Combination of Daptomycin plus Ceftriaxone Is More Active than Vancomycin plus Ceftriaxone in Experimental Meningitis after Addition of Dexamethasone

We examined the cerebrospinal fluid penetration of daptomycin after the addition of dexamethasone and its bactericidal efficacy with and without ceftriaxone in an experimental rabbit model of pneumococcal meningitis. The combination of daptomycin with ceftriaxone was the most efficacious regimen for pneumococcal meningitis. The previous addition of dexamethasone affected the antibacterial activity of daptomycin only marginally, either as monotherapy or combined with ceftriaxone, although the penetration of daptomycin into inflamed meninges was significantly reduced from 6 to 2%. Daptomycin with ceftriaxone might be a potential candidate for the empirical therapy of bacterial meningitis, although the activity of this regimen against Listeria monocytogenes remains to be demonstrated.The worldwide continuous spread of penicillin-resistant pneumococci represents one of the major challenges for clinicians and infectiologists. The epidemiological situation in Europe varies considerably with a global tendency of increasing penicillin resistance rates from 6% in 1997 to 22% in 1999 (8). In the United States and Canada, the combined rate of penicillin intermediate plus resistant strains varied between 24% and 67% as reported in the multinational SENTRY antimicrobial resistance surveillance program (5). Based on a recent study, the rates of highly resistant strains were 14.7%, 12.7%, and 15.9% for Europe, Latin America, and North America, respectively (9). In adults, pneumococci are the most frequent pathogens causing meningitis (1, 11). In meningitis due to highly resistant strains, high-dose vancomycin has been recommended, either alone or in combination with third-generation cephalosporins (1, 14). In adults, the addition of steroids, now established as standard adjunctive therapy, reduces the penetration of vancomycin into the cerebrospinal fluid (CSF) by 29% (10, 12).We have previously shown that daptomycin, a cyclic lipopeptide, was very efficacious in experimental pneumococcal meningitis due to a penicillin-resistant strain and managed to sterilize the CSF samples of all rabbits at the end of the experimental period. Little is known about the effect of dexamethasone, as standard adjunctive treatment in bacterial meningitis, on the meningeal penetration and efficacy of daptomycin. The aim of this study was first to test the effect of dexamethasone on the penetration of daptomycin into inflamed meninges and second to compare the activity of the different regimens after the addition of dexamethasone in pneumococcal meningitis due to a penicillin-resistant strain. The comparator regimen was ceftriaxone combined with vancomycin, which is the standard empirical regimen.     

Activity of Telavancin against Staphylococci and Enterococci Determined by MIC and Resistance Selection Studies

This study used CLSI broth microdilution to test the activity of telavancin and comparator antimicrobial agents against 67 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates. Twenty-six vancomycin-intermediate S. aureus (VISA) strains were among the isolates tested; all strains were susceptible to telavancin at ≤1 μg/ml, whereas 12/26 (46%) of these isolates were nonsusceptible to daptomycin at the same concentration. All strains were susceptible to quinupristin-dalfopristin, while resistance was found to all other drugs tested. Telavancin demonstrated potent activity against all vancomycin-susceptible isolates as well as against heterogeneously VISA and VISA resistance phenotypes. In multistep resistance selection studies, telavancin yielded one stable mutant after 43 days in one MRSA strain out of the 10 MRSA strains tested with the MIC rising eightfold from 0.25 μg/ml (parent) to 2 μg/ml. MICs for this clone did not increase further when passages were continued for the maximum 50 days. In contrast, daptomycin selected stable resistant clones (MIC increase of >4×) after 14 to 35 days in 4 of 10 MRSA strains with MICs increasing from 1 to 2 μg/ml (parents) to 4 to 8 μg/ml (resistant clones). Sequencing analysis of daptomycin resistance determinants revealed point mutations in the mprF genes of all four stable daptomycin-resistant clones. Teicoplanin gave rise to resistant clones after 14 to 21 days in 2 of 10 MRSA strains with MICs rising from 1 to 2 μg/ml (parents) to 4 to 16 μg/ml (stable resistant clones). Linezolid selected stable resistant clones after 22 to 48 days in 2 of 10 MRSA strains with MICs rising from 2 to 4 μg/ml (parents) to 32 μg/ml (resistant clones). Vancomycin yielded no resistant clones in 10 MRSA strains tested; however, MICs increased two- to fourfold from 1 to 8 μg/ml to 2 to 16 μg/ml after 50 days. No cross-resistance was found with any clone/antimicrobial combination. The two enterococci developed resistance to daptomycin, and one developed resistance to linezolid. Single-step mutation frequencies for telavancin (<4.0 × 10⁻¹¹ to <2.9 × 10⁻¹⁰ at 2× MIC) were lower than the spontaneous mutation frequencies obtained with the comparators.Staphylococcus aureus is becoming increasingly resistant to antibiotics. Methicillin (meticillin)-resistant S. aureus (MRSA) strains are increasingly encountered all over the world and cannot be treated with existing ß-lactams. Additionally, the majority of hospital-acquired methicillin-resistant (and also some methicillin-susceptible) strains are resistant to all currently available quinolones. The situation has become more complicated by the appearance of heterogeneously vancomycin-intermediate S. aureus (hVISA) strains, vancomycin-intermediate S. aureus (VISA) strains, and recently nine reported vancomycin-resistant S. aureus (VRSA) strains (1). Two recent papers emphasize the recent spread of VISA strains in Turkey (47) and France (15), and a recent alert from the New York City Department of Health (11) has documented six cases of VISA infections in New York City, NY, between February and October 2007, which led to four fatalities. It seems clear that VISA phenotypes occur everywhere but that they are not being routinely detected due to lack of standardized methodology (1, 20). Recently, Rybak et al. (43) have indicated, with Etest macromethod and population analysis testing, that the incidence of hVISA strains has increased over the past 22 years to an overall incidence of 2.2%. Yusof et al. (55) have recently described the utility of the Etest macromethod using a double-sided vancomycin-teicoplanin Etest strip which accurately differentiates between hVISA and VISA strains. Utilization of the latter method will surely increase reports of the incidence of hVISA and VISA strains. As of this time, the pathogenicity of VRSA strains awaits confirmation.The situation has become further complicated by the appearance and rapid spread, especially in the United States, of community-acquired MRSA strains that are especially virulent, possibly by virtue of production of Panton-Valentine leukocidin (4, 10, 16, 28, 34, 37). Although these strains are currently more susceptible to antimicrobial agents than hospital-acquired strains are, this situation will surely change. Additionally, treatment of the community-acquired MRSA strains with glycopeptides will increase the selective pressure leading to nonsusceptibility to vancomycin and teicoplanin. Recently, we and others have documented clinical development of daptomycin resistance after daptomycin therapy (25), and not all VISA strains are daptomycin susceptible (1, 19, 25, 26). There is an urgent need for new agents to treat MRSA infections.Telavancin is an investigational lipoglycopeptide active (MICs of ≤1 μg/ml) against gram-positive organisms including MRSA (17, 18, 22, 29, 31, 38, 48). Barcia-Macay and coworkers (3) have reported telavancin MICs of 0.5 μg/ml against two strains of VISA and of 2 to 4 μg/ml against two strains of VRSA. Leuthner and coworkers in a study of 50 glycopeptide-nonsusceptible staphylococci and 3 VRSA strains showed telavancin to be potent against all 37 hVISA and VISA strains (13 coagulase-negative strains; resistance phenotypes not differentiated from one another) with MICs of <1 μg/ml, with higher MICs of 2 to 4 μg/ml against the 3 VRSA strains (33). The antibacterial mode of action of telavancin results from inhibition of bacterial cell wall synthesis and interference with the barrier function of the bacterial cell membrane (22). The mode of action of daptomycin also involves targeting the bacterial cell membrane to initiate antimicrobial activity (2, 24, 26). This fact is supported by the involvement of staphylococcal genes regulating cell membrane surface charge (e.g., mprF) (39) and fatty acid synthesis (e.g., yycG) (36) in the development of daptomycin nonsusceptibility.In an effort to expand the comparative activity of telavancin against MRSA strains of various resistance phenotypes, we have investigated the activity of telavancin against MRSA strains by determining the activities of telavancin and comparator agents against 67 vancomycin-susceptible and -nonsusceptible MRSA strains using broth microdilution and also by testing the potential of telavancin and the comparator agents vancomycin, teicoplanin, daptomycin, and linezolid to select for resistance in 10 MRSA strains as well as two strains of enterococci by single-step and multistep selection methodology. Additionally, we conducted molecular genetic studies to characterize the mechanism(s) of daptomycin resistance in isolated clones.(Part of this study was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy and the 46th Annual Meeting of the Infectious Disease Society of America, a joint meeting held in Washington, DC, in 2008 [12].)     

