Activity of a New Antipseudomonal Cephalosporin,CXA-101 (FR264205), against Carbapenem-Resistant and Multidrug-Resistant Pseudomonas aeruginosa Clinical Strains

The activity of the new cephalosporin CXA-101 (CXA), previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, was evaluated against a collection of 236 carbapenem-resistant P. aeruginosa isolates, including 165 different clonal types, from a Spanish multicenter (127-hospital) study. The MICs of CXA were compared to the susceptibility results for antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. The MIC of CXA in combination with tazobactam (4 and 8 μg/ml) was determined for strains with high CXA MICs. The presence of acquired β-lactamases was investigated by isoelectric focusing and PCR amplification followed by sequencing. Additional β-lactamase genes were identified by cloning and sequencing. The CXA MIC₅₀/MIC₉₀ for the complete collection of carbapenem-resistant P. aeruginosa isolates was 1/4 μg/ml, with 95.3% of the isolates showing an MIC of ≤8 μg/ml. Cross-resistance with any of the antibiotics tested was not observed; the MIC₅₀/MIC₉₀ of CXA-101 was still 1/4 when multidrug-resistant (MDR) strains (42% of all tested isolates) or AmpC-hyperproducing clones (53%) were analyzed. Almost all (10/11) of the strains showing a CXA MIC of >8 μg/ml produced a horizontally acquired β-lactamase, including the metallo-β-lactamase (MBL) VIM-2 (one strain), the extended-spectrum β-lactamase (ESBL) PER-1 (one strain), several extended-spectrum OXA enzymes (OXA-101 [one strain], OXA-17 [two strains], and a newly described OXA-2 derivative [W159R] designated OXA-144 [four strains]), and a new BEL variant (BEL-3) ESBL (one strain), as identified by cloning and sequencing. Synergy with tazobactam in these 11 strains was limited, although 8 μg/ml reduced the mean CXA MIC by 2-fold. CXA is highly active against carbapenem-resistant P. aeruginosa isolates, including MDR strains. Resistance was restricted to still-uncommon strains producing an acquired MBL or ESBL.The increasing prevalence of nosocomial infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa strains severely compromises the selection of appropriate antibacterial treatments and is therefore associated with significant morbidity and mortality (13, 20). The growing threat of antimicrobial resistance in P. aeruginosa results from the extraordinary capacity of this microorganism to develop resistance to almost any available antibiotic by the selection of mutations in chromosomal genes and from the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (or metallo-β-lactamases [MBLs]) or extended-spectrum β-lactamases (ESBLs) (14, 18). VIM and IMP MBLs and OXA and PER ESBLs have disseminated among P. aeruginosa strains from diverse geographic areas (6, 11, 16, 22, 29, 32, 39). These resistance genes are frequently carried in integrons, together with aminoglycoside resistance determinants that are often located in transferable elements such as plasmids or transposons (12, 24, 25, 26, 31). Noteworthy among the mutation-mediated resistance mechanisms are those leading to the repression or inactivation of the porin OprD, conferring resistance to carbapenems (5, 8, 23, 30, 41), or those leading to the hyperproduction of the chromosomal cephalosporinase AmpC, such as AmpD or PBP4 inactivation (10, 19), determining resistance to penicillins and cephalosporins. In addition, mutations leading to the upregulation of one of several efflux pumps encoded in the P. aeruginosa genome may confer resistance or reduced susceptibility to multiple agents, including almost all β-lactams, fluoroquinolones, and aminoglycosides (2, 17, 28). The accumulation of these chromosomal mutations can lead to the emergence of MDR (or even pan-antibiotic-resistant) strains which eventually may be responsible for outbreaks in the hospital setting (4, 9).Unfortunately, in the last 2 decades, little progress has occurred in developing novel antipseudomonal agents that can overcome MDR in P. aeruginosa. CXA-101, previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, is a new promising cephalosporin intended for the treatment of P. aeruginosa infections that appears to be stable against the most common resistance mechanisms driven by mutation in this species (35, 36). The objective of this study was to evaluate the activity of CXA-101 against a collection of well characterized carbapenem-resistant and MDR P. aeruginosa isolates obtained in a large multicenter study in Spain (8, 33). Additionally, this study also aimed to investigate the possible mechanisms of resistance to CXA-101.(This study was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2009.)     

