfomycin alone and in combination with vancomycin, daptomycin, rifampin, and tigecycline against MRSA (ATCC 43300) in a foreign-body (implantable cage) infection model. The MICs of the individual agents were as follows: fosfomycin, 1 μg/ml; daptomycin, 0.125 μg/ml; vancomycin, 1 μg/ml; rifampin, 0.04 μg/ml; and tigecycline, 0.125 μg/ml. Microcalorimetry showed synergistic activity of fosfomycin and rifampin at subinhibitory concentrations against planktonic and biofilm MRSA. In time-kill curves, fosfomycin exhibited time-dependent activity against MRSA with a reduction of 2.5 log₁₀ CFU/ml at 128 × the MIC. In the animal model, planktonic bacteria in cage fluid were reduced by <1 log₁₀ CFU/ml with fosfomycin and tigecycline, 1.7 log₁₀ with daptomycin, 2.2 log₁₀ with fosfomycin-tigecycline and fosfomycin-vancomycin, 3.8 log₁₀ with fosfomycin-daptomycin, and >6.0 log₁₀ with daptomycin-rifampin and fosfomycin-rifampin. Daptomycin-rifampin cured 67% of cage-associated infections and fosfomycin-rifampin cured 83%, whereas all single drugs (fosfomycin, daptomycin, and tigecycline) and rifampin-free fosfomycin combinations showed no cure of MRSA cage-associated infections. No emergence of fosfomycin resistance was observed in animals; however, a 4-fold increase in fosfomycin MIC (from 2 to 16 μg/ml) occurred in the fosfomycin-vancomycin group. In summary, the highest eradication of MRSA cage-associated infections was achieved with fosfomycin in combination with rifampin (83%). Fosfomycin may be used in combination with rifampin against MRSA implant-associated infections, but it cannot replace rifampin as an antibiofilm agent.     

Antimicrobial Activity of Fosfomycin-Tobramycin Combination against Pseudomonas aeruginosa Isolates Assessed by Time-Kill Assays and Mutant Prevention Concentrations

The antibacterial activity of fosfomycin-tobramycin combination was studied by time-kill assay in eight Pseudomonas aeruginosa clinical isolates belonging to the fosfomycin wild-type population (MIC = 64 μg/ml) but with different tobramycin susceptibilities (MIC range, 1 to 64 μg/ml). The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined in five of these strains (tobramycin MIC range, 1 to 64 μg/ml) in aerobic and anaerobic conditions simulating environments that are present in biofilm-mediated infections. Fosfomycin-tobramycin was synergistic and bactericidal for the isolates with mutations in the mexZ repressor gene, with a tobramycin MIC of 4 μg/ml. This effect was not observed in strains displaying tobramycin MICs of 1 to 2 μg/ml due to the strong bactericidal effect of tobramycin alone. Fosfomycin presented higher MPC values (range, 2,048 to >2,048 μg/ml) in aerobic and anaerobic conditions than did tobramycin (range, 16 to 256 μg/ml). Interestingly, the association rendered narrow or even null MSWs in the two conditions. However, for isolates with high-level tobramycin resistance that harbored aminoglycoside nucleotidyltransferases, time-kill assays showed no synergy, with wide MSWs in the two environments. glpT gene mutations responsible for fosfomycin resistance in P. aeruginosa were determined in fosfomycin-susceptible wild-type strains and mutant derivatives recovered from MPC studies. All mutant derivatives had changes in the GlpT amino acid sequence, which resulted in a truncated permease responsible for fosfomycin resistance. These results suggest that fosfomycin-tobramycin can be an alternative for infections due to P. aeruginosa since it has demonstrated synergistic and bactericidal activity in susceptible isolates and those with low-level tobramycin resistance. It also prevents the emergence of resistant mutants in either aerobic or anaerobic environments.     

