clinicaltrials.gov Identifier: NCT00272675.

The number of infections due to resistant gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and vancomycinresistant Enterococcus faecium, continues to increase (8,10,12,17). The development of effective antimicrobial agents to treat these infections is an area of intense research. Oxazolidinone antimicrobial agents represent a promising new class of compounds which act by inhibiting protein synthesis and have demonstrated potency against these emerging pathogens (13, 16; R. N. Jones, M. A. Pfaller, M. E. Erwin, and M. L. Beach, Abstr. 37th Infect. Dis. Soc. Am. Annu. Meet., abstr. 97, 1999).Linezolid is a new oxazolidinone that has demonstrated both in vitro activity and in vivo efficacy in animal models and clinical trials (2, 13, 15; Jones et al., Abstr. 37th Infect. Dis. Soc. Am. Annu. Meet.). The goals of our studies were (i) to characterize the in vivo time course of antimicrobial activity of linezolid against susceptible and resistant S. pneumoniae and S. aureus strains and (ii) to determine the PK/PD parameter and the magnitude of the PK/PD parameter predictive of efficacy.(Part of this work was presented at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., September 1998.) MATERIALS AND METHODSBacteria, media, and antibiotic. Eight strains of S. pneumoniae (one penicillinsusceptible, two penicillin-intermediate, and five penicillin-resistant S. pneumoniae strains) and four strains of S. aureus (two methicillin-susceptible and two methicillin-resistant S. aureus strains) were used for these experiments. S. aureus and S. pneumoniae organisms were grown, subcultured, and quantified in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.), on Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.), and on sheep blood agar plates (Remel, Milwaukee, Wis.). Linezolid was supplied by Pharmacia & Upjohn Company, Kalamazoo, Mich.In vitro susceptibility studies. The MICs of linezolid, penicillin, and methicillin for the various isolates were determined by using standard National Committee for Clinical Laboratory Standards microdilution methods.Murine infection model. Six-week-old, specific-pathogen-free, female ICR/ Swiss mice weighing 23 to 27 g (Harlan Sprague-Dawley, Madison, Wis.) were used for all studies. All animal studies were approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital. Mice were rendered neutropenic (neutrophils at Ͻ100/mm 3 ) by injecting cyclophosphamide (Mead Johnson Pharmaceuticals, Evansville, Ind.) intraperitoneally 4 days (150 mg/kg) and 1 day (100 mg/kg) before experimental infection. Previous studies have shown that this regimen produces neutropenia in this model for 5 days (1). Broth cultures of freshly plated bacteria were grown to logarithmic phase overnight to an absorbance of 0.3 at 580 nm (Spectronic 88; Bausch and Lomb, Rochester, N.Y.). After a 1:10 dilution into fresh Mueller-Hinton broth, bacterial counts of the ...