Syndecan-Fc Hybrid Molecule as a Potent In Vitro Microbicidal Anti-HIV-1 Agent

In the absence of a vaccine, there is an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of human immunodeficiency virus type 1 (HIV-1). In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested the syndecan-Fc hybrid molecule for various in vitro microbicidal anti-HIV-1 properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and dendritic cells (DC); (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug resistant HIV-1 isolates, HIV-2, and simian immunodeficiency virus (SIV); (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells; (iv) it prevents HIV-1 transfer from dendritic cells to CD4⁺ T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it prevents herpes simplex virus infection. The heparan sulfate chains of the syndecan-Fc hybrid molecule are absolutely required for HIV-1 neutralization. Several lines of evidence suggest that the highly conserved Arg298 in the V3 region of gp120 serves as the locus for the syndecan-Fc hybrid molecule neutralization. In conclusion, this study suggests that the syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.The dominant cell surface heparan sulfate proteoglycans (HSPG) (25, 29, 30, 33) are syndecans, which are transmembrane receptors highly expressed on adherent cells (macrophages and epithelial and endothelial cells) but poorly expressed on suspension cells (T cells) (2, 3, 4, 10, 35). Their ectodomain bears three linear heparan sulfate (HS) chains, which are composed of a repetition of a sulfated disaccharide motif (1). The sulfation pattern of HSs dictates the ligand specificity of syndecans (1). HSPG, including syndecans, serve as receptors for human deficiency virus type-1 (HIV-1) (16), herpes simplex virus (HSV) (7), human papillomavirus (HPV) (13, 37), and human T-lymphotropic virus type 1 (HTLV-1) (19, 20). Pretreatment of target cells such as macrophages with heparinase, an enzyme that removes HS moieties from syndecans, significantly reduces HIV-1 infectivity (35). Although syndecans do not alleviate the requirement for CD4 and chemokine receptors for viral entry (35), these in cis attachment receptors amplify HIV-1 infection by promoting viral adsorption to the surface of permissive cells. Syndecans also serve as in trans receptors for HIV-1 (2, 16). HIV-1 binds syndecans richly expressed on the endothelium and remains infectious for a week, whereas cell-free virus loses its infectivity after a single day (2). Moreover, HIV-1 attached onto the endothelium via syndecans represents an in trans source of infection for circulating T cells (2). Primary HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates produced from peripheral blood mononuclear cells (PBMCs) exploit syndecans (2). Furthermore, syndecans on microvascular endothelial cells play a significant role in cell-free HIV-1 transmigration through the blood-brain barrier (3). Thus, HIV-1 has maximized its utilization of syndecans in the body.A single conserved arginine (Arg298) in the V3 region of gp120 governs HIV-1 binding to syndecans (11). An amine group on the side chain of this residue is absolutely required for syndecan utilization by HIV-1 (11). HIV-1 binds syndecans via a 6-O sulfation (11) within the HS chains, demonstrating that this binding is not the result of random interactions between basic residues and negative charges but the result of specific contacts between gp120 and a well-defined sulfation in syndecans. Surprisingly, the Arg298 in gp120 that mediates HIV-1 binding to syndecans also mediates HIV-1 binding to CCR5 (42), suggesting that HIV-1 recognizes similar motifs on syndecans and CCR5 (11). Supporting this hypothesis, the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5 (11). The finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.More recent work suggests that syndecans play a critical role in HIV-1 transmission (4). HIV-1 transmission includes transmigration of HIV-1 through the genital epithelium and subsequent capture and transfer of infectious particles from dendritic cells (DC) and/or Langerhans cells (LC) to T cells (31, 34, 38). Importantly, human cervical and vaginal mucosal epithelia richly express syndecans in vivo (4). HS chain removal by heparinase treatment prevents HIV-1 transcytosis through primary human cervical and vaginal mucosal epithelia (4). The Arg298 V3 HIV-1 mutant, which cannot interact with syndecans (11), fails to transcytose (4). Thus, syndecans facilitate HIV-1 epithelial transmigration in vitro. More recently, we identified syndecan-3 as an HIV-1 receptor on DC (12). Syndecan-3 stabilizes the captured virus, enhances DC infection in cis, and promotes transfer to T cells (12). Removal of the HS from the cell surface by heparinase or by silencing syndecan-3 by small interfering RNA (siRNA) partially inhibited HIV-1 transmission by immature DC (12), whereas neutralizing both syndecan-3 and DC-SIGN abrogated HIV-1 capture and subsequent transmission (12). Our findings that syndecans modulate both HIV-1 epithelial transmigration and HIV-1 transfer from DC to CD4⁺ T cells suggest that syndecans represent new targets for the development of topical microbicides.Nearly 40 million people currently live with HIV-1/AIDS; the majority of them reside in developing countries. It has been estimated that there are over 14,000 new HIV-1 infections per day in adults aged 15 to 50 years, with 50% of the newly infected being women aged 15 to 24 years (21, 22). In the absence of a vaccine, there is thus an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of HIV-1 (14, 26). Topical microbicides are currently defined as vaginally or rectally applied products that prevent male-to-female or female-to-male transmission of HIV-1 infectious particles (39).In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested this syndecan-Fc hybrid molecule for its in vitro microbicidal properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and DC; (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug-resistant HIV-1 isolates, HIV-1, and SIV; (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells (PGEC); (iv) it prevents HIV-1 transfer from DC to CD4⁺ T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it inhibits HSV infection. Thus, this syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.     

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.     

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).     

Inhibition of Envelope-Mediated CD4+-T-Cell Depletion by Human Immunodeficiency Virus Attachment Inhibitors

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) binding induces proapoptotic signals in CD4⁺ T cells without a requirement of infection. Defective virus particles, which represent the majority of HIV-1, usually contain a functional Env and therefore represent a potentially significant cause of such CD4⁺-T-cell loss. We reasoned that an HIV-1 inhibitor that prohibits Env-host cell interactions could block the destructive effects of defective particles. HIV-1 attachment inhibitors (AIs), which potently inhibit Env-CD4 binding and subsequent downstream effects of Env, display low-nanomolar antiapoptotic potency and prevent CD4⁺-T-cell depletion from mixed lymphocyte cultures, also with low-nanomolar potency. Specific Env amino acid changes that confer resistance to AI antientry activity eliminate AI antiapoptotic effects. We observed that CD4⁺-T-cell destruction is specific for CXCR4-utilizing HIV-1 strains and that the fusion blocker enfuvirtide inhibits Env-mediated CD4⁺-T-cell killing but is substantially less potent than AIs. These observations, in conjunction with observed antiapoptotic activities of soluble CD4 and the CXCR4 blocker AMD3100, suggest that this AI activity functions through a mechanism common to AI antientry activity, e.g., prevention of Env conformation changes necessary for specific interactions with cellular factors that facilitate viral entry. Our study suggests that AIs, in addition to having potent antientry activity, could contribute to immune system homeostasis in individuals infected with HIV-1 that can engage CXCR4, thereby mitigating the increased risk of adverse clinical events observed in such individuals on current antiretroviral regimens.CD4⁺-T-cell levels decrease in patients throughout the course of human immunodeficiency virus type 1 (HIV-1) infection (12, 13, 16, 31). Eventually this decline leads to the disruption of effective immune responses, numerous opportunistic infections, AIDS-defining illnesses, and death (10, 41). Antiretroviral therapy (ART) has been successful in mitigating these effects (15). However, even among ART responders, those who harbor HIV-1 that engages CXCR4 have an increased risk of adverse clinical outcome, including an increased incidence of both AIDS-related and non-AIDS-related diseases (1, 25, 33). This pattern suggests a need for an antiretroviral strategy that blocks the harmful effects of viruses that can signal through CXCR4. A source of these effects is CXCR4-mediated signal transduction induced by Env interactions, which could provide an explanation for the observed correlation between the presence of CXCR4-utilizing HIV-1 and accelerated disease progression (35, 38). Recent studies indicate that up to 18% of asymptomatic ART-naive and 47% of asymptomatic ART-experienced individuals harbor HIV-1 that exploits CXCR4 for cell entry (6, 22, 34, 54), suggesting that this interaction is pertinent throughout the course of infection in many individuals.Clearly, the high level of ongoing HIV-1 production is a primary contributor to the loss of CD4⁺ T cells, and it has been estimated that the rate of production of HIV-1 RNA approximates the rate of CD4⁺-T-cell destruction (17, 52). Replication-competent HIV-1 is cytotoxic to these cells, and it is thought that the viral proteins Vpr, Tat, and Nef (in addition to Env) contribute to this effect (11, 26, 45, 53). However, the infectious titer of HIV-1 is low in comparison to viral genome levels (7, 36), suggesting that the effects of replication-competent HIV-1 alone cannot account for the rate of CD4⁺-T-cell loss. Consequently, replication-defective virus likely contributes substantially to this effect. Such particles usually contain a functional Env, which can act as a signaling partner for a variety of lymphocyte surface factors, thereby influencing the physiology of HIV-uninfected (bystander) cells (4, 24, 28, 38, 47). The relevance of bystander killing to primate lentivirus-mediated disease progression is supported by observations of simian immunodeficiency virus-infected sooty mangabeys. These monkeys display substantially lower levels of bystander killing than are observed in HIV-infected humans (39, 40). This characteristic is thought to contribute to their capacity to carry high viral loads (comparable to HIV-1 loads found in humans with progressive AIDS) while sustaining an asymptomatic state of infection. Moreover, by maintaining consistently high CD4⁺-T-cell counts sooty mangabeys seem to avoid the potential consequences of immune dysfunction encountered by HIV-infected humans.Based on this information, it would seem that an HIV-1 inhibitor that prohibits Env-host cell interactions and Env-mediated CD4⁺-T-cell destruction would increase the likelihood of a favorable clinical outcome. Fuzion (enfuvirtide) and maraviroc are FDA-approved drugs that inhibit the HIV-cell fusion process or block the HIV coreceptor CCR5, respectively (2, 20, 23, 29, 43). In addition, attachment inhibitors (AIs) are antiretrovirals currently in development that target HIV-1 Env-mediated cell entry. AIs operate by potently inhibiting Env interactions with CD4, meaning that they operate upstream of CCR5 engagement or the virus-cell fusion process (14, 18, 27, 49, 50). In this study we tested whether these different types of HIV-1 entry inhibitors could prevent Env-induced cytopathicity and reveal that only AIs provide low-nanomolar protection against this effect, which was not unexpected since it seems to be driven through CXCR4 engagement.     