Efficacy of High Doses of Daptomycin versus Alternative Therapies against Experimental Foreign-Body Infection by Methicillin-Resistant Staphylococcus aureus

Since the currently approved dose of daptomycin (6 mg/kg of body weight/day) has been associated with clinical failures and resistance development, higher doses for some difficult-to-treat infections are being proposed. We studied the efficacy of daptomycin at high doses (equivalent to 10 mg/kg/day in humans) and compared it to that of reference and alternative treatments in a model of foreign-body infection with methicillin (meticillin)-resistant Staphylococcus aureus. In vitro studies were conducted with bacteria in the log and stationary phases. For the in vivo model, therapy with daptomycin at 100 mg/kg/day, vancomycin at 50 mg/kg/12 h, rifampin (rifampicin) at 25 mg/kg/12 h, or linezolid at 35 mg/kg/12 h was administered for 7 days. Antibiotic efficacy was evaluated using either bacteria from tissue cage fluids or those attached to coverslips. We screened for the emergence of linezolid- and rifampin-resistant strains and analyzed the surviving population from the daptomycin-treated group. Only daptomycin was bactericidal in both the log- and stationary-phase studies. Daptomycin (decrease in the log number of CFU per milliliter of tissue cage fluid, 2.57) and rifampin (decrease, 2.6 log CFU/ml) were better (P < 0.05) than vancomycin (decrease, 1.1 log CFU/ml) and linezolid (decrease, 0.9 log CFU/ml) in the animal model. Rifampin-resistant strains appeared in 60% of cases, whereas no linezolid resistance emerged. No daptomycin-resistant subpopulations were detected at frequencies of 10⁻⁷ or higher. In conclusion, daptomycin at high doses proved to be as effective as rifampin, and the two were the most active therapies for this experimental foreign-body infection. These high doses ensured a profile of safety from the development of resistance.Daptomycin is a lipopeptide drug with bactericidal activity toward methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) in a concentration-dependent manner (27, 32). It is currently approved for use at 4 mg/kg of body weight/day for skin and soft-tissue infections (1) and at 6 mg/kg/day for bacteremia and right-side endocarditis (12, 14).In recent years, reports of clinical failures and the emergence of resistant strains following daptomycin treatment have raised great concern (6, 18, 31). As a result, higher doses of daptomycin are being proposed as an alternative for some difficult-to-treat infections such as complicated bacteremia and endocarditis. Recently, doses of 10 mg/kg/day were studied using an in vitro model of staphylococcal endocarditis, with the results being promising in terms of efficacy and resistance prevention (25, 26). To date, clinical experience with the activity of the drug at doses higher than 6 mg/kg/day is limited (15, 28), whereas good safety and tolerance profiles for daptomycin at up to 12 mg/kg/day in volunteers have been reported (4).Foreign-body infections are difficult to treat because of the presence of bacterial biofilm and tolerance to antibiotics (10, 39). MRSA is commonly involved in such infections, and daptomycin may be a promising drug (39). However, clinical experience in this area is again scarce, and the recommended doses are not clearly established, there being reports of clinical failures with doses of 4 to 6 mg/kg/day (13, 24).The rat model of tissue cage infection is a well-standardized model that reasonably mimics human device infections (17, 20, 35). In this model, the efficacy of daptomycin has been partially studied (29, 34).Taken together, the available experimental and clinical data seem to indicate that high doses of daptomycin are required in the setting of foreign-body infection.In the present study, we aimed to test the efficacy of daptomycin at doses equivalent to 10 mg/kg/day in humans in a model of foreign-body infection with MRSA, comparing it with the efficacy of the current reference or main alternative treatments such as vancomycin, rifampin (rifampicin), and linezolid. We also sought to analyze the protection offered against the emergence of resistant strains.     

Underestimation of Vancomycin and Teicoplanin MICs by Broth Microdilution Leads to Underdetection of Glycopeptide-Intermediate Isolates of Staphylococcus aureus

Broth microdilution was compared with tube macrodilution and a simplified population analysis agar method for evaluating vancomycin and teicoplanin MICs and detecting glycopeptide-intermediate isolates of Staphylococcus aureus. Modal vancomycin and teicoplanin MICs recorded by tube macrodilution and the agar plate assay, which both used inocula of 10⁶ CFU, were significantly higher (2 μg/ml) against a panel of borderline glycopeptide-susceptible and glycopeptide-intermediate methicillin-resistant S. aureus (MRSA) bloodstream isolates compared to broth microdilution (1 μg/ml). Vancomycin and teicoplanin MIC distributions by tube macrodilution and agar testing were also markedly different from those evaluated by broth microdilution. The 20-fold-lower inoculum size used for broth microdilution compared to macrodilution and agar MIC assays explained in part, but not entirely, the systematic trend toward lower vancomycin and teicoplanin MICs by microdilution compared to other methods. Broth microdilution assay led to underdetection of the vancomycin-intermediate S. aureus (VISA) phenotype, yielding only three VISA isolates, for which vancomycin MICs were 4 μg/ml compared to 8 and 19 VISA isolates detected by macrodilution and agar testing, respectively. While macrodilution and agar testing detected 7 and 22 isolates with elevated teicoplanin MICs (8 μg/ml), respectively, broth microdilution failed to detect such isolates. Detection rates of isolates with elevated vancomycin and teicoplanin MICs by macrodilution and agar testing assays were higher at 48 h than at 24 h. In conclusion, the sensitivity of broth microdilution MIC testing is questionable for reliable detection and epidemiological surveys of glycopeptide-intermediate resistance in S. aureus isolates.Since 1997, two major categories of vancomycin resistance in Staphylococcus aureus have been defined. The first category refers to vancomycin-resistant S. aureus (VRSA) clinical isolates with exogenously acquired, vanA-mediated high-level resistance (vancomycin MICs, ≥16 μg/ml) (7, 45); the second category includes vancomycin-intermediate S. aureus (VISA) isolates that developed low-level resistance (vancomycin MICs, ≥4 to <16 μg/ml) via complex, incompletely defined endogenous mechanisms (6, 10, 21, 51). Since VISA isolates are almost uniformly cross-resistant to teicoplanin (21, 30), they are frequently designated glycopeptide-intermediate S. aureus (GISA) (50). In contrast to vancomycin, widely different teicoplanin susceptibility breakpoints have been proposed by different national or international committees, varying from 2 (13) to 8 (10) μg/ml, which leads to a confusing situation.Soon after their initial discovery in Japan (23), it was realized that a large proportion of VISA isolates, referred to as hVISA, show heterogeneous expression of vancomycin-intermediate resistance, including a minority population (perhaps as few as 10⁻⁶ cells) for which the vancomycin MIC is ≥4 μg/ml, while the majority of bacteria are still vancomycin susceptible (vancomycin MICs, ≤2 μg/ml) (10, 21, 22, 24, 51). No mechanistic model explaining heterogeneous expression of glycopeptide resistance has been provided. hVISA/hGISA are assumed to be precursors of VISA/GISA strains, with glycopeptides providing the selective pressure for conversion (2, 14, 22, 24, 33, 39, 44, 55). On the other hand, serial passages on antibiotic-free media frequently lead to gradual dilution and eventual elimination of the resistant subpopulation (2, 21, 24). These data potentially challenge the previously established distinction between hGISA and GISA (21, 29, 51).Despite repeated efforts to create one, there is no standard molecular or phenotypic assay allowing reliable detection of GISA and hGISA clinical or laboratory isolates (5, 30). This situation can be explained by (i) the multifactorial molecular basis of hGISA/GISA phenotypes, which did not reveal any ubiquitous, single, specific molecular marker for their detection (24-27, 41), and (ii) the variable, phenotypic expression of low-level glycopeptide resistance, which is significantly influenced by several technical parameters, including the compositions of liquid or solid test media and varying time frames and inoculum sizes.Standard CLSI-recommended broth microdilution and agar MIC-testing methods (9) were reported to have suboptimal sensitivity for detecting some hGISA isolates (21, 51) because they use relatively small inocula (5 × 10⁴ CFU/well and 1 × 10⁴ CFU/spot, respectively). Accordingly, specifically designed agar screening or population analysis profiles, as well as modified Etest methods, were developed for improved detection of hGISA and GISA by integrating requirements for larger bacterial inocula and longer incubation periods (5, 17, 24, 48, 51, 54, 58, 60). Nevertheless, standardization of these elaborated, labor-intensive susceptibility test methods is difficult (17, 48, 58, 60), and their relationships with standard glycopeptide MIC breakpoints are not well defined. Finally, the recent revisions of vancomycin MIC breakpoints by CLSI (10) and of both teicoplanin and vancomycin MIC breakpoints by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (13), which were based on glycopeptide susceptibility surveys of S. aureus clinical isolates (15, 51, 59), hamper analysis of hGISA/GISA prevalence data reported before 2006.Despite the lack of standardized hGISA detection methods, a number of clinical reports have linked vancomycin therapeutic failure of methicillin-resistant S. aureus (MRSA) infections with the presence of VISA or hVISA isolates or with emergence of vancomycin-intermediate resistance during glycopeptide therapy (3, 8, 24, 25, 33, 36-38, 44, 51, 52). Even higher rates of vancomycin treatment failures were reported for bacteremic patients infected with MRSA isolates for which vancomycin MICs (2 μg/ml) were still in the susceptible range than for those with lower vancomycin MICs (<2 μg/ml) (3, 11, 18-20, 31, 32, 34, 35, 43, 46, 51). An emerging creep of vancomycin and teicoplanin MICs against MRSA in the last decade, which was suggested by large-scale epidemiological studies (19, 28, 47, 51, 56), has been challenged by more recent data (1, 42). Collectively, most of the discrepancies in the clinical and epidemiological results might have resulted from the lack of reliable, sensitive detection methods for hGISA and GISA.During a retrospective surveillance study that explored the prevalence of intermediate glycopeptide resistance in MRSA bloodstream isolates from our institution, we discovered that vancomycin MICs, assayed by the reference macrodilution (tube) method (9), were 2 μg/ml for a vast majority of our nosocomial isolates. Since these MIC estimates were significantly higher than those currently reported in clinical and epidemiological MRSA surveillance studies, in which the modal vancomycin MIC assayed by the broth microdilution (1, 18, 24, 42, 51) or agar dilution (40, 59) method was 1 μg/ml, we evaluated the impacts of three different susceptibility-testing methods, namely, broth microdilution, tube macrodilution, and a simplified population analysis assay, on glycopeptide MIC distributions for our panel of MRSA isolates. A detailed analysis of parameters that potentially contributed to assay-dependent differences in vancomycin and teicoplanin MIC estimates, such as the inoculum size, time of incubation, and medium composition, was performed. A novel approach, combining broth macrodilution and agar testing, is proposed for discriminating glycopeptide-susceptible from hGISA and GISA isolates.     