Pharmacokinetics and Pharmacodynamics of Continuous-Infusion Meropenem in Pediatric Hematopoietic Stem Cell Transplant Patients

This study explored the pharmacokinetics and the pharmacodynamics of continuous-infusion meropenem in a population of pediatric hematopoietic stem cell transplant (HSCT) patients who underwent therapeutic drug monitoring. The relationship between meropenem clearance (CL_M) and estimated creatinine clearance (CL_CR) was assessed by nonlinear regression. A Monte Carlo simulation was performed to investigate the predictive performance of five dosing regimens (15 to 90 mg/kg of body weight/day) for the empirical treatment of severe Gram-negative-related infections in relation to four different categories of renal function. The optimal target was defined as a probability of target attainment (PTA) of ≥90% at steady-state concentration-to-MIC ratios (C_SS/MIC) of ≥1 and ≥4 for MICs of up to 8 mg/liter. A total of 21 patients with 44 meropenem C_SS were included. A good relationship between CL_M and estimated CL_CR was observed (r² = 0.733). Simulations showed that at an MIC of 2 mg/liter, the administration of continuous-infusion meropenem at doses of 15, 30, 45, and 60 mg/kg/day may achieve a PTA of ≥90% at a C_SS/MIC ratio of ≥4 in the CL_CR categories of 40 to <80, 80 to <120, 120 to <200, and 200 to <300 ml/min/1.73 m², respectively. At an MIC of 8 mg/liter, doses of up to 90 mg/kg/day by continuous infusion may achieve optimal PTA only in the CL_CR categories of 40 to <80 and 80 to <120 ml/min/1.73 m². Continuous-infusion meropenem at dosages up to 90 mg/kg/day might be effective for optimal treatment of severe Gram-negative-related infections in pediatric HSCT patients, even when caused by carbapenem-resistant pathogens with an MIC of up to 8 mg/liter.     

Antimicrobial susceptibility of Enterococcus strains used in clinical practice as probiotics

The aim of this study was to evaluate the antimicrobial susceptibilities of probiotic strains that are suggested to be effective for preventing antibiotics-associated diarrhea (AAD). The minimum inhibitory concentrations (MICs) of 17 antibiotics against probiotic strains were tested by the agar plate dilution method or broth microdilution method. In all, eight probiotic strains containing Enterococcus faecalis, Bifidobacterium spp., Clostridium butyricum, and Lactobacillus acidophilus were tested. Although the MIC range was wide, from less than 0.0625 to more than 1,024 μg/ml, the MICs of 11 beta-lactams were high for three of four enterococci, with a range of 32 to more than 1,024 μg/ml. In contrast, fluoroquinolones and vancomycin showed potent activities against all enterococci, of which MICs were 0.25–8 μg/ml. Two Bifidobacterium strains and one Lactobacillus strain showed low MICs against many of the beta-lactams, fluoroquinolones, macrolides, and vancomycin, with MICs of 8 μg/ml or less. Fosfomycin showed generally mild activity against enterococci (MIC, 8–32 μg/ml) and anaerobic strains (MIC, 32 to >1,024 μg/ml), respectively. The probiotics strains with high MIC values may survive in the intestinal tract, even if the patient was concomitantly using the antibiotics in clinical practice. Therefore, our results suggest that adequate combinations of probiotics strains and antibiotics should be important for preventing AAD. Further study is needed to determine the efficacy of probiotics in clinical practice.     

Reevaluation of the critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide using wild-type MIC distributions and pncA gene sequencing

Pyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistant Mycobacterium tuberculosis strains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates of Mycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinical M. tuberculosis isolates and compared the results to pncA sequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n = 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤ 8 to 64 mg/liter), and no pncA gene mutations were detected. In strains resistant in both Bactec systems (n = 18), the PZA MICs ranged from 256 to ≥ 1,024 mg/liter. The discordances between pncA sequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated to pncA mutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤ 64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.     

Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

Ceftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200 Enterobacteriaceae and 25 Pseudomonas aeruginosa strains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistant Enterobacteriaceae strains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBL Escherichia coli (MIC₉₀ of 0.25 mg/liter), ESBL Klebsiella pneumoniae (MIC₉₀ of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC₉₀ of 1 mg/liter), non-ESBL E. coli (MIC₉₀ of ≤0.125 mg/liter), non-ESBL K. pneumoniae (MIC₉₀ of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC₉₀ of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistant P. aeruginosa strains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC₉₀ of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtained in vitro from two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC₉₀ values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains of Enterobacteriaceae and P. aeruginosa were ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affect Enterobacteriaceae and P. aeruginosa susceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.     