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.     

Pharmacokinetics and Buccal Mucosal Concentrations of a 15 Milligram per Kilogram of Body Weight Total Dose of Liposomal Amphotericin B Administered as a Single Dose (15 mg/kg), Weekly Dose (7.5 mg/kg), or Daily Dose (1 mg/kg) in Peripheral Stem Cell Transplant Patients

The pharmacokinetics and safety of extended-interval dosing of prophylactic liposomal amphotericin B (L-AMB) in peripheral stem cell transplant recipients were evaluated. The patients received L-AMB daily at 1 mg/kg of body weight or weekly at 7.5 mg/kg or received L-AMB as a single dose (15 mg/kg). The buccal mucosal tissue concentrations of L-AMB were measured. Of the 24 patients enrolled, 5 withdrew after the initial dose due to an infusion-related reaction (n = 2) or significant increases in the serum creatinine (Scr) levels (n = 3). Weekly L-AMB dosing (7.5 mg/kg) produced mean plasma concentrations of >0.300 μg/ml for the first 7 days and >0.220 μg/ml for 7 days after the second dose. A single L-AMB dose (15 mg/kg) produced mean plasma concentrations of >0.491 μg/ml for at least 7 seven days. These concentrations are within the range of the MICs reported in the literature for susceptible strains of Candida and are at the lower limits of the MICs for Aspergillus spp. Extended-interval dosing produced buccal mucosal tissue concentrations well in excess of the MICs reported in the literature for susceptible strains of Candida and Aspergillus spp. Infusion-related reactions occurred in 24% of the patients. Baseline and end-of-study Scr, electrolyte (K⁺, Mg²⁺, PO₄), and serum transaminase levels were similar across the dosage groups. Five (31%) patients met the nephrotoxicity definition prior to completion of the study. Patients in the weekly or single-dose groups experienced nephrotoxicity significantly faster than the patients in the daily dosing cohort. A weekly L-AMB dose (7.5 mg/kg) or a single L-AMB dose (15 mg/kg) produced sufficient concentrations in plasma and highly vascular tissue to warrant further studies of the safety, efficacy, and practicality of the weekly prophylactic administration of L-AMB.Amphotericin B (AMB) exhibits concentration-dependent fungicidal activity against many common opportunistic fungal pathogens (19, 20). A dose-fractionation study with a murine disseminated candidiasis model demonstrated that maximization of the maximum drug concentration in plasma (C_max)/MIC ratio optimizes the efficacy of AMB (2). Theoretically, this should enable the intermittent administration of large amounts of AMB, which could make its use by ambulatory patients more practical. However, in practice, maximization of the concentration-dependent activity of AMB deoxycholate is difficult due to its dose-related nephrotoxicity. However, lipid AMB formulations are safer and demonstrate subtle pharmacokinetic differences in disposition from that of AMB deoxycholate, which allows the administration of higher doses. Lipid AMB formulations such as liposomal amphotericin B (L-AMB) should enable clinicians to optimize the pharmacodynamic properties of AMB and facilitate their use by ambulatory patients.Data from studies with neutropenic animals suggest that the administration of a single dose or intermittent doses of L-AMB up to 20 mg/kg of body weight can be employed to prevent or manage infections due to yeasts or molds (1, 22). Higher L-AMB doses produce increased intravascular concentrations, which may facilitate its penetration into certain tissues and which may produce reductions in the fungal burdens in those tissues more marked than those achieved with other AMB formulations (17, 28). In mice, L-AMB administered at 15 mg/kg thrice weekly produced sufficiently high and sustained kidney and spleen concentrations to provide prophylactic efficacy against systemic challenge with a Candida sp. 3 and 6 weeks posttreatment (30). In a murine model, the administration of a single prophylactic L-AMB dose (1, 5, 10, or 20 mg/kg) 7 to 9 days prior to challenge with Candida albicans or Histoplasma capsulatum produced increased L-AMB kidney and spleen concentrations which correlated with dose-dependent increases in efficacy (14).In humans, L-AMB has the pharmacokinetic profile of a small unilamellar vesicle. L-AMB is slowly cleared from the bloodstream; has a long circulation half-life (t_1/2), and achieves a high C_max and a high level of systemic drug exposure (area under the concentration-time curve [AUC]) but has a small volume of distribution (V) (4, 5). L-AMB exhibits a triphasic plasma concentration profile (5-7). The mononuclear phagocytic system (MPS) is primarily responsible for the tissue uptake and distribution of lipid AMB formulations (16, 29). L-AMB is not efficiently cleared by the MPS due to its small particle size distribution (diameter, <100 nm) (11, 12). The incorporation of AMB into a small unilamellar vesicle liposome changes its MPS uptake and alters its distribution and subsequent excretion (5).A nonlinear relationship between the L-AMB dosage and the plasma AUC from time zero to 24 h (AUC_0-24), the plasma AUC from time zero to infinity (AUC_0-∞), and C_max was observed in immunocompromised adults (32). The values of the pharmacokinetic parameters increased following the administration of up to 10 mg/kg/day but declined with subsequent increases in the dosage to 15 mg/kg/day, suggesting an alteration in elimination at higher doses (32). However, the disposition in tissue was not assessed in that study. Liposomal drugs sequestered within tissues of the MPS may not be able to diffuse freely back into the plasma prior to elimination and may be slowly eliminated from those tissue compartments (5, 12). Thus, whether changes in the elimination of L-AMB from the plasma reflect altered elimination or enhanced tissue penetration in humans is unknown.Animal studies have demonstrated the importance of including tissue drug concentrations in the assessment of the pharmacokinetics of L-AMB in plasma (14, 17, 30). However, for technical and ethical reasons, such data from analyses of the pharmacokinetics in humans are often lacking (1, 4).The purpose of the pilot study described here was to investigate the pharmacokinetics and safety of extended-interval dosing of prophylactic L-AMB in medically stable peripheral stem cell transplant (PSCT) recipients. Moreover, by using a readily accessible tissue (buccal mucosal tissue), this study sought to assess the persistence of L-AMB in the tissues of these patients.(Preliminary findings of this work were presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, November 2004, Washington, DC [abstr. A-35].)     