Addition of Gentamicin or Rifampin Does Not Enhance the Effectiveness of Daptomycin in Treatment of Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

This study evaluated the activity of daptomycin combined with either gentamicin or rifampin against three methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates in vitro and one isolate in vivo against a representative strain (MRSA-572). Time-kill experiments showed that daptomycin was bactericidal against these strains at concentrations over the MIC. Daptomycin at sub-MIC concentrations plus gentamicin at 1× and 2× the MIC yielded synergy, while the addition of rifampin at 2 to 4 μg/ml resulted in indifference (two strains) or antagonism (one strain). The in vivo activity of daptomycin (6 mg/kg of body weight once a day) was evaluated ± gentamicin (1 mg/kg intravenously [i.v.] every 8 h [q8h]) or rifampin (300 mg i.v. q8h) in a rabbit model of infective endocarditis by simulating human pharmacokinetics. Daptomycin plus gentamicin (median, 0 [interquartile range, 0 to 2] log₁₀ CFU/g vegetation) was as effective as daptomycin alone (0 [0 to 2] log₁₀ CFU/g vegetation) in reducing the density of bacteria in valve vegetations (P = 0.83), and both were more effective than daptomycin plus rifampin (3 [2 to 3.5] log₁₀ CFU/g vegetation; P < 0.05) for the strain studied. In addition, daptomycin sterilized a ratio of vegetations that was similar to that of daptomycin plus gentamicin (10/15 [67%] versus 9/15 [60%]; P = 0.7), and both regimens did so more than daptomycin plus rifampin (3/15 [20%]; P = 0.01 and P = 0.02, respectively). No statistical difference was noted between daptomycin plus gentamicin and daptomycin alone for MRSA treatment. In the combination arm, all isolates from vegetations remained susceptible to daptomycin, gentamicin, and rifampin. Sixty-one percent of the isolates (8/13) acquired resistance to rifampin during monotherapy. In the daptomycin arm, resistance was detected in only one case, in which the daptomycin MIC rose to 2 μg/ml among the recovered bacteria. In conclusion, the addition of gentamicin or rifampin does not enhance the effectiveness of daptomycin in the treatment of experimental endocarditis due to MRSA.Staphylococcus aureus is a common cause of infective endocarditis (IE), with methicillin-resistant S. aureus (MRSA) strains found in up to one-third of all cases (11, 28). Due to multidrug resistance among many strains, vancomycin is the standard therapy for IE caused by MRSA (1). However, vancomycin therapy has been associated with poor outcomes that may be explained by the drug''s slow bactericidal activity and insufficient diffusion into valve vegetations (5, 10, 23).Daptomycin is a cyclic lipopeptide that is rapidly bactericidal against gram-positive pathogens such as MRSA, including strains that exhibit resistance to vancomycin. It is approved for the treatment of skin and soft tissue infections, S. aureus bacteremia, and right-sided native valve endocarditis (6). However, there is limited information regarding the efficacy of daptomycin in the treatment of left-sided native valve IE caused by MRSA. In a randomized clinical trial (10), none of the patients with left-sided endocarditis treated with daptomycin at 6 mg/kg of body weight/day were cured, and postmarketing registry data (24) revealed a successful clinical outcome in only 9 out of 15 cases (60%). Therefore, given the lack of efficacy data with daptomycin monotherapy in left-sided MRSA endocarditis, the continued evaluation of methods to enhance the activity of daptomycin is warranted. It is unknown whether daptomycin''s activity against MRSA may be improved by combining it with one or more additional antibiotics to produce a potentially additive or synergistic effect. Gentamicin has been shown to augment daptomycin''s activity against strains of MRSA in vitro (4, 20, 35). The combination of daptomycin plus rifampin has demonstrated additive activity against MRSA in vitro (4) and has enhanced activity against MRSA in vivo (4, 32). The aim of this study was to evaluate the in vitro activity of daptomycin combined with gentamicin or rifampin against MRSA and compare treatment with daptomycin alone to treatment with both combinations in experimental MRSA aortic valve endocarditis using a human-adapted pharmacokinetic model.(This work was previously presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy [ICAAC], Chicago, IL, 17 to 20 September 2007 [29a] and at the 48th Annual ICAAC-IDSA Annual Meeting, Washington, DC, 25 to 28 October 2008 [29b].)     

Efficacy Profiles of Daptomycin for Treatment of Invasive and Noninvasive Pulmonary Infections with Streptococcus pneumoniae

Daptomycin is a novel lipopeptide antibiotic with excellent activity against Gram-positive bacterial pathogens, but its therapeutic value for the treatment of invasive pneumococcal disease compared to that for the treatment of pneumococcal pneumonia is incompletely defined. We investigated the efficacy of daptomycin in two models of Streptococcus pneumoniae-induced lung infection, i.e., pneumococcal pneumonia and septic pneumococcal disease. Mice were infected with a bioluminescent, invasive serotype 2 S. pneumoniae strain or a less virulent serotype 19 S. pneumoniae strain and were then given semitherapeutic or therapeutic daptomycin or ceftriaxone. Readouts included survival; bacterial loads; and septic disease progression, as determined by biophotonic imaging. Semitherapeutic daptomycin treatment fully protected the mice against the progression of septic disease induced by serotype 2 S. pneumoniae, while therapeutic treatment of the mice with daptomycin or ceftriaxone led to ∼70% or ∼60% survival, respectively. In contrast, mice infected with serotype 19 S. pneumoniae developed severe pneumonia and lung leakage even in the presence of increased intra-alveolar daptomycin levels, resulting in only 40% survival, whereas the ceftriaxone-treated mice had 100% survival. Together, although daptomycin demonstrates little efficacy in the treatment of pneumococcal pneumonia, daptomycin is highly effective in preventing S. pneumoniae-induced septic death, thus possibly offering a therapeutic option for patients with life-threatening septic pneumococcal disease.The Gram-positive bacterium Streptococcus pneumoniae is the most prevalent pathogen in community-acquired pneumonia (CAP). CAP is known to frequently progress to invasive pneumococcal disease, thereby causing significant morbidity and mortality worldwide. In developed countries, death from pneumococcal disease occurs primarily among elderly individuals, in whom bacteremic pneumonia is associated with case-fatality rates of 10 to 20% and in whom pneumococcal bacteremia is associated with a case-fatality rate of up to 60% (33). As such, S. pneumoniae causes more deaths from invasive infections than any other bacterium and is the fifth leading cause of death worldwide (12). The worldwide increase in the rates of resistance of S. pneumoniae to frequently used antibiotics such as beta-lactams and macrolides and the rapid global spread of multidrug-resistant clones require both the development of novel immunization strategies and the search for novel antibiotic substances that are active against S. pneumoniae strains resistant to other antimicrobial drugs (1, 9, 24). The clinical situation with S. pneumoniae is further complicated by the fact that pathogenicity profiles vary considerably between and among different serotypes of S. pneumoniae. For example, serotypes 19 and 23 of S. pneumoniae are considered less virulent in humans and mice, whereas serotypes 2 and 4 frequently cause invasive pneumococcal disease and multiorgan failure (MOF) (14). It is just the progression from local infection to sepsis and MOF that contributes to the unacceptably high mortality rates in intensive care units. In view of the distinct pathogenicity profiles of S. pneumoniae serotypes and strains that result in different clinical courses, novel antibiotic substances with the potential to cure infections with S. pneumoniae need to be rigorously validated in experimental model systems specifically representing the major clinical phenotypes of pneumococcal lung infections, i.e., localized pneumococcal pneumonia or invasive pneumococcal disease.Daptomycin is a novel, transmembrane pore-forming 13-amino-acid compound that is produced by Streptomyces roseosporus (21). It belongs to the group of cyclic lipopeptides (for a review, see reference 25), which have particular efficacy against Gram-positive bacterial pathogens, whereas Gram-negative bacterial pathogens are not affected, probably due to their outer membrane (28). Daptomycin inserts into the bacterial cell membrane in a calcium-dependent manner and causes oligomerization of the daptomycin peptide into transmembrane pores, thereby triggering membrane leakage, the release of intracellular ions, and rapid cell death (28, 29). Daptomycin has potent bactericidal activity against a wide range of Gram-positive bacteria and antibiotic-resistant Gram-positive pathogens, such as vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate S. aureus, and penicillin-resistant Streptococcus pneumoniae, for which there are few therapeutic alternatives (4, 27, 30). Importantly, the spontaneous bacterial acquisition of daptomycin resistance has rarely been observed (11, 27, 31). Until now, daptomycin has been approved for use for the treatment of complicated skin and soft tissue or skin structure infections caused by Gram-positive bacteria and for the treatment of endocarditis and staphylococcal sepsis (10). However, in a mouse model of S. pneumoniae or MRSA pneumonia, daptomycin failed to significantly purge bacterial loads at 24 h after infection, although the findings of time-response and survival studies were not reported (26). In contrast, daptomycin was found to be effective in a rat model of hematogenous pneumonia caused by S. aureus (26). Of note, in a phase III clinical trial for the treatment of patients with community-acquired pneumonia, daptomycin (applied at a dose of 4 mg/kg of body weight) failed to achieve superiority over ceftriaxone (efficacies, 79 and 87%, respectively) (23). Thus, the currently available experimental and clinical data for mice and humans suggest that daptomycin has little efficacy against Gram-positive bacterial infections of the lung, possibly due to interactions of the compound with pulmonary surfactant components (26), and is therefore currently not recommended for use for the monotherapy of pneumonia.To the best of our knowledge, no detailed experimental data are available from investigations of the efficacy of daptomycin in defined mouse models of S. pneumoniae-induced lung infections either causing pneumonia in the absence of sepsis or causing fatal invasive pneumococcal disease. Therefore, in the current study, we made use of self-glowing, bioluminescent strains of S. pneumoniae with different virulence profiles to determine the efficacy of daptomycin compared to that of ceftriaxone in two models: a model of pneumococcal pneumonia caused by a less virulent serotype 19 S. pneumoniae strain and a model of septic pneumococcal disease caused by a highly invasive serotype 2 S. pneumoniae strain.     