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.     

Efficacy of High-Dose Amoxicillin-Clavulanate against Experimental Respiratory Tract Infections Caused by Strains of Streptococcus pneumoniae

The purpose of the present investigation was to determine if the efficacy of amoxicillin-clavulanate against penicillin-resistant Streptococcus pneumoniae could be improved by increasing the pediatric amoxicillin unit dose (90 versus 45 mg/kg of body weight/day) while maintaining the clavulanate unit dose at 6.4 mg/kg/day. A rat pneumonia model was used. In that model approximately 6 log₁₀ CFU of one of four strains of S. pneumoniae (amoxicillin MICs, 2 μg/ml [one strain], 4 μg/ml [two strains], and 8 μg/ml [one strain]) were instilled into the bronchi of rats. Amoxicillin-clavulanate was given by computer-controlled intravenous infusion to approximate the concentrations achieved in the plasma of children following the administration of oral doses of 45/6.4 mg/kg/day or 90/6.4 mg/kg/g/day divided every 12 h or saline as a control for a total of 3 days. Infusions continued for 3 days, and 2 h after the cessation of infusion, bacterial numbers in the lungs were significantly reduced by the 90/6.4-mg/kg/day equivalent dosage for strains for which amoxicillin MICs were 2 or 4 μg/ml. The 45/6.4-mg/kg/day equivalent dosage was fully effective only against the strain for which the amoxicillin MIC was 2 μg/ml and had marginal efficacy against one of the two strains for which amoxicillin MICs were 4 μg/ml. The bacterial load for the strain for which the amoxicillin MIC was 8 μg/ml was not reduced with either dosage. These data demonstrate that regimens which achieved concentrations in plasma above the MIC for at least 34% of a 24-h dosing period resulted in significant reductions in the number of viable bacteria, indicating that the efficacy of amoxicillin-clavulanate can be extended to include efficacy against less susceptible strains of S. pneumoniae by increasing the amoxicillin dose.     

Effectiveness of fosfomycin combined with other antimicrobial agents against multidrug-resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolates using the efficacy time index assay

We investigated the effectiveness of fosfomycin combined with other antibiotics, such as piperacillin, cefepime, ceftazidime, imipenem, meropenem, aztreonam, gentamicin, or levofloxacin, against 30 Pseudomonas aeruginosa strains, including multidrug-resistant strains, isolated from clinical specimens, using the efficacy time index (ETI) assay. The assay refers to the result of pharmacokinetics obtained from adult men volunteers, and yields an ETI to evaluate the effect of a combination of antimicrobial agents. With the ETI, based on serum concentration 3 h after the administration of two antimicrobial agents, the effectiveness of antimicrobial combinations was evaluated as follows: poor, ETI < 0.5; fair, 0.5 ≤ ETI < 1; good, 1 ≤ ETI < 8; and excellent, ETI ≥ 8. The combination of fosfomycin and cefepime (efficacy rate [excellent plus good], 76.7%) and fosfomycin/aztreonam (efficacy rate, 76.7%) appeared to be the most effective, followed by fosfomycin/meropenem (efficacy rate, 76.6%), fosfomycin/imipenem (efficacy rate, 73.3%), fosfomycin/ceftazidime (efficacy rate, 70%), fosfomycin/gentamicin (efficacy rate, 70%), fosfomycin/piperacillin (efficacy rate, 66.7%), and fosfomycin/levofloxacin (efficacy rate, 66.7%). Fosfomycin/cefepime, fosfomycin/aztreonam, and fosfomycin/meropenem may be clinically useful in selected patients, particularly for P. aeruginosa. The ETI assay provided information on the minimum inhibitory concentration (MIC) of many pairs of combined antimicrobial agents simultaneously. The ETI assay may be a useful technique with which to investigate the effect of combinations of antimicrobial agents against P. aeruginosa, including multidrug-resistant strains. Received: July 5, 2001 / Accepted: October 29, 2001     

In Vivo Activities of Amoxicillin and Amoxicillin-Clavulanate against Streptococcus pneumoniae: Application to Breakpoint Determinations