NB-002, a Novel Nanoemulsion with Broad Antifungal Activity against Dermatophytes,Other Filamentous Fungi,and Candida albicans

NB-002 is an oil-in-water emulsion designed for use for the treatment of skin, hair, and nail infections. The activity of NB-002 was compared to the activities of the available antifungal drugs against the major dermatophytes responsible for cutaneous infections, Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, and Microsporum spp., as well as 12 other genera of filamentous fungi. NB-002 consistently displayed fungicidal activity against all dermatophytes. The comparator compounds were either fungistatic or fungicidal, and for some strain-drug combinations, tolerance was observed. Assessment of the development of spontaneous resistance to NB-002 in different dermatophyte species yielded few stably resistant mutants. For filamentous nondermatophyte fungi, the MIC range varied from 0.06 to 0.5 μg/ml for Alternaria spp. to 2 to 8 μg/ml for Paecilomyes spp. NB-002 had activity against both azole-susceptible and -resistant Candida albicans yeast isolates, with MIC₉₀s of 2 μg/ml, respectively, and minimum fungicidal concentrations at which 90% of isolates are inhibited of 4 and 8 μg/ml, respectively. The kinetics of the fungicidal activity of NB-002 against T. rubrum isolates were compared to those of the other antifungal drugs. NB-002 killed both mycelia and microconidia even when the fungal forms were dormant or not actively growing. Electron micrographs of mycelia and spores treated with NB-002 showed the significant disruption of the fungal structure. The in vitro broad coverage of NB-002 against filamentous fungi, dermatophytes, and C. albicans, as well as its rapid fungicidal activity, warrants further investigations to ascertain if NB-002 would be useful for the treatment of cutaneous mycoses.Superficial fungal infections are found in the top layers of the skin and mucous membranes, the hair, and nails. Examples of fungal infections of the skin and other external surfaces include athlete''s foot, jock itch, ringworm, and other tinea infections. Most of these infections are caused by three genera of dermatophytes: Trichophyton, Epidermophyton, and Microsporum spp. (3, 4, 20, 29, 45, 47).Filamentous fungi that are normal soil saprophytes have also emerged as major opportunistic fungi, especially in immunosuppressed patients (34, 53). Such organisms include Aspergillus spp. (1, 18, 34, 42, 49), Fusarium spp. (18, 22, 28, 34, 36, 49), Scedosporium spp. (18, 34), Paecilomyces spp. (23, 24), Scopulariopsis spp. (18), Scytalidium spp. (11), Chaetomium spp. (13), Alternaria spp. (2, 18, 49), Acremonium spp. (18), and Curvularia spp. (49). Yeasts such as Candida albicans also cause skin infections, generally at sites between the fingers and toes, around the anus, and on the penis or at sites of abrasion where the skin is continuously moist (46).Most cutaneous infections are treated with topical antifungals containing naftidine, tolnaftate, terbinafine, or itraconazole. Oral therapies of griseofulvin, terbinafine, and itraconazole are used to treat tinea capitis. Nail infections can be treated with orally administered agents as well as the topical agent ciclopirox. Terbinafine and azole-like compounds carry the risk of liver and cardiac side effects and drug-drug interactions (14, 31, 38). Topical therapies for inflammatory dermatomycoses often combine an azole and a corticosteroid to rapidly reduce inflammatory symptoms and to increase the bioavailability of the antifungal agent (30).Antimicrobial nanoemulsions are highly stable oil-in-water emulsions composed of nanometer-sized, positively charged droplets that have broad-spectrum activity against enveloped viruses, fungi, and bacteria (5, 17, 25-27, 35, 43, 48). NB-002 contains the cationic quaternary ammonium compound cetylpyridinium chloride (CPC) oriented at its oil-water interface, which stabilizes the nanoemulsion droplets, contributes to the anti-infective activity, and serves as a marker of activity.Studies with NB-002 containing fluorescein have shown that the nanodroplets permeate human cadaver skin by a transfollicular route (6). By the use of a modified Franz cell apparatus, NB-002 was also shown to diffuse laterally from hair follicles and sebaceous glands along tissue planes to reach concentrations in excess of 200 μg/g in human cadaver epidermis as far as 1 cm from the site of application (7). This concentration is significantly above the MIC₉₀ and the minimum fungicidal concentration at which 90% of isolates are inhibited (MFC₉₀) or the ranges of MICs and MFCs determined in this work: ≤4 μg/ml for Trichophyton spp., Microsporum spp., and Epidermophyton floccosum.The studies described here assessed the microbiological activity of NB-002 against fungal pathogens that cause cutaneous infections. Furthermore, we show that the fungicidal activity of NB-002 is rapid and that it kills both the microconidia and mycelia of the dermatophyte Trichophyton rubrum. Consistent with this kill-on-contact mechanism of action, the MICs for the majority of dermatophyte isolates spontaneously resistant to NB-002 were plus or minus twofold of the initial MIC.     