Inhibitory and Bactericidal Activities of Daptomycin,Vancomycin, and Teicoplanin against Methicillin-Resistant Staphylococcus aureus Isolates Collected from 1985 to 2007

The inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against a collection of 479 methicillin-resistant Staphylococcus aureus isolates were assessed. The isolates were collected from U.S. and European hospitals from 1985 to 2007 and were primarily from blood and abscess cultures. The MICs and minimum bactericidal concentrations (MBCs) of the three agents were determined, and the MBC/MIC ratios were calculated to determine the presence or absence of tolerance. Tolerance was defined as an MBC/MIC ratio of ≥32 or an MBC/MIC ratio of ≥16 when the MBC was greater than or equal to the breakpoint for resistance. Tolerance to vancomycin and teicoplanin was observed in 6.1% and 18.8% of the strains, respectively. Tolerance to daptomycin was not observed.Although vancomycin and teicoplanin are the standard therapies for staphylococcal bacteremia, tolerance to vancomycin and teicoplanin has been demonstrated in both coagulase-negative staphylococci and Staphylococcus aureus as well as in various Streptococcus species (2, 3, 7, 10, 13, 15, 20, 21, 23, 25). Daptomycin, a lipopeptide antibiotic, has been demonstrated to have rapid bactericidal activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and tolerance to this drug has not been demonstrated (2, 9, 10, 19, 21, 24, 26, 28).The issue of antibiotic tolerance is a complicated one. Some studies have suggested that infections caused by tolerant strains may be more difficult to treat, especially when they cause complicated infections such as endocarditis, meningitis, or osteomyelitis or cause infections in immunocompromised patients (7, 8, 14, 15, 16, 18, 20, 22, 23, 25). Other investigators'' expert analyses do not agree that there is proof of a correlation between tolerant strains and treatment failures or that bactericidal activity is required for the treatment of serious MRSA infections (17, 25, 26, 27, 28). Controversy concerning the appropriate methods for the determination of tolerance in clinical isolates and in the practicality of testing isolates for tolerance in the clinical laboratory also exists.This study looked at MRSA isolates obtained primarily from blood and abscess cultures collected between 1985 and 2007. The main purpose of the study was to determine the in vitro inhibitory and bactericidal activities and the level of tolerance to the three drugs observed by standardized MIC and minimum bactericidal concentration (MBC) tests (4, 5, 19).(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

Impact of sarA on Daptomycin Susceptibility of Staphylococcus aureus Biofilms In Vivo

We used a murine model of catheter-associated biofilm formation to determine whether the mutation of the staphylococcal accessory regulator (sarA) has an impact on the susceptibility of established Staphylococcus aureus biofilms to treatment with daptomycin in vivo. The experiments were done with two clinical isolates, one of which (UAMS-1) was obtained from the bone of a patient suffering from osteomyelitis, while the other (UAMS-1625) is an isolate of the USA300 clonal lineage of community-acquired methicillin (meticillin)-resistant S. aureus. UAMS-1625 had a reduced capacity to form a biofilm in vivo compared to that of UAMS-1 (P = 0.0015), but in both cases the mutation of sarA limited biofilm formation compared to that of the corresponding parent strain (P ≤ 0.001). The mutation of sarA did not affect the daptomycin MIC for either strain, but it did result in increased susceptibility in vivo in the context of an established biofilm. Specifically, daptomycin treatment resulted in the clearance of detectable bacteria from <10% of the catheters colonized with the parent strains, while treatment with an equivalent daptomycin concentration resulted in the clearance of 46.4% of the catheters colonized with the UAMS-1 sarA mutant and 69.1% of the catheters colonized with the UAMS-1625 sarA mutant. In the absence of daptomycin treatment, mice with catheters colonized with the UAMS-1625 parent strain also developed skin lesions in the region adjacent to the implanted catheter. No such lesions were observed in any other experimental group, including untreated mice containing catheters colonized with the UAMS-1625 sarA mutant.A primary concern with all bacterial pathogens is the continued emergence of antibiotic-resistant strains. This is perhaps most apparent in Staphylococcus aureus, as evidenced by the current predominance of methicillin (meticillin)-resistant strains even among community-acquired isolates (27). However, many S. aureus infections are recalcitrant to antimicrobial therapy even in the absence of acquired resistance (14). One factor that contributes to this recalcitrance is the formation of a bacterial biofilm on host tissues and indwelling medical devices (20, 33). This biofilm not only limits the efficacy of antimicrobial therapy but also provides some level of intrinsic resistance to host defenses (12, 32). For this reason, the effective treatment of biofilm-associated S. aureus infections often requires surgical intervention to remove infected tissues and/or indwelling devices (6, 19).The growth of bacteria in a biofilm is a lifestyle option rather than a necessity, and in this context it is unlikely that any intervention strategy targeting biofilm formation per se would be therapeutically effective in and of itself. Nevertheless, such strategies have the potential to limit the intrinsic resistance associated with the biofilm mode of growth and thereby enhance the efficacy of conventional antimicrobial therapy. The studies described in this report were aimed at testing this hypothesis. We placed a specific emphasis on daptomycin based on reports concluding that it retains activity against slowly or even nongrowing bacteria (21) and has greater activity than other antibiotics in the context of an established biofilm (18, 25, 42).Biofilm formation in S. aureus is a complex process that is influenced by numerous genes. However, in many cases, the impact of individual genes is strain dependent (10, 23). For this reason, we chose to focus our efforts on the staphylococcal accessory regulator (sarA), the mutation of which has been shown to limit biofilm formation both in vivo and in vitro not only in S. aureus but also in S. epidermidis (1, 11, 17, 28, 34, 35, 37, 39). To avoid any bias associated with strain choice, we included two genetically distinct S. aureus clinical isolates and their isogenic sarA mutants. One of these (UAMS-1) is a well-characterized, biofilm-positive osteomyelitis isolate (2, 8, 30), while the other (UAMS-1625) is a methicillin-resistant S. aureus (MRSA) isolate of the USA300 clonal lineage that was isolated from a patient with a fatal brain abscess (29). The experiments employed three different biofilm models, with a primary emphasis in the context of daptomycin susceptibility on an in vivo, murine model of catheter-associated biofilm formation (8, 26). The results confirmed that the mutation of sarA limited biofilm formation in both strains and that this limitation was correlated with increased daptomycin susceptibility in vivo in the specific context of an established biofilm.     

Comparison of Tigecycline and Vancomycin for Treatment of Experimental Foreign-Body Infection Due to Methicillin-Resistant Staphylococcus aureus