The in vivo activities of amoxicillin and amoxicillin-clavulanate against 17 strains of Streptococcus pneumoniae with penicillin MICs of 0.12–8.0 mg/liter were assessed in a cyclophosphamide-induced neutropenic murine thigh infection model. Renal impairment was produced by administration of uranyl nitrate to prolong the amoxicillin half-life in the mice from 21 to 65 min, simulating human pharmacokinetics. Two hours after thigh infection with 10⁵ to 10⁶ CFU, groups of mice were treated with 7 mg of amoxicillin per kg of body weight alone or combined with clavulanate (ratio, 4:1) every 8 h for 1 and 4 days. There was an excellent correlation between the MIC of amoxicillin (0.03 to 5.6 mg/liter) and (i) the change in log₁₀ CFU/thigh at 24 h and (ii) survival after 4 days of therapy. Organisms for which MICs were 2 mg/liter or less were killed at 1.4 to 4.2 and 1.6 to 4.1 log₁₀ CFU/thigh at 24 h by amoxicillin and amoxicillin-clavulanate, respectively. The four strains for which MICs were >4 mg/liter grew 0.2 to 2.6 and 0.6 to 2.3 logs at 24 h despite therapy with amoxicillin and amoxicillin-clavulanate, respectively. Infection was uniformly fatal by 72 h in untreated mice. Amoxicillin therapy resulted in no mortality with organisms for which MICs were 1 mg/liter or less, 20 to 40% mortality with organisms for which MICs were 2 mg/liter, and 80 to 100% mortality with organisms for which MICs were 4.0–5.6 mg/liter. Lower and higher doses (0.5, 2, and 20 mg/kg) of amoxicillin were studied against organisms for which MICs were near the breakpoint. These studies demonstrate that a reduction of 1 log₁₀ or greater in CFU/thigh at 24 h is consistently observed when amoxicillin levels exceed the MIC for 25 to 30% of the dosing interval. These studies would support amoxicillin (and amoxicillin-clavulanate) MIC breakpoints of 1 mg/liter for susceptible, 2 mg/liter for intermediate, and 4 mg/liter for resistant strains of S. pneumoniae.     

In Vitro Activity of Ceftolozane Alone and in Combination with Tazobactam against Extended-Spectrum-β-Lactamase-Harboring Enterobacteriaceae

Ceftolozane, formally CXA-101, is a new antipseudomonal cephalosporin that is also active in vitro against Enterobacteriaceae but is vulnerable to extended-spectrum β-lactamases (ESBLs). The addition of tazobactam is intended to broaden coverage to most ESBL-producing Escherichia coli and Klebsiella pneumonia as well as other Enterobacteriaceae. The in vitro activities of ceftolozane-tazobactam combinations against 67 clinically and molecularly characterized ESBL-producing isolates were examined by checkerboard MIC testing to evaluate their potential clinical feasibility and to assess the optimal tazobactam concentrations to be used in MIC determinations of ceftolozane. Isolates included those from E. coli (n = 32), K. pneumoniae (n = 19), Enterobacter cloacae (n = 15), and Citrobacter freundii (n = 1). Checkerboard experiments were performed to study interactions over the range of 0.008 to 64 mg/liter ceftolozane and 0.063 to 32 mg/liter tazobactam using 2-fold-dilution series. The MIC₅₀ and MIC₉₀ of ceftolozane alone for all isolates were 16 and ≥64 mg/liter, respectively. Increasing concentrations of tazobactam resulted in decreasing MICs of ceftolozane. The 50th and 90th percentile concentrations of tazobactam required to reduce the MIC of ceftolozane to 8 mg/liter for all organisms in this ESBL collection were 0.5 and 4 mg/liter, respectively. For E. coli, K. pneumoniae, and E. cloacae, these values were 0.5 and 2, 1 and 16, and 0.5 and 4 mg/liter, respectively. When combined with a fixed amount of 4 mg/liter tazobactam (current CLSI concentration used for susceptibility testing), 90% of the isolates would have an MIC of ≤4 mg/liter. The combination ceftolozane-tazobactam is a promising alternative option for treating infections due to ESBL-harboring isolates.     