Intracellular Activity of Antibiotics against Staphylococcus aureus in a Mouse Peritonitis Model

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log₁₀ number of CFU/ml (Δlog₁₀ CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog₁₀ CFU/ml) > cefuroxime (3.56 Δlog₁₀ CFU/ml) > rifampin (1.86 Δlog₁₀ CFU/ml) > gentamicin (0.61 Δlog₁₀ CFU/ml) > azithromycin (0.21 Δlog₁₀ CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.Staphylococcus aureus is a major human pathogen that causes both community- and hospital-acquired infections (35). It causes a diverse array of infections ranging from relatively minor skin and wound infections to more serious and life-threatening diseases such as pneumonia (20, 46), endocarditis (48), osteomyelitis (17, 29), arthritis (1), and meningitis (40). Some of these types of S. aureus infections, e.g., endocarditis, are associated with high rates of mortality (25 to 50%), despite antimicrobial treatment (48, 49, 57). Furthermore, S. aureus infections are often persistent and are associated with treatment difficulties, such as a slow response to antibiotic treatment and recurrences, that lead to an extended duration of antimicrobial therapy (11, 13, 31). The antimicrobial treatment of S. aureus infections has also become more difficult due to the emergence of multidrug-resistant strains (3, 4).Several factors may help explain the capacity of staphylococci to avoid the actions of antibiotics. Biofilm formation might be the main reason for a deficient antibiotic effect when foreign bodies are involved in the staphylococci infections (12, 15, 53). Otherwise, the intracellular presence of the bacteria could offer a good explanation for the slow response to antibiotics, since bacteria located intracellularly might be protected from the effects of antibiotics (55).S. aureus has classically been classified as an extracellular pathogen (21). Conversely, several reports have established that S. aureus internalizes and survives within professional and even nonprofessional mammalian phagocytes (7, 19, 24, 25, 26, 27). The attitude is therefore changing toward classifying S. aureus as a facultative/opportunistic intracellular pathogen (13, 36, 41, 42, 55).Having an intracellular target for antimicrobial therapy is more complex than having an extracellular target, because intracellular antimicrobial activity further depends on the penetration into and accumulation in the cell, cellular metabolism, the subcellular disposition, and the bioavailability of the drug. The bacterial responsiveness to antibiotics can also change intracellularly (54, 55). Antimicrobial activity is therefore often impaired intracellularly (6, 56).To date, this knowledge of the intracellular presence of S. aureus has not influenced the choice of antibiotic to be used for the treatment for S. aureus infections. Penicillinase-stable penicillins, for instance, are considered the mainstay of treatment for methicillin-susceptible S. aureus infections (5, 23, 35), even though penicillins are usually considered not to penetrate cells (8, 30, 50).Recurrent S. aureus infections may also, at least partly, be explained by the intracellular presence of the bacteria. Gresham et al. demonstrated that polymorphonuclear neutrophils with intracellular S. aureus isolated from the peritoneums of infected mice could cause a new infection by intraperitoneal injection of these cells into healthy mice (24). They also demonstrated that intracellular survival was linked to the global regulator sar, which regulates multiple virulence factors in S. aureus. These two observations could together indicate that intracellular survival is a part of the pathogenesis of S. aureus.Appropriate models for the testing of the intracellular activities of antimicrobials against S. aureus are needed. Several in vitro models that use various cells and cell lines are available for the study of intracellular S. aureus (6, 10, 18, 24, 44, 51), but only a few in vivo models have been developed.Here we present an in vivo model that can be used to study the intracellular activities of antimicrobials against S. aureus.(Part of this study was presented at the 16th European Congress of Clinical Microbiology and Infectious Diseases, Nice, France.)     

Voriconazole Pharmacokinetics in Liver Transplant Recipients

The objective of this study was to evaluate the pharmacokinetics of voriconazole and the potential correlations between pharmacokinetic parameters and patient variables in liver transplant patients on a fixed-dose prophylactic regimen. Multiple blood samples were collected within one dosing interval from 15 patients who were initiated on a prophylactic regimen of voriconazole at 200 mg enterally (tablets) twice daily starting immediately posttransplant. Voriconazole plasma concentrations were measured using high-pressure liquid chromatography (HPLC). Noncompartmental pharmacokinetic analysis was performed to estimate pharmacokinetic parameters. The mean apparent systemic clearance over bioavailability (CL/F), apparent steady-state volume of distribution over bioavailability (V_ss/F), and half-life (t_1/2) were 5.8 ± 5.5 liters/h, 94.5 ± 54.9 liters, and 15.7 ± 7.0 h, respectively. There was a good correlation between the area under the concentration-time curve from 0 h to infinity (AUC_0-∞) and trough voriconazole plasma concentrations. t_1/2, maximum drug concentration in plasma (C_max), trough level, AUC_0-∞, area under the first moment of the concentration-time curve from 0 h to infinity (AUMC_0-∞), and mean residence time from 0 h to infinity (MRT_0-∞) were significantly correlated with postoperative time. t_1/2, λ, AUC_0-∞, and CL/F were significantly correlated with indices of liver function (aspartate transaminase [AST], total bilirubin, and international normalized ratio [INR]). The C_max, last concentration in plasma at 12 h (C_last), AUMC_0-∞, and MRT_0-∞ were significantly lower in the presence of deficient CYP2C19*2 alleles. Donor characteristics had no significant correlation with any of the pharmacokinetic parameters estimated. A fixed dosing regimen of voriconazole results in a highly variable exposure of voriconazole in liver transplant patients. Given that trough voriconazole concentration is a good measure of drug exposure (AUC), the voriconazole dose can be individualized based on trough concentration measurements in liver transplant patients.Due to chronic immunosuppression, infections are common life-threatening complications in organ transplant patients (7). Invasive aspergillosis is one of the most dreaded complications after organ transplantation (21) due to its high mortality rate, which can range up to 88.1% (18).Voriconazole (V-Fend [Pfizer]; formerly known as UK-109496), (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1,2,4-triazol-1-yl)butan-2-ol, is a novel broad-spectrum triazole systemic antifungal agent and an ideal drug to prevent invasive aspergillosis. Compared with other azole antifungal agents, it has potent activity against a broader spectrum of clinically significant fungal pathogens, including Aspergillus, Candida, Cryptococcus neoformans, and some unusual organisms, such as Fusarium and Pseudallescheria boydii (11, 24, 37, 39, 43).Voriconazole is extensively metabolized hepatically, primarily via the cytochrome P450 (CYP) isoenzymes CYP2C19 and, secondarily, CYP2C9 and CYP3A4 (15, 25, 39) to inactive metabolites. Large inter- and intraindividual variabilities in voriconazole plasma concentrations regardless of the route of administration or the type of patient population have been documented and discussed in the literature (2, 17, 19, 22, 23, 33, 38, 40, 42, 45). Factors associated with interindividual variability of voriconazole exposure include liver dysfunction, alcohol abuse in the past (47), concomitant use of potent CYP450 inducers or inhibitors (10, 12), CYP2C19 genetic polymorphisms (including poor as well as ultrarapid metabolizers) (13, 20, 46), gastrointestinal abnormalities (e.g., mucositis or diarrhea) (38) impairing drug absorption, and intake with or without food (15).Voriconazole is approved at our institution for prophylaxis in all liver transplant patients. The pharmacokinetics of voriconazole in liver transplant patients has not been evaluated, and there is limited information about the pharmacokinetics of voriconazole in other solid organ transplant patient populations (3). We hypothesized that the use of a fixed-dosing regimen of voriconazole would lead to a large degree of variability in voriconazole exposure in liver transplant patients. Given that a low voriconazole plasma level of less than 0.25 μg/ml is associated with a poor outcome in patients with aspergillosis (4, 8, 22, 31, 38, 40) and with ultimately death of the patients, while a high voriconazole plasma concentration of over 5.5 μg/ml is correlated with an increased risk for toxicity, including visual disturbances, elevated transaminase levels, central nervous system (CNS) disorders (e.g., encephalopathy), and electrolyte disturbances (2, 4, 38, 41), it is important to optimize the use of voriconazole in this patient population.The objective of this prospective single-center observational study was to characterize the pharmacokinetics of voriconazole in liver transplant patients on a fixed-dose prophylactic regimen in order to determine the extent of interpatient variability in voriconazole exposure among liver transplant patients and to evaluate the potential correlations between pharmacokinetic parameters and certain patient variables that could potentially explain the large interindividual variability in voriconazole pharmacokinetics in liver transplant patients.     