Twice-daily 7-day regimens of tigecycline (7 mg/kg) and vancomycin (50 mg/kg) were compared in a rat tissue cage model of chronic foreign-body infection due to methicillin (meticillin)-resistant Staphylococcus aureus strain MRGR3. Subcutaneously administered tigecycline reached levels in tissue cage fluid that were nearly equivalent or slightly superior to the antibiotic MIC (0.5 μg/ml) for strain MRGR3. After 7 days, equivalent, significant reductions in bacterial counts were recorded for tigecycline-treated and vancomycin-treated rats, compared with those for untreated animals.Antimicrobial therapy for foreign-body infections due to Staphylococcus aureus is challenging (38), in particular for multidrug-resistant hospital-associated and community-acquired isolates of methicillin (meticillin)-resistant S. aureus (MRSA) (3, 12, 15, 16). Tigecycline is a novel injectable glycylcycline broad-spectrum antibiotic that demonstrates excellent in vitro and in vivo activity against MRSA and other multiresistant organisms (9, 11, 22, 28, 32) and can overcome both major tetracycline resistance mechanisms, namely ribosomal protection (10, 23) and efflux (4, 27). Tigecycline has shown good activity in various animal models of serious MRSA infections (21, 39, 40), as well as against biofilm-embedded bacteria (14, 26).We previously used a rat tissue cage model of S. aureus chronic foreign-body infections for evaluating a number of antimicrobial agents, namely vancomycin (17), teicoplanin (31), imipenem (30), ceftobiprole (37), daptomycin (29, 35), and several fluoroquinolones (2, 17, 36). This study reports the activity of tigecycline compared to that of the reference anti-MRSA agent vancomycin in a tissue cage model of MRSA chronic foreign-body infection.(This study was presented in part at the 18th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, April 2008.)MRSA strain MRGR3, whose properties were previously described (2, 5, 17, 29-31, 36, 37), was used for in vitro and in vivo studies. Strain MRGR3 is resistant to methicillin, gentamicin, erythromycin, tetracycline, and chloramphenicol (17).MICs of freshly prepared (1, 13, 25) tigecycline (Wyeth Research, Collegeville, PA) or vancomycin (Vancocin; Teva Pharma AG, Switzerland) for MRSA strain MRGR3 or quality control S. aureus ATCC 29213 were determined by broth macrodilution in cation-adjusted Mueller-Hinton broth (CAMHB), according to Clinical and Laboratory Standards Institute guidelines (7).The animal protocol used for evaluating the in vivo activities of tigecycline and vancomycin was previously described in detail (17, 37) and approved by the Ethics Committee of the Faculty of Medicine, University of Geneva, and the Veterinary Office of the State of Geneva. Three weeks after subcutaneous implantation of four tissue cages per animal in anesthetized Wistar rats (37), tissue cage fluids were checked for sterility (17).Pilot pharmacokinetic studies were performed using groups of noninfected rats to find an adequate dosing regimen of tigecycline for therapy of tissue cage infections as described previously (37). Tigecycline levels in cage fluids (and blood) were estimated by a microbiological assay (21), with a detection limit of 0.25 μg/ml. To account for protein binding, all plasma or tissue cage fluid samples were diluted with 1 volume of phosphate-buffered saline and assayed in duplicate, with reference to duplicate standard concentrations (0.25 to 8 μg/ml) of tigecycline, in phosphate-buffered saline supplemented with 50% plasma or pooled tissue cage fluids, respectively.Each tissue cage was chronically infected by inoculating 5 × 10⁵ CFU of log-phase MRGR3 (37). Two weeks later, all rats whose cage fluids contained ≥10⁵ CFU/ml received twice-daily doses (by the subcutaneous route for 7 days) of tigecycline (7 mg/kg), vancomycin (50 mg/kg), or no antibiotic (control group). Differences in CFU counts of cage fluid quantitative cultures, performed at day 1 (before treatment) and day 8 (12 h after the last injection of either tigecycline or vancomycin), were expressed as the change in number of log₁₀ CFU/ml (37) and evaluated by one-way analysis of variance and post-analysis of variance pairwise comparisons between individual groups via the Tukey HSD test (http://faculty.vassar.edu/lowry/VassarStats.html), using P values of <0.05 with two-tailed significance levels.Tigecycline resistance was screened by plating 10-fold-diluted cage fluids (100 μl) onto MH agar supplemented with 2 μg/ml tigecycline. No single colony grew on tigecycline-supplemented plates inoculated with 10⁸ CFU of in vitro-grown cultures of strain MRGR3.The MIC of tigecycline in CAMHB for MRSA strain MRGR3 was 0.5 μg/ml, namely at the upper limit of susceptibility breakpoints (7), and was unaffected by supplementation of CAMHB with 50% tissue cage fluid (data not shown). Since tigecycline did not produce a 3-log₁₀ reduction in the number of MRGR3 CFU/ml, it was not considered bactericidal. Nevertheless, supra-MIC levels (1, 2, and 4 μg/ml) of tigecycline produced a 2- to 3-log₁₀ decrease in the number of MRGR3 CFU/ml at 24 h. The vancomycin MIC and minimal bactericidal concentration for strain MRGR3 were 1 and 2 μg/ml, respectively (17).Average tigecycline levels, scored for tissue cage fluids (n = 6) from 0 to 12 h after subcutaneous administration, remained quite constant over time, showing ≤3-fold variations between results at different time points and moderate animal-to-animal differences (Fig. ​(Fig.1).1). A 7-mg/kg twice-daily regimen yielded cage fluid levels of 0.39 to 0.70 μg/ml tigecycline at day 4 and 0.33 to 1.01 μg/ml at day 7, such results thus being nearly equivalent or slightly superior to the antibiotic MIC for MRGR3. Tigecycline plasma levels at 2 h on day 4 were 1.87 ± 0.66 μg/ml, in agreement with other reports (8, 21). A 14-mg/kg twice-daily regimen led to plasma and tissue cage fluid tigecycline levels ca. twofold higher than the 7-mg/kg regimen (Fig. ​(Fig.1).1). Average peak and trough cage fluid levels of vancomycin were previously determined (17) as 12 and 2 μg/ml at 4 and 12 h, respectively.Open in a separate windowFIG. 1.Pharmacokinetic levels of tigecycline in tissue cage fluids of rats on day 4 (open symbols) or day 7 (closed symbols) of therapy every 12 h with 7 mg/kg (○) or 14 mg/kg (▵) of tigecycline. Each value is the mean result of six determinations.At day 1, mean bacterial counts for MRGR3-infected cages were not significantly different (P = 0.65) in controls (6.85 ± 0.19 log₁₀ CFU/ml; n = 28), tigecycline-treated rats (6.92 ± 0.13 log₁₀ CFU/ml; n = 29), or vancomycin-treated rats (6.70 ± 0.18 log₁₀ CFU/ml; n = 27). At day 8, significant (P < 0.01 versus controls) reductions were recorded in bacterial counts in cage fluids of both tigecycline-treated (−0.62 ± 0.17 CFU/ml; n = 29) and vancomycin-treated (−0.76 ± 0.18 log₁₀ CFU/ml; n = 27) rats, whereas the bacterial counts for controls slightly increased (+0.18 ± 0.19 log₁₀ CFU/ml; n = 28) (Fig. ​(Fig.2).2). The reductions in CFU counts for vancomycin-treated and tigecycline-treated rats were not significantly different. Finally, no MRGR3 isolate showing increased tigecycline MIC was observed in any posttherapy cage fluid sample (n = 29). The lack of emergence of MRGR3 derivates with diminished susceptibility to tigecycline is consistent with the difficulty in selecting laboratory-derived, tigecycline-resistant mutants of S. aureus (18), and it contrasts with the emergence of resistant subpopulations during low-dose daptomycin therapy of S. aureus-infected tissue cages (35).Open in a separate windowFIG. 2.Decrease in viable counts of MRSA MRGR3 in tissue cage fluids of rats treated for 7 days with tigecycline or vancomycin.Several studies performed with the rat tissue cage model demonstrated the low initial in vivo response of foreign-body-associated chronic MRSA infections (2, 5, 6, 17, 20, 29-31, 35-37). A much greater reduction of viable MRSA counts in cage fluids requires longer periods of antibiotic therapy (5), as found in clinical situations with foreign-body infections (38). Major pharmacokinetic properties of tigecycline, observed in human and animal studies, are very low plasma levels, long half-lives, and high volumes of distribution indicating extensive tigecycline distribution into the tissues (8, 11, 19, 28, 32, 40). In line with previous observations that showed a requirement for active, preferentially bactericidal, antibiotic levels for obtaining significant reductions of CFU counts in MRSA-infected cage fluids (29, 37), we selected for therapy a twice-daily 7-mg/kg regimen yielding cage fluid tigecycline levels above the MIC for strain MRGR3 for >50% of the dosing interval (32, 33), while minimizing the occurrence of side effects previously observed with higher-dose regimens (39). Our regimen is similar to those required for activity in other animal models of hard-to-treat S. aureus infections, such as endocarditis or osteomyelitis (21, 39), although its relevance to human therapy is not fully defined (32). In addition, the incomplete in vitro killing activity of tigecycline, namely a <3-log₁₀ reduction in number of MRGR3 CFU at 24 h, prevents a pharmacodynamic analysis of tigecycline in vivo activity more detailed than those of previously evaluated bactericidal antibiotics in MRSA-infected cages (29, 37). We can also speculate that other properties of tigecycline, namely its in vivo activity against intracellular, slowly growing, or biofilm-forming bacteria, might significantly contribute to tigecycline activity in MRSA-infected cages (34). Indeed, high intracellular levels of tigecycline were shown to accumulate in human polymorphonuclear neutrophils and prevent growth of phagocytized bacteria (24). Further studies are needed to elucidate the mechanisms of tigecycline activity against hard-to-treat MRSA infections.     

Rapamycin Protects Mice from Staphylococcal Enterotoxin B-Induced Toxic Shock and Blocks Cytokine Release In Vitro and In Vivo

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-2, gamma interferon (IFN-γ), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.Staphylococcal exotoxins are among the most common etiological agents that cause toxic shock syndrome (28-30, 38, 44). The disease is characterized by fever, hypotension, desquamation of skin, and dysfunction of multiple organ systems (8, 38, 41). These toxins bind directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and subsequently stimulate T cells expressing specific Vβ elements on T-cell receptors (9, 15, 24, 29, 35, 42). Staphylococcal enterotoxin B (SEB) and the distantly related toxic shock syndrome toxin 1 are also called superantigens because they induce massive proliferation of T cells (29). In vitro and in vivo studies show that these superantigens induce high levels of various proinflammatory cytokines, and these potent mediators cause lethal shock in animal models (1, 6, 22, 27, 37, 39, 45, 51, 55). SEB also causes food poisoning (4, 21, 52) and is a potential bioterrorism threat agent, as humans are extremely sensitive to this superantigen, especially by inhalation (28). There is currently no effective therapeutic treatment for SEB-induced shock except for the use of intravenous immunoglobulins (11). Various in vitro experiments identified inhibitors to counteract the biological effects of SEB, only some of which were successful in ameliorating SEB-induced shock in experimental models (1, 25-27, 51).Rapamycin is a relatively new FDA-approved drug used to prevent graft rejection in renal transplantation, as it shows less nephrotoxicity than do calcineurin inhibitors (14, 40, 43, 48). Recent studies reveal other uses in animal models of cancer (23, 34), diabetic nephropathy (36), bleomycin-induced pulmonary fibrosis (31), liver fibrosis (5), and tuberous sclerosis (32). Rapamycin binds intracellularly to FK506-binding proteins, specifically FKBP12; the rapamycin-FKBP12 complex then binds to a distinct molecular target called mammalian target of rapamycin (mTOR) (reviewed in reference 48). Rapamycin inhibits mTOR activity, prevents cyclin-dependent kinase activation, and affects G₁-to-S-phase transition (16, 48). Other studies identified mTOR as the conserved serine-threonine kinase for sensing cellular stress, and rapamycin promotes anabolic cellular processes in response to stress signals (20, 47, 50, 54). The mTOR pathway regulates myogenesis (13), cell cycle arrest (20), adipocyte differentiation (3), and insulin signaling (47, 50). The immunological effects of rapamycin include regulation of T-cell activation (48); differentiation, expansion, and preservation of regulatory T cells (2, 10, 19, 46); downregulation of dendritic cells (12, 53); and granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced neutrophil migration (17). Rapamycin impairs dendritic cell maturation and function by inhibiting the expression of adhesion molecule ICAM-1 (12, 53). Thus, rapamycin has a broad spectrum of effects and interferes with the activation of multiple cell types of the immune system.Based on the potent immunosuppressive effects of rapamycin, we investigated the therapeutic impact of rapamycin on SEB-mediated toxic shock. The therapeutic efficacy of rapamycin in SEB-induced toxic shock was investigated by using a lethal murine model with intranasal delivery of SEB (22). This “double-hit” murine model relies on two low doses of SEB without the use of sensitizing agents such as lipopolysaccharide (LPS) or galactosamine to induce lethal shock (6, 27, 33, 37, 45). In this “SEB-only” toxic shock model, SEB was administered intranasally (i.n.) and another dose of SEB was strategically given intraperitoneally (i.p.) 2 h later to induce systemic cytokine release and pulmonary inflammation with lethality as an endpoint. We examined the effect of rapamycin on proinflammatory cytokines and chemokines induced by SEB in vitro using human peripheral blood mononuclear cells (PBMC) as a first step to test its immunological effects on SEB activation.     

Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a K_m of 1,750 ± 210 μM. Introduction of 1.0 or 10 nM maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a K_i of 3.0 ± 0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.Human cytomegalovirus (HCMV), a betaherpesvirus, is a widespread pathogen infecting between 40 and 80% of the population. Although immunocompetent individuals rarely manifest any symptoms, HCMV can result in severe disease, such as interstitial pneumonia, mental retardation, and hearing loss in immunocompromised and immunologically immature individuals (26, 50). Currently, therapeutic agents such as ganciclovir (GCV), foscarnet (PFA), cidofovir, and fomivirsen are used for the treatment or prophylaxis of HCMV disease (1, 7, 20, 22, 39, 50). However, long-term therapy is generally required due to recurrence of infection upon cessation of therapy, leading to the development of drug resistance and severe adverse effects (4, 13, 16, 23, 33, 41). With the increased use of immunosuppression for cancer chemotherapy and organ transplantation, there is an increasing need for more effective and less toxic drugs to treat HCMV.We have demonstrated previously that cyclopropavir (CPV) (Fig. ​(Fig.1),1), a bis-hydroxymethyl methylenecyclopropane guanosine nucleoside analog, is approximately 10-fold more active in vitro (50% effective concentration [EC₅₀] of 0.46 μM) than GCV (EC₅₀ of 4.1 μM) (51, 52). In addition, CPV is active against several HCMV mutants that are resistant to GCV or PFA (29). Further experimentation in vivo with CPV demonstrated 2- to 5-log₁₀ reductions in titers of murine cytomegalovirus (MCMV), resulting in reduced mortality in severe combined immunodeficient (SCID) mice, and reduced viral replication in human fetal tissue implanted in SCID mice infected with HCMV (28).Open in a separate windowFIG. 1.Structures of cyclopropavir (CPV) and ganciclovir (GCV).Previous studies have established that the mechanism of action of CPV involves inhibition of viral DNA synthesis (29). Furthermore, the activity of CPV was reduced approximately 20-fold against an HCMV UL97 deletion mutant (29), thereby indicating the importance of this gene product in the action of CPV. Taken together, these results suggest that the mechanism of action of CPV resembles that of GCV, in which the drug is first phosphorylated by viral pUL97, a protein kinase that can phosphorylate nucleoside analogs (32, 36, 46, 47). Upon further phosphorylation by endogenous cellular kinases, the triphosphate of GCV inhibits HCMV DNA polymerase, resulting in inhibition of viral replication (5, 9, 19, 34, 37, 38, 45). Therefore, we investigated the possibility that viral pUL97 also phosphorylates CPV to its monophosphate (CPV-MP). In addition, our previous results demonstrating the enantioselective phosphorylation of (+)-CPV-MP (31) allowed us to determine whether CPV phosphorylation by pUL97 is stereoselective.     

Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg²⁺-four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This “dione resistance-determining region” overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg²⁺ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg²⁺ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg²⁺-dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.The established and emerging resistance of gram-positive pathogens to fluoroquinolones, macrolides, and beta-lactams has underlined the need for new antimicrobial agents (2, 6, 28, 32, 35, 46, 52). Recent work has shown that quinazolinediones constitute a promising new class of antibacterial inhibitors of DNA gyrase and topoisomerase IV (topo IV) that share structural similarity with fluoroquinolones (14, 50, 51). Among this group of inhibitors, the 3-aminoquinazolinedione PD 0305790 (3-amino-7-[(R)-3-[(S)-1-aminoethyl]-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-8-methyl-1H-quinazoline-2,4-dione) (Fig. ​(Fig.1)1) has particularly potent activity against susceptible and drug-resistant gram-positive species, especially Streptococcus pneumoniae (23). The drug differs most notably from quinolones, e.g., ciprofloxacin, in having the 3-aminoquinazoline 2,4-dione system in place of the 3-carboxy-substituted 4-quinolone ring (Fig. ​(Fig.1).1). Surprisingly, these subtle differences in drug structure confer activity against quinolone-resistant strains (23).Open in a separate windowFIG. 1.Structures of PD 0305970 and ciprofloxacin.Initial work with Escherichia coli gyrase has shown that, like quinolones, 3-aminodiones, such as PD 0305790, stabilize an enzyme “cleavage complex” on DNA (50). This complex is thought to reflect the trapping of an intermediate in the topoisomerase reaction cycle in which the gyrase GyrA₂GyrB₂ (or the topo IV ParC₂ParE₂) complex promotes the ATP-dependent passage of one DNA duplex through a transient double-strand break in a second DNA segment (8, 30). DNA scission in the cleavage complex is Mg²⁺-dependent and involves the covalent phosphotyrosyl linkage of the two gyrase GyrA subunits (ParC in topo IV) to the 5′ phosphate ends of an enzyme-bridged double-stranded DNA break (5, 7-9, 16). Until recently, there was no high-resolution structure of a gyrase or topo IV cleavage complex, although information was available for some individual domains (3, 7-9, 22, 24, 31, 47, 55) and for yeast topo II (4, 12, 15, 29). By analogy with the yeast enzyme, it was reasoned that the N-terminal breakage-reunion domain of GyrA (ParC) (carrying the catalytic tyrosine) and the C-terminal Toprim metal binding domain of the GyrB (ParE) subunit come together to mediate DNA cleavage on each strand (7-9, 12, 13, 18, 34). Indeed, we have recently shown that the S. pneumoniae ParC breakage-reunion and ParE Toprim domains are sufficient to form a cleavage complex stabilized by moxifloxacin and clinafloxacin (25). Moreover, we have solved the crystal structures of these complexes, revealing two quinolone drugs intercalated at the highly bent DNA gate (25). It is known that different quinolones can preferentially target gyrase or topo IV in Streptococcus pneumoniae (1, 17, 20, 33, 38, 41), and these structures provide new insight into the nature of the cleavage complex.Biochemical evidence points to the participation of two Mg²⁺ ions in DNA strand scission (11, 34, 49, 54). One Mg²⁺ ion is suggested to bind the 3′ bridging oxygen of the scissile DNA phosphodiester bond, thereby promoting DNA cleavage by stabilizing the leaving 3′-OH group (11, 34). The function of the putative second ion is unclear but may involve potentiation of the catalytic tyrosyl OH of GyrA (ParC), facilitating nucleophilic attack on the DNA phosphate (11, 34). Quinolones chelate free Mg²⁺, but it is presently unclear if the drugs interact similarly with enzyme-bound Mg²⁺ (reviewed in reference 27). Quinolone resistance mutations occur in the so-called “quinolone resistance-determining regions” (QRDRs) located in the GyrA/ParC breakage reunion domain (10, 36, 57) and in the GyrB (ParE) Toprim fold (58).To date, efforts with 3-hydroxy- and 3-amino-quinazolinediones have established structure-activity relationships in vivo and inhibition of E. coli DNA gyrase in vitro (14, 19, 23, 50, 51). A preliminary report has identified three mutations in PD 0305970-resistant pneumococci that map in GyrB and ParE (23). However, little is known about how 3-aminodiones stabilize the topoisomerase cleavage complex vis-à-vis quinolones and how they overcome resistance. To initiate studies of this area, we have used a variety of genetic approaches in concert with functional enzyme and biochemical assays to examine the interaction of PD 0305970 with its topoisomerase targets in S. pneumoniae.     