The in vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY 152,288) were assessed in an experimental murine thigh infection model in neutropenic mice. Mice were infected with one of several strains of Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, or Klebsiella pneumoniae. Most infections were treated with a twice-daily dosing schedule, with administration of 0.75 to 192 mg of GAR-936 or WAY 152,288 per kg of body weight. A maximum-effect dose-response model was used to calculate the dose that produced a net bacteriostatic effect over 24 h of therapy. This dose was called the bacteriostatic dose. More extensive dosing studies were performed with S. pneumoniae 1199, E. coli ATCC 25922, and K. pneumoniae ATCC 43816, with doses being given as one, two, four, or eight equal doses over a period of 24 h. The dosing schedules were designed in order to minimize the interrelationship between the various pharmacokinetic and pharmacodynamic parameters studied. These parameters were time above 0.03 to 32 times the MIC, area under the concentration-time curve (AUC), and maximum concentration of drug in serum (C max ). The bacteriostatic dose remained essentially the same, irrespective of the dosing frequency, for S. pneumoniae 1199 (0.3 to 0.9 mg/kg/day). For E. coli ATCC 25922 and K. pneumoniae ATCC 43816, however, more frequent dosing led to lower bacteriostatic doses. Pharmacokinetic studies demonstrated dose-dependent elimination half-lives of 1.05 to 2.34 and 1.65 to 3.36 h and serum protein bindings of 59 and 71% for GAR-936 and WAY 152,288, respectively. GAR-936 and WAY 152,288 were similarly effective against the microorganisms studied, with small differences in maximum effect and 50% effective dose. The glycylcyclines were also similarly effective against tetracycline-sensitive and tetracycline-resistant bacteria. Time above a certain factor (range, 0.5 to 4 times) of the MIC was a better predictor of in vivo efficacy than C max or AUC for most organism-drug combinations. The results demonstrate that in order to achieve 80% maximum efficacy, the concentration of unbound drug in serum should be maintained above the MIC for at least 50% of the time for GAR-936 and for at least 75% of the time for WAY 152,288. The results of these experiments will aid in the rational design of dose-finding studies for these glycylcyclines in humans. 288 are members of the class of glycylcyclines, a new group of antibiotics derived from minocycline. These drugs have potent activity against a variety of tetracycline-sensitive and tetracycline-resistant bacteria (1-3, 5, 8, 10, 14, 15).The objective of the present study was to determine the effects of various dosing regimens on the in vivo efficacy of GAR-936 and WAY 152,288 and identify which pharmacokinetic or pharmacodynamic parameter best correlated with efficacy. The in vivo antibacterial activities of GAR-936 and WAY 152,288 against several isolates of common human pathogens (Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, ...

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Objectives: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. Methods: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). Results: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n ¼ 388, 43%), Escherichia coli (n ¼ 264, 30%) and Enterobacter cloacae complex (n ¼ 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with bla OXA-48 being predominant (38%, 336/892), followed by bla NDM-1 (16%, 145/892). For the first time in the Netherlands, bla OXA-181 , bla OXA-232 and bla VIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. Conclusions: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are bla OXA-48 and bla NDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.

The prevalence of ESBL was determined among isolates of Escherichia coli (n = 571) and Klebsiella spp. (n = 196) collected during a 1-week study period in 8 university and 3 large regional laboratories all over the Netherlands. 18 isolates were positive for at least one of the screening tests used, i.e., VITEK-ESBL, E-test ESBL and MIC ratio of ceftazidime/ceftazidime-clavulanic acid, cefotaxime/cefotaxime-clavulanic acid. In 5 of these 18 putative ESBLs no betalactamase production was detectable. A TEM type was found in three E. coli and two Klebsiella spp. An SHV type was present in five Klebsiella spp. In one E. coli and one Klebsiella pneumoniae both enzymes were present. In one Klebsiella oxytoca neither of the two enzymes was present. Using PCR for both ESBL TEM and ESBL SHV, an SHV ESBL was found in one E. coli and four Klebsiella isolates. The mutations at position 238 and 240 were already described. In one E. coli isolate a TEM ESBL was found with three mutations, at position 21, 164 and 265. These mutations were already described in other ESBLs but not in this combination suggesting a new TEM ESBL. The overall prevalence of ESBL producing E. coli and Klebsiella spp. was less than 1% (6 out of 767).