Fosfomycin-Daptomycin and Other Fosfomycin Combinations as Alternative Therapies in Experimental Foreign-Body Infection by Methicillin-Resistant Staphylococcus aureus

The efficacy of daptomycin, imipenem, or rifampin with fosfomycin was evaluated and compared with that of daptomycin-rifampin in a tissue cage model infection caused by methicillin-resistant Staphylococcus aureus (MRSA). Strain HUSA 304 was used. The study yielded the following results for MICs (in μg/ml): fosfomycin, 4; daptomycin, 1; imipenem, 0.25; and rifampin, 0.03. The study yielded the following results for minimum bactericidal concentration (MBC) (in μg/ml): fosfomycin, 8; daptomycin, 4; imipenem, 32; and rifampin, 0.5. Daptomycin-rifampin was confirmed as the most effective therapy against MRSA foreign-body infections. Fosfomycin combinations with high doses of daptomycin and rifampin were efficacious alternative therapies in this setting. Fosfomycin-imipenem was relatively ineffective and did not protect against resistance.     

Species-Specific and Drug-Specific Differences in Susceptibility of Candida Biofilms to Echinocandins: Characterization of Less Common Bloodstream Isolates

Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.     

Fosfomycin Synergy In Vitro with Amoxicillin,Daptomycin, and Linezolid against Vancomycin-Resistant Enterococcus faecium from Renal Transplant Patients with Infected Urinary Stents

Fosfomycin is a potential option for vancomycin-resistant enterococcus (VRE) infections despite limited in vitro and clinical data. In this study, 32 VRE isolates from renal transplant patients with urinary stent infections were susceptible to fosfomycin, daptomycin, and linezolid and resistant to amoxicillin, minocycline, and nitrofurantoin based on their MIC₅₀s and MIC₉₀s. Fosfomycin was bacteriostatic at 0.5 to 16× the MIC (32 to 2,048 μg/ml); synergy occurred when fosfomycin was combined with daptomycin (2.8 to 3.9 log₁₀ CFU/ml kill; P < 0.001) or amoxicillin (2.6 to 3.4; P < 0.05). These combinations may be potent options to treat VRE urinary infections pending investigation of clinical efficacy.     

In Vivo Activities of Simulated Human Doses of Cefepime and Cefepime-AAI101 against Multidrug-Resistant Gram-Negative Enterobacteriaceae

The combination of cefepime with AAI101, a novel extended-spectrum β-lactamase inhibitor, possesses potent in vitro activity against many resistant Gram-negative pathogens. Against a panel of 20 mostly carbapenemase-producing cefepime-nonsusceptible strains of the family Enterobacteriaceae, we evaluated the MICs of cefepime in the presence of various fixed AAI101 concentrations (1, 2, 4, 8, and 16 mg/liter) and the in vivo efficacy of simulated human doses of cefepime and cefepime-AAI101 in a neutropenic murine thigh infection model. At 2 h after inoculation, mice were dosed with regimens that provided a profile mimicking the free drug concentration-time profile observed in humans given cefepime at 2 g every 8 h (q8h; as a 30-min infusion) or cefepime-AAI101 at 2 g/0.5 g q8h (as a 30-min infusion). Efficacy was determined by calculation of the change in thigh bacterial density (log₁₀ number of CFU) after 24 h relative to the starting inoculum (0 h). After 24 h, bacterial growth of 2.7 ± 0.1 log₁₀ CFU (mean ± standard error) was observed in control animals. Efficacy for cefepime monotherapy was observed against only 3 isolates, whereas increases in bacterial density similar to that in the control animals were noted for the remaining 17 strains (all with cefepime MICs of ≥64 mg/liter). The humanized cefepime-AAI101 dosing regimen resulted in bacterial reductions of ≥0.5 log₁₀ CFU for 12 of the 20 strains. Evaluation of efficacy as a function of the fraction of the dosing interval during which free drug concentrations were above the MIC determined with different fixed concentrations of AAI101 suggested that a fixed concentration of 8 mg/liter AAI101 is most predictive of in vivo activity for the studied regimen.