Evaluation of a DNA Microarray,the Check-Points ESBL/KPC Array,for Rapid Detection of TEM,SHV, and CTX-M Extended-Spectrum β-Lactamases and KPC Carbapenemases

Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ß-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ß-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ß-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemase (KPC) are reported increasingly in Gram-negative bacilli (GNB) (5, 6, 17, 18, 25, 30). KPC producers, initially identified in the United States, are now reported worldwide, and illnesses caused by them have become endemic in some regions (25). Isolates expressing KPC enzymes may be reported as susceptible to carbapenems due to heterogeneous and variable levels of expression of β-lactam resistance.The vast majority of ESBLs belong to the TEM, SHV, and CTX-M types (5, 18, 28). These ß-lactamases are encoded by plasmid-located genes and therefore can very easily spread among Enterobacteriaceae (6, 14, 16). More than 160 TEM-type and 110 SHV-type ß-lactamases have been identified worldwide. Amino acid substitutions at many sites in TEM-1 ß-lactamases have been documented, but those at positions 104, 164, 238, and 240 most often lead to an ESBL phenotype (5, 28). As with TEM, SHV-type ESBLs have one or more amino acid substitutions located around the active site compared to SHV-1: substitutions at positions 238 and/or 240 are the most common and are associated with resistance to ceftazidime, cefotaxime, and aztreonam. Less commonly, an alteration at positions 146 or 179 provides ceftazidime resistance (28).Unlike TEM/SHV enzymes, all the CTX-M enzymes are ESBLs (6, 28). More than 80 CTX-M-variants, sharing 71 to 98% amino acid sequence identities, have now been described and are divided now into five groups (groups CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-8, and CTX-M-25) based on amino acid sequence identity (5).Detection of ESBLs is primarily based on phenotypic testing, such as evidencing a synergy image using the double-disk synergy test performed with expanded-spectrum cephalosporins (ESC) and ticarcillin-clavulanic acid disks (3, 10, 23). This test is not always obvious and is usually time-consuming since it requires subculturing or the use of cloxacillin-containing plates to inhibit the naturally occurring and plasmid-mediated cephalosporinases. Unambiguous identification of KPCs by phenotypic methods is relatively difficult (25). Over the last 20 years, alternative strategies aimed at replacing or complementing traditional phenotypic methods have been proposed. Standard PCR and gene sequencing is still the most widely used technique. Other molecular detection techniques for ESBLs and KPC genes have been proposed, but none have been really suited for routine detection (1, 4, 8, 9, 11, 13, 15, 19, 20, 22, 24, 26, 27, 29, 31, 39), since usually only one ESBL/KPC gene is detected at a time. Finally, the presence of narrow-spectrum variants of TEM and SHV types may complicate significantly the molecular detection of TEM/SHV-type ESBLs (28).Microarray technology has recently been developed for the typing of Salmonella isolates (37, 38). This technology has the potential to detect an almost unlimited number of genes within one reaction mixture. Here, a new commercial DNA-based test, the Check-Points ESBL/KPC array, aimed at identifying TEM-, SHV-, and CTX-M-type ESBLs as well as KPC-type carbapenemases, was evaluated by comparing its performance with that of standard PCR on well-characterized reference strains and on 40 ESBL producers isolated at the Bicetre hospital from January to March 2009.     

Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest,Disk Diffusion,and Agar Dilution Methods with RPMI and IsoSensitest Media

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC₅₀) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.Three echinocandin class drugs, anidulafungin, caspofungin, and micafungin, are licensed for the treatment of invasive candidiasis. They are among the preferred agents for invasive candidiasis, as a number of recent fungemia surveys have reported a considerable proportion of cases involving species with reduced susceptibility to fluconazole (3, 4, 24, 28, 31, 37, 44). Additionally, anidulafungin has been associated with an improved success rate, even in cases involving fluconazole-susceptible species (39). Following increased use, sporadic cases of failures associated with elevated MICs have been reported. In the majority of cases, these failures have been associated with mutations in two hot spot regions of FKS genes, which encode the target and major subunit of the 1,3-ß-d-glucan synthase complex (5, 7, 22, 25, 26, 33, 34). Consequently, close monitoring and robust susceptibility testing methods have become increasingly important.EUCAST and CLSI have developed standard methods based on broth dilution for the susceptibility testing of yeasts (9, 41). Methodological differences include glucose concentration, inoculum size, shape of microtiter wells (flat or round), and end-point reading (visual or spectrophotometric), but the methods are more alike than different and in general generate similar results (11, 42). Recently, CLSI proposed an S value of ≤2 μg/ml as a tentative susceptibility breakpoint for caspofungin, micafungin, and anidulafungin for Candida spp., taking into account analysis of mechanisms of resistance, an epidemiological MIC population distribution, parameters associated with success in pharmacodynamic models, and results of clinical efficacy studies (9, 38). As no significant differences in clinical response were noted among the various species, results for all species were merged, and a susceptibility breakpoint of 2 μg/ml was found to encompass the vast majority of isolates, while not bisecting the population of Candida parapsilosis. The crucial issue is whether current susceptibility testing methods and breakpoints clearly and reliably identify isolates with resistance mechanisms associated with treatment failures (5, 7, 8, 13, 14, 16, 18, 22, 25, 26, 33, 40). Not only have cases involving isolates classified as susceptible using the reference methods been shown to contain resistance mutations (5, 7, 13, 14, 22, 25), but also recent studies suggest that a breakpoint of an S value of ≤2 μg/ml may be too high for anidulafungin and micafungin, considering the 1,3-ß-d-glucan synthase kinetic inhibition data of wild-type and mutant enzymes from resistant strains (17, 18). Finally, we recently reported a resistant Candida albicans isolate that failed to be identified as resistant when the reference methodologies were used, while Etest, agar dilution, and disk diffusion methods correctly identified it (5).We therefore undertook a comparative study of the two references methods, a modified EUCAST microdilution method using IsoSensitest medium, agar dilution, and disk and Etest diffusion using RPMI-1640 as well as IsoSensitest medium to evaluate their ability to reliably discriminate between a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. The semisynthetic IsoSensitest medium was chosen as an alternative medium due to this medium having previously been shown to be appropriate for amphotericin B MIC testing (10).     

In Vivo Protection Provided by a Synthetic New Alpha-Galactosyl Ceramide Analog against Bacterial and Viral Infections in Murine Models

Alpha-galactosyl ceramide (α-GalCer) has been known to bind to the CD1d receptor on dendritic cells and activate invariant natural killer T (iNKT) cells, which subsequently secrete T-helper-cell 1 (Th1) and Th2 cytokines, which correlate with anti-infection activity and the prevention of autoimmune diseases, respectively. α-GalCer elicits the secretion of these two cytokines nonselectively, and thus, its effectiveness is limited by the opposing effects of the Th1 and Th2 cytokines. Reported here is the synthesis of a new α-GalCer analog (compound C34), based on the structure of CD1d, with a 4-(4-fluorophenoxy) phenyl undecanoyl modification of the N-acyl moiety of α-GalCer. Using several murine bacterial and viral infection models, we demonstrated that C34 has superior antibacterial and antiviral activities in comparison with those of several other Th1-selective glycolipids and that it is most effective by administering it to mice in a prophylactic manner before or shortly after infection.Natural killer T (NKT) cells contribute to a variety of immunological processes through the recognition of NKT cell receptors by lipid and glycolipid antigens presented by CD1d molecules (2, 35). CD1d molecules are major histocompatibility complex class I-like proteins and are expressed by most monocytes, macrophages, dendritic cells, and B cells as well as by nonlymphoid cells. CD1d presents glycolipid antigens to CD1d-restricted T cells (or NKT cells), which are implicated in the host innate defense system through the production of T-helper-cell 1 (Th1) and Th2 types of cytokines, such as gamma interferon (IFN-γ) and interleukin-4 (IL-4), respectively (32, 39). The production of Th1 cytokines is shown to correlate with the antitumor, antibacterial, and antiviral effects of glycolipids, while the ability to induce the Th2 cytokines is thought to correlate with the amelioration of certain autoimmune diseases, such as type 1 diabetes (9, 10, 28).Among the variety of ligands that bind to CD1d, the most well studied one is alpha-galactosyl ceramide (α-GalCer) (7, 17, 30), which is a synthetic, structurally optimized compound of the natural product lead identified from the marine sponge (Agelas mauritianus) as agelasphin-9b (24). The synthetic analog (2S,3S,4R)-1-O-(α-d-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol was made as KRN7000 at Kirin Brewery, Japan (23), and has generally been referred to as α-GalCer. Mice treated with α-GalCer were shown to be protected against a variety of infections (13, 15, 16, 22, 33). However, the effectiveness of α-GalCer is limited by the opposing effects of the Th1 and Th2 cytokines induced by this glycolipid. In order to develop selective Th1 or Th2 activators, many α-GalCer analogs were synthesized (1, 3, 5, 8, 12, 14, 21, 34, 36), and their immune-modulating activities were shown to be related to the affinity of binding to CD1d (8). Recent studies using a CD1d array established that the secretion of IFN-γ and IL-4 by NKT cells is determined by the binding of α-GalCer analogs to CD1d (21), and a higher affinity for CD1d would shift the cytokine release profile toward a stronger Th1 response (3, 21).This report described the synthesis of the 4-(4-fluorophenoxy)phenyl undecanoyl derivative of α-GalCer as a selective Th1 activator. This analog was designed on the basis of the structure of CD1d, where the N-acyl group of the glycolipid interacts most favorably with the lipid binding moiety of CD1d (36). Its efficacies for protection against both bacterial and viral infections were compared with those of α-GalCer and several other analogs using murine models of infection. Our study found that among the synthetic α-GalCer analogs tested, compound C34 is the most effective in offering protection against all of the bacterial and viral infection models tested, especially when it is given in a prophylactic manner or shortly after infection.     