A thigh muscle infection induced with Escherichia coli in irradiated mice was used as a model to compare the in vivo pharmacodynamics of the antibacterial effect of four cephalosporins (i.e., cefepime, ceftriaxone, ceftazidime, and cefoperazone) with the in vitro antibacterial pharmacodynamics of these drugs. The following in vitro pharmacodynamic parameters were determined: the maximum effect as a measure for efficacy, the 50% effective concentration as a parameter for potency, and the slope of the concentration-effect relationship. For analysis of the in vivo antibacterial pharmacodynamics, the same parameters were applied for the dose instead of the concentration. For the detection of a relationship between concentration and antibacterial effect in vivo, we determined the pharmacokinetics of the four cephalosporins in the plasma of mice. The results showed that, in general, there is a direct relationship between the in vivo and in vitro pharmacodynamics of these cephalosporins. The maximum effects of cefepime, ceftazidime, and cefoperazone were approximately similar in vivo and in vitro. The sequence of potency of these drugs was, in descending order, cefepime, ceftazidime, and cefoperazone. Ceftriaxone differed from the other three cephalosporins in that it displayed unexpected in vivo pharmacodynamics. Ceftriaxone was just as efficacious as the other three in vitro, but its maximum effect in vivo was much lower. This relatively low maximum effect of ceftriaxone in vivo was not explained by the pharmacokinetic characteristics of the drug. From the present results it can be concluded that the in vitro efficacy of cephalosporins does not necessarily have a predictive value for the in vivo efficacy.Cephalosporins are a group of antibacterial agents used increasingly for the empiric treatment of suspected infections as well as for the treatment of gram-negative infections in granulocytopenic patients (4).The usual method for the in vitro comparison of antibacterial agents is to determine the MICs of the agents under study for several groups of bacteria. The MIC is, however, a static parameter of antibacterial effect because it does not take into account the pharmacodynamic characteristics of drug action (10), such as the potency of the drug, the slope of the curve of the concentration-effect relationship, and the maximum effect on bacterial growth. The MIC alone can be considered to be a measure of the in vitro potency of the antibiotic, but as a quantitative parameter it is rather inaccurate, because it is usually determined on the basis of twofold dilution steps.For a valid comparison of in vitro and in vivo antibacterial pharmacodynamics, it is essential to determine the pharmacokinetics of the antimicrobial agents as well, because such data provide information on the relationship between the plasma concentration and the in vivo effect. The thigh infection model used in this study has the advantage that the concentration of the antimicrobial agent in the interstitial fluid of the thigh muscle corresponds with...

Although combination therapy with antimicrobial agents is often used, no available method explains or predicts the efficacies of these combinations satisfactorily. Since the efficacies of antimicrobial agents can be described by pharmacodynamic indices (PDIs), such as area under the concentration-time curve (AUC), peak level, and the time that the concentration is above the MIC (time>MIC), it was hypothesized that the same PDIs would be valid in explaining efficacy during combination therapy. Twenty-four-hour efficacy data (numbers of CFU) for Pseudomonas aeruginosa in a neutropenic mouse thigh model were determined for various combination regimens: ticarcillin-tobramycin (n = 41 different regimens), ceftazidime-netilmicin (n = 60), ciprofloxacin-ceftazidime (n = 59), netilmicin-ciprofloxacin (n = 38) and for each of these agents given singly. Multiple regression analysis was used to determine the importance of various PDIs (time>MIC, time>0.25× the MIC, time>4× the MIC, peak level, AUC, AUC/MIC, and their logarithmically transformed values) during monotherapy and combination therapy. The PDIs that best explained the efficacies of single-agent regimens were time>0.25× the MIC for beta-lactams and log AUC/MIC for ciprofloxacin and the aminoglycosides. For the combination regimens, regression analysis showed that efficacy could best be explained by the combination of the two PDIs that each best explained the response for the respective agents given singly. A regression model for the efficacy of combination therapy was developed by use of a linear combination of the regression models of the PDI with the highestR 2 for each agent given singly. The model values for the single-agent therapies were then used in that equation, and the predicted values that were obtained were compared with the experimental values. The responses of the combination regimens could best be predicted by the sum of the responses of the single-agent regimens as functions of their respective PDIs (e.g., time>0.25× the MIC for ticarcillin and log AUC/MIC for tobramycin). The relationship between the predicted response and the observed response for the combination regimens may be useful for determination of the presence of synergism. We conclude that the PDIs for the individual drugs used in this study are class dependent and predictive of outcome not only when the drugs are given as single agents but also when they are given in combination. When given in combination, there appears to be a degree of synergism independent of the dosing regimen applied.