Novel Ribosomal Mutations in Staphylococcus aureus Strains Identified through Selection with the Oxazolidinones Linezolid and Torezolid (TR-700)

TR-700 (torezolid), the active moiety of the novel oxazolidinone phosphate prodrug TR-701, is highly potent against gram-positive pathogens, including strains resistant to linezolid (LZD). Here we investigated the potential of Staphylococcus aureus strains ATCC 29213 (methicillin-susceptible S. aureus [MSSA]) and ATCC 33591 (methicillin-resistant S. aureus [MRSA]) to develop resistance to TR-700. The spontaneous frequencies of mutation of MSSA 29213 and MRSA 33591 resulting in reduced susceptibility to TR-700 at 2× the MIC were 1.1 × 10⁻¹⁰ and 1.9 × 10⁻¹⁰, respectively. These values are ∼16-fold lower than the corresponding LZD spontaneous mutation frequencies of both strains. Following 30 serial passages in the presence of TR-700, the MIC for MSSA 29213 remained constant (0.5 μg/ml) while increasing eightfold (0.25 to 2.0 μg/ml) for MRSA 33591. Serial passage of MSSA 29213 and MRSA 33591 in LZD resulted in 64- and 32-fold increases in LZD resistance (2 to 128 μg/ml and 1 to 32 μg/ml, respectively). Domain V 23S rRNA gene mutations (Escherichia coli numbering) found in TR-700-selected mutants included T2500A and a novel coupled T2571C/G2576T mutation, while LZD-selected mutants included G2447T, T2500A, and G2576T. We also identified mutations correlating with decreased susceptibility to TR-700 and LZD in the rplC and rplD genes, encoding the 50S ribosomal proteins L3 and L4, respectively. L3 mutations included Gly152Asp, Gly155Arg, Gly155Arg/Met169Leu, and ΔPhe127-His146. The only L4 mutation detected was Lys68Gln. TR-700 maintained a fourfold or greater potency advantage over LZD against all strains with ribosomal mutations. These data bring to light a variety of novel and less-characterized mutations associated with S. aureus resistance to oxazolidinones and demonstrate the low resistance potential of torezolid.Staphylococcus aureus infections pose a serious health threat worldwide. Increasing antibiotic resistance and the prevalence of methicillin (meticillin)-resistant S. aureus (MRSA) in clinical settings have created a demand for novel therapeutic agents. Linezolid (LZD) has a broad spectrum of activity against a variety of gram-positive pathogens, including MRSA, and was the first oxazolidinone antibiotic to gain FDA approval (1). LZD acts through inhibition of protein synthesis via binding to the peptidyl transferase center (PTC) of the 50S ribosomal subunit (37, 65, 68). Despite in vitro studies demonstrating a low resistance potential for LZD (31, 79), soon after its approval in 2000, LZD-resistant (LZD^r) MRSA (72) and LZD^r, vancomycin (VAN)-resistant enterococci (22) emerged in the clinic. Although rare, resistance has most commonly occurred in patients undergoing long-term LZD therapy (10, 17, 22, 45, 72, 74). Three classes of oxazolidinone resistance mechanisms have been previously characterized: mutations in the domain V region of 23S rRNA genes (69), acquisition of the ribosomal methyltransferase gene cfr (43), and mutations in the rplD gene encoding the 50S ribosomal protein L4 (76).A variety of 23S rRNA mutations conferring resistance to LZD in S. aureus have been identified, including C2192T (26), G2447T (69), T2500A (45), A2503G (39), T2504C (39), G2505A (50), G2766T (38), and G2576T (72) (Escherichia coli numbering), the most common mutation observed clinically. To date, T2500A and G2576T are the only 23S rRNA mutations that have been documented in clinical S. aureus isolates. 23S rRNA-mediated resistance occurs in a gene dose-dependent fashion, with higher copy numbers of 23S mutant alleles associated with increased resistance (5, 38, 66, 78). Despite evidence of fitness costs associated with some 23S rRNA mutations (5, 8, 44), highly LZD^r 23S rRNA homozygous mutant strains of S. aureus (72), Staphylococcus epidermidis (71), and Enterococcus faecalis (60) have been recovered clinically.A second mechanism of LZD resistance has been identified in staphylococci possessing cfr, a gene encoding a ribosomal methyltransferase (32, 70) which catalyzes methylation at position 8 of nucleotide A2503 in the 23S rRNA domain V region (21). Cfr methylation confers resistance to five classes of 50S ribosomal subunit-targeted antibiotics defined by the PhLOPS_A phenotype, including phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (43). Recent outbreaks of LZD^r staphylococci possessing the transposon- and plasmid-associated cfr methyltransferase (11, 16a, 32, 46, 47, 70) are particularly problematic due to the potential for rapid gene transfer between strains of both human and animal origins, driven by the use of antibiotics in any of the classes to which resistance is conferred.The third and least common class of LZD resistance involves mutations in ribosomal protein L4, which have been associated with reduced LZD susceptibility in streptococci (19, 76). L4 interacts closely with the PTC, and mutations are thought to confer antibiotic resistance by perturbing 23S rRNA structure (23).Despite the characterization of these three primary resistance classes, there have been a number of reports describing LZD^r staphylococci, streptococci, enterococci, and mycobacteria of clinical (28, 29, 58) and laboratory (12, 25, 40, 57, 62) origins possessing “unknown” resistance mechanisms. A recent report documents mutations in an endogenous ribosomal methyltransferase (modifying G2445) and altered efflux via upregulation of ABC transporters linked to LZD resistance (19). Such mutations may account for the resistance phenotype of some of the undefined LZD^r strains previously observed.Increasing incidences of clinical resistance to LZD and the potential for rapid horizontal dissemination of cfr have contributed to the demand for expanded-spectrum oxazolidinones (4) such as TR-700 (torezolid), the active antibacterial moiety of the prodrug TR-701 (torezolid phosphate) (Fig. ​(Fig.1)1) (64). TR-700 demonstrates 4- to 16-fold greater potency than LZD against LZD^s and LZD^r strains (30, 38, 64). Notably, TR-700 has been shown to be 16-fold more active than LZD against S. aureus Cfr strains (64). Molecular modeling suggests that this is likely due to the smaller size of the hydroxymethyl group compared to the acetamide group of LZD, as well as additional C and D ring interactions with the PTC, allowing TR-700 to bind in the presence of the methylated A2503 base (64). Enhanced PTC binding properties are also thought to contribute to its potency advantage over LZD and may translate into lower frequencies of resistance (64). Preliminary reports suggested that resistance to TR-700 is very low (13, 30), although further work to characterize the frequency of resistance and resistance mechanisms was warranted.Open in a separate windowFIG. 1.TR-700 and LZD chemical structures.In this study, we investigated the potential for two S. aureus strains to develop resistance to TR-700 and LZD through serial passage and analysis of spontaneous mutation frequencies. Underlying resistance mechanisms were elucidated by sequencing of genes encoding 23S rRNA and ribosomal proteins L3, L4, and L22. Mutations were analyzed structurally by using LZD-bound 50S ribosomal subunit crystallographic data.(Portions of this work were presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases [J. B. Locke, M. Hilgers, and K. J. Shaw, posters P1103 and P1104], Helsinki, Finland, 16 to 19 May 2009.)     