Correlation between Vancomycin MIC Values and Those of Other Agents against Gram-Positive Bacteria among Patients with Bloodstream Infections Caused by Methicillin-Resistant Staphylococcus aureus

An increase in the distribution of vancomycin MIC values among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates has been noted. It is postulated that the shift in vancomycin MIC values may be associated with a concurrent rise in the MIC values of other anti-MRSA agents. Scant data are available on the correlation between vancomycin MIC values and the MIC values of other anti-MRSA agents. This study examined the correlation between vancomycin MIC values and the MIC values of daptomycin, linezolid, tigecycline, and teicoplanin among 120 patients with bloodstream infections caused by MRSA at a tertiary care hospital between January 2005 and May 2007. For each included patient, the MIC values of the antibiotics under study were determined by the Etest method and were separated into the following two categories: day 1 (index) and post-day 1 (subsequent). For subsequent isolates, the MIC values for each antibiotic from the post-day 1 terminal isolate were used. Among the index isolates, there was a significant correlation (P value, <0.01) between the MIC values for vancomycin and daptomycin and between the MIC values for vancomycin and teicoplanin. The MIC values for daptomycin were significantly correlated with linezolid, tigecycline, and teicoplanin MIC values. Among the 48 patients with subsequent isolates, vancomycin MIC values were significantly correlated with MIC values for daptomycin, linezolid, and teicoplanin (ρ value of ≥0.38 for all comparisons). This study documented an association between vancomycin MIC values and the MIC values of other anti-MRSA antibiotics among patients with bloodstream infections caused by MRSA primarily treated with vancomycin.An increase in the distribution of vancomycin MIC values among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates has been noted in several recent reports (3, 11, 14). This shift is a concern because a growing number of studies have shown that patients with infections caused by MRSA with vancomycin MIC values at the higher end of the Clinical and Laboratory Standards Institute (CLSI) and Food and Drug Administration (FDA) susceptibility range are less responsive to vancomycin (3, 4, 5, 7, 12, 13). Clinicians must now consider using alternative therapies for such patients. However, it is unclear whether the observed shift in the distribution of vancomycin MIC values for MRSA is associated with similar shifts in the distribution of MIC values of other anti-MRSA agents.It is postulated that the shift in vancomycin MIC values may be associated with a concurrent rise in the MIC values of other anti-MRSA agents. Scant data are available on the correlation between vancomycin MIC values and the MIC values of other anti-MRSA agents. To date, analyses have been limited to the index MRSA isolate and have primarily focused on the correlation between vancomycin and daptomycin (6, 10, 11).This study examined the correlation between vancomycin MIC values and the MIC values of daptomycin, linezolid, tigecycline, and teicoplanin among patients with bloodstream infections caused by MRSA. Given the recent reports describing the emergence of resistance during therapy, our analyses included both the index (day 1) and subsequent (post-day 1) isolates.     

Increased Temperature Enhances the Antimicrobial Effects of Daptomycin,Vancomycin, Tigecycline,Fosfomycin, and Cefamandole on Staphylococcal Biofilms

Implant-related infections are serious complications of trauma and orthopedic surgery and are most difficult to treat. The bacterial biofilms of 34 clinical Staphylococcus sp. isolates (Staphylococcus aureus, n = 14; coagulase-negative staphylococci, n = 19) were incubated with daptomycin (DAP; 5, 25, or 100 mg/liter), vancomycin (VAN; 5, 25, or 100 mg/liter), tigecycline (TGC; 1, 5, or 25 mg/liter), fosfomycin (FOM; 100, 250, or 1,000 mg/liter), and cefamandole (FAM; 50, 100, or 500 mg/liter) for 24 h at three different ambient temperatures: 35°C, 40°C, and 45°C. To quantify the reduction of the biomass, the optical density ratio (ODr) of stained biofilms and the number of growing bacteria were determined. Increasing the temperature to 45°C or to 40°C during incubation with FAM, FOM, TGC, VAN, or DAP led to a significant but differential reduction of the thickness of the staphylococcal biofilms compared to that at 35°C (P < 0.05). Growth reduction was enhanced for DAP at 100 mg/liter at 35°C, 40°C, and 45°C (log count reductions, 4, 3.6, and 3.3, respectively; P < 0.05). A growth reduction by 2 log counts was detected for FAM at a concentration of 500 mg/liter at 40°C and 45°C (P = 0.01). FOM at 1,000 mg/liter reduced the bacterial growth by 1.2 log counts (not significant). The antibacterial activity of antimicrobial agents is significantly but differentially enhanced by increasing the ambient temperature and using high concentrations. Adjuvant hyperthermia may be of value in the treatment of biofilm-associated implant-related infections.Implant-related infections are severe complications of trauma and orthopedic surgery that frequently require long-term antimicrobial treatment, supportive management, and multiple additional surgical procedures (53). Staphylococcus aureus and coagulase-negative staphylococci (CoNS), primarily Staphylococcus epidermidis, are the most common organisms associated with implant-related infections after trauma and orthopedic surgery (13).Bacterial biofilms develop on the surfaces of the implants (12, 13, 30, 41). The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix that adheres to an inert or living surface. The bacteria embedded in the biofilm are quasiprotected by this self-made polymeric matrix; thus, resistance to antimicrobials is increased such that the concentrations needed to kill the biofilm bacteria are 500 to 5,000 times higher than the levels needed to kill planktonic bacteria (8, 12, 13). Although removal of the colonized foreign material may be the most effective means to treat biofilm-associated infections (14), the implant must sometimes be retained because of technical or physiological complications. In this case, any hope for cure or at least the stability of the patient lies in antimicrobial treatment alone.Antimicrobial agents for the treatment of staphylococcal infections include beta-lactam antibiotics and, in the case of methicillin resistance, vancomycin (VAN), fosfomycin (FOM), tigecycline (TGC), or daptomycin (DAP) (32, 38). FOM is a small-molecule antibiotic with a wide antibacterial spectrum and excellent tissue penetration, representing an excellent alternative antimicrobial agent for the treatment of deep-seated infections (18, 27). Both TGC and DAP are highly active against Gram-positive cocci resistant to commonly used antibiotics, including methicillin-resistant staphylococci (18, 43). Antimicrobial agents may reduce biofilm and bacterial growth (35, 39). In a previous study, we demonstrated that the antibiotic concentrations achieved under normal physiological conditions did not decrease the growth of established staphylococcal biofilms (21). Bacterial biofilm growth was significantly reduced by the use of antimicrobials at excessive concentrations or when antimicrobials were used in combination with azithromycin (21, 37).Because the effects of antibiotics on established biofilms are unsatisfactory, other measures to reduce biofilm thickness and to kill biofilm bacteria may be helpful (37). Adjunctive therapy to enhance the activity of an antimicrobial to save an infected but unremovable implant or to improve the engraftment of a new implant after reimplantation may be a benefit for the patient (13, 16). Experimental measures taken to reduce bacterial biofilms included antibiotic combination therapy, particularly with rifampin; therapy with a combination of antibiotics and chemicals like EDTA or N-acytylcysteine; or therapy with a combination of antibiotics and physical measures, such as ultrasound or an electric current (7, 15, 39, 51). Low-frequency ultrasound was used to prevent uncontrolled heating and associated tissue damage (5, 6). For physical therapy, however, ultrasound of 1 MHz (unpulsed) had been described to have a beneficial effect on osteoarthritis (45). Further, application of heat has traditionally been used in physical therapy with various rates of success and minimal harm (9).Increasing the temperature induced the formation of thicker biofilms (40). Although the amount of heating was never measured in the ultrasound or electric current experiments, heating may have an influence on the effects of antibiotics on biofilms. Thus, we investigated the effects of stepwise increases in the ambient temperature together with antibiotic treatment on biofilm thickness and bacterial growth. Static biofilms of clinical isolates from orthopedic implant infections or preoperative skin isolates, including S. aureus and CoNS (S. epidermidis, S. lugdunensis, S. hominis, and S. capitis), were incubated at an ambient temperature of 35, 40, or 45°C for 24 h with DAP, VAN, FOM, TGC, or FAM. Three concentrations of each of the antimicrobial agents were investigated.(Some of the data presented here were presented as poster K2069 at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America 46th Annual Meeting, San Francisco, CA, 2009.)     

Development,Validation, and Routine Application of a High-Performance Liquid Chromatography Method Coupled with a Single Mass Detector for Quantification of Itraconazole,Voriconazole, and Posaconazole in Human Plasma

We have developed and validated a high-performance liquid chromatography method coupled with a mass detector to quantify itraconazole, voriconazole, and posaconazole using quinoxaline as the internal standard. The method involves protein precipitation with acetonitrile. Mean accuracy (percent deviation from the true value) and precision (relative standard deviation percentage) were less than 15%. Mean recovery was more than 80% for all drugs quantified. The lower limit of quantification was 0.031 μg/ml for itraconazole and posaconazole and 0.039 μg/ml for voriconazole. The calibration range tested was from 0.031 to 8 μg/ml for itraconazole and posaconazole and from 0.039 to 10 μg/ml for voriconazole.The incidence of mycoses has continued to increase over the past 2 decades, especially in immunocompromised patients. Notwithstanding the fact that in the last decades new antifungal agents have been approved, there is still a therapeutic need for azole compounds, such as itraconazole (ITC), posaconazole (PSC), and voriconazole (VRC), which inhibit 14a-demethylase, a key enzyme in the ergosterol biosynthesis of yeasts and molds (40).Antifungal prophylaxis, empirical therapy, and treatment of established fungal infections in the hematology patient population may be associated with significant toxicity or drug interactions, leading to subtherapeutic antifungal drug concentrations and poorer clinical outcomes (47). For example, a relationship between plasma concentrations and antifungal efficacy was shown for ITC (19), and the ratio between the area under the concentration-time curve (AUC) and MIC was identified to be predictive for the treatment efficacy of voriconazole and posaconazole, as well (1, 2). Antifungal therapeutic drug monitoring (TDM) could be an important tool in clinical practice if compliance is poor, the therapeutic window is narrow, or drug interactions and toxicity are common adverse effects. Therefore, quantification of drug in plasma samples is an important issue in clinical practice to improve efficacy and to decrease toxicity.Many methods to individually quantify ITZ (6, 7, 9, 17, 18, 26, 31-34, 37, 45, 46), PSC (8, 35, 39), and VRC (11, 20, 24, 25, 28, 29, 36, 38) in human plasma have been published. Only one method described the quantification of the three triazoles plus fluconazole, ITC metabolite, and ketokonazole in human plasma using a solid-phase extraction procedure.The aim of this study was to develop and validate a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method useful in routine TDM for quantitation of ITC, PSC, and VRC in human plasma using a protein precipitation extraction procedure and direct injection in an HPLC system.