In Vitro Antiviral Activity of Favipiravir (T-705) against Drug-Resistant Influenza and 2009 A(H1N1) Viruses

Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay with MDCK cells, and a subset was also tested in both yield reduction and focus inhibition (FI) assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 μM (0.5 μg/ml) (50% effective concentrations [EC₅₀s] of 0.19 to 22.48 μM and 0.03 to 3.53 μg/ml), and for all viruses, with the exception of a single dually resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 μM (0.5 μg/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.In the United States alone, seasonal influenza is responsible annually for infecting between 5 and 20% of the American population, resulting in more than 200,000 hospitalizations and 36,000 deaths (8). Globally, seasonal influenza causes between 250,000 and 500,000 deaths every year (60). Influenza is not only a disease of great medical importance but also of economic importance. Despite available vaccines, a recent study predicted that in the United States influenza results in direct medical costs of the order of $10.4 billion each year, with the total economic burden for the United States being projected at $87.1 billion each year (44). It is widely accepted that vaccination remains the most effective approach for the prevention of viral infections (48). Although there is a safe and effective annual trivalent influenza vaccine, a large proportion of the global population does not receive the yearly influenza vaccine. This can be due to a variety of reasons, including the lack of access to adequate health care, unavailability of vaccine supply, allergies, and adverse reactions. During the 2009 pandemic (pdm), in addition to the vaccination and epidemiological control measures being exerted by health care officials, antivirals targeting influenza offer an essential tool in treating infected patients, in addition to protecting those at high risk of infection, such as the young, elderly, and health care workers.Currently, there are two classes of anti-influenza drugs licensed in the United States for use in the treatment and management of influenza infections in humans: M2 ion channel blockers (also known as adamantanes) and neuraminidase (NA) inhibitors (NAIs) (30). Influenza antivirals are highly effective in the treatment of influenza infections if used promptly following the onset of symptoms or following exposure (45, 46). Both the M2 blockers amantadine and rimantadine are taken by the patient orally (45). However, of the two available NAIs, only oseltamivir is available as an oral formulation (zanamivir has to be inhaled [14, 53]), although other routes of administration have been investigated (31). The use of the M2 blockers amantadine and rimantadine is limited due to the rapid emergence of transmissible drug-resistant mutant viruses and the fact that they offer protection only against influenza A virus infections (32). The high prevalence of adamantane resistance in seasonal A(H3N2) viruses and oseltamivir resistance in seasonal A(H1N1) viruses is reflected in the CDC recommendations for the use of influenza antivirals (6).The majority of adamantane-resistant A(H3N2) and A(H1N1) viruses circulating globally in recent years share the same mutation, S31N, in the M2 protein (20), although other resistance-conferring mutations have been detected also (including A30T, L26F, and V27A) (20, 49). The globally spread oseltamivir-resistant seasonal A(H1N1) viruses share the same mutation, H275Y (H274Y in N2 subtype amino acid numbering), in the drug-targeted enzyme neuraminidase, although other mutations are known to cause reduced susceptibility in vitro (19, 47, 50).Seasonal A(H1N1) viruses resistant to both the adamantanes and the NAI oseltamivir have previously been reported, without an apparent link to treatment (12, 50). Currently, zanamivir is the only drug effective against both adamantane-resistant and/or oseltamivir-resistant influenza viruses, but due to the fact that it has to be inhaled, it is less suitable for use with several high-risk groups, including the severely ill (41), infants (33), and the elderly (22). Furthermore, zanamivir may decrease pulmonary function, so it is not recommended for the treatment of infections in individuals with chronic underlying lung and heart disease conditions (23).Since 1997, there have been several outbreaks of highly pathogenic avian influenza A(H5N1) infections in poultry, with a substantial number of infections occurring in humans (1). The overall case fatality of A(H5N1) infections in humans is over 60% and, unlike seasonal influenza, is most deadly in the young and healthy (ages 10 to 19 years) (59). Oseltamivir is the medication of choice for treating individuals infected with A(H5N1) (17). However, resistance in A(H5N1) viruses has been detected following the treatment of patients with oseltamivir (18, 38). In addition, naturally occurring reduced susceptibility to oseltamivir (35, 40) and possibly to zanamivir (29) has been documented for circulating A(H5N1) viruses, including novel mutations in the NA (29, 35). Adamantane resistance is widely spread among A(H5N1) viruses that carry mutations at amino acid residues 26, 27, and 31 in the M2 protein (13, 35) and among swine viruses circulating in Eurasia (27).In April 2009, a novel reassortant A(H1N1) virus was first identified as circulating in humans in both Mexico and the United States (7, 9). Since April, the virus has continued to transmit among humans, and on 11 June 2009 the World Health Organization classified the outbreak as the first influenza pandemic of the 21st century (58). The 2009 A(H1N1) pandemic viruses consist of a unique combination of gene segments, including those of the North American (triple reassortants) and Eurasian swine lineages (27, 54). The 2009 A(H1N1) pandemic viruses are resistant to the adamantanes and sensitive to the NAIs (3, 16). Yet, concerns exist about the possibility of acquisition of resistance to the NAI oseltamivir, since the majority of A(H1N1) viruses which have been circulating predominantly worldwide during the 2008-2009 influenza season are oseltamivir resistant due to the resistance-conferring H275Y mutation in the NA. Such an acquisition of resistance by the 2009 A(H1N1) pandemic viruses would be a major setback and would further limit the already sparse therapeutic options (15, 57). There have been laboratory-confirmed cases of oseltamivir-resistant 2009 A(H1N1) pandemic viruses (each carrying the H275Y resistance-conferring mutation in the NA) in the United States (5).Collectively, these recent findings emphasize not only the need for new effective antivirals to control and treat influenza infections but also the need to identify new molecular targets (47).One such compound which is currently being investigated and undergoing clinical trials for the treatment of influenza infections is favipiravir (T-705), a pyrazine derivative (2, 26, 31). Favipiravir targets the RNA-dependent RNA polymerase (RdRp), a component of influenza virus different from that of currently licensed influenza antivirals (24, 25). It was shown that favipiravir can inhibit the viral replication of influenza type A, B, and C viruses (24, 25, 55). Favipiravir reduces influenza virus replication by selectively inhibiting the viral RdRp, since it does not affect the synthesis of host cellular DNA and RNA (25). Favipiravir has also shown great potential to act as a broad-spectrum antiviral against many RNA viruses, as reviewed by Furuta and coworkers (26).The purpose of this study was to evaluate the ability, in vitro, of favipiravir to inhibit the viral replication of contemporary influenza viruses as well as viruses with pandemic potential, including viruses resistant to the currently available and licensed anti-influenza drugs. In this report we demonstrate that favipiravir is a potent inhibitor of seasonal influenza A and B virus replication, including that of drug-resistant and drug-sensitive viruses. In addition, favipiravir was shown to effectively inhibit influenza A viruses of other antigenic subtypes, including A(H2N2), viruses of avian origin [A(H4N2), A(H7N2), and A(H5N1)], and viruses of swine origin [A(H1N1) and A(H1N2)], as well as the 2009 A(H1N1) pandemic viruses.     

In Vitro Antifungal Susceptibilities and Amplified Fragment Length Polymorphism Genotyping of a Worldwide Collection of 350 Clinical,Veterinary, and Environmental Cryptococcus gattii Isolates

The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC₉₀ (0.125 μg/ml) for all C. gattii isolates, followed by a 1 log₂ dilution step increase (MIC₉₀, 0.25 μg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC₉₀ of 0.5 μg/ml, but fluconazole and flucytosine had relatively high MIC₉₀s of 8 μg/ml.The basidiomycetous yeast Cryptococcus gattii is responsible for life-threatening invasive disease in apparently healthy humans and animals (7, 19). A typical C. gattii infection is acquired through the respiratory tract, from which it can further disseminate to the central nervous system, resulting in fatal meningitis (7, 19, 32). Cryptococcosis caused by the primary pathogenic yeast C. gattii was, until a decade ago, a rarely encountered infection outside tropical and subtropical regions (17, 26, 27). However, this changed due to an unprecedented outbreak that emerged in the temperate climate of Vancouver Island (British Columbia, Canada) that subsequently expanded farther into the Pacific Northwest (1, 8, 10, 16). Its sibling species, Cryptococcus neoformans, differs ecologically and epidemiologically from C. gattii since it occurs on a global scale and is linked with disease occurring in immunocompromised individuals, such as HIV-positive patients and transplant patients who receive immune-suppressive medicines (7, 10, 18, 19, 31).Cryptococcus gattii can be discerned from C. neoformans using a wide range of microbiological and molecular techniques (7, 20). A convenient method is the use of canavanine-glycine-bromothymol blue (CGB) medium, which allows C. gattii but not C. neoformans to grow and which changes the pH indicator in the medium from green-yellowish to blue (18). With the increasing use of molecular techniques, such as PCR fingerprinting, restriction fragment length polymorphism (RFLP) analysis of the PLB1 and URA5 loci, and amplified fragment length polymorphism (AFLP) fingerprint analysis, as well as several multilocus sequence typing (MLST) approaches, it became clear that C. gattii could be divided into five distinct genotypes, named AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV, and AFLP10 (the last one of which is a recently observed novel genotype) (2, 6, 7, 13, 16, 20, 21, 23). Until recently, a serotype agglutination assay was widely used to distinguish C. neoformans (serotypes A and D) from C. gattii (serotypes B and C) (7, 27). In general, serotype B strains are found in each of the five C. gattii AFLP genotypes, but it seems that C. gattii serotype C strains are restricted to genotypes AFLP5/VGIII and AFLP7/VGIV (2, 6, 16, 21, 27).In addition, it was found that C. gattii and C. neoformans can form interspecies hybrids, named genotype AFLP8 (C. neoformans var. neoformans AFLP2/VNIII serotype D × C. gattii AFLP4/VGI serotype B) and AFLP9 (C. neoformans var. grubii AFLP1/VNI serotype A × C. gattii AFLP4/VGI serotype B). These interspecies hybrids have, until now, been isolated only from clinical samples, and they might have a higher virulence potential than regular C. gattii or C. neoformans isolates (4, 5; F. Hagen, K. Tintelnot, and T. Boekhout, unpublished data).Treatment of cryptococcosis depends on, besides the immune status of the patient, the severity and localization of the infection (11). Severe cases of cryptococcosis in immunocompetent and -compromised patients are treated according to the guidelines of the Infectious Diseases Society of America, according to which treatment consists of an induction therapy for 2 weeks with a combination of amphotericin B and flucytosine, followed by a 10-week consolidation therapy with fluconazole (11, 24).Cryptococcus neoformans has been extensively studied for its in vitro susceptibility to a wide variety of antifungal compounds, including the new triazoles posaconazole, voriconazole, ravuconazole, and isavuconazole (12, 14, 28, 29, 33). Despite the ongoing C. gattii outbreak, only a few studies using relatively small sets of C. gattii isolates have been performed to investigate their in vitro susceptibilities to amphotericin B, flucytosine, fluconazole, and the new triazole antifungals (12, 15, 28-30). A few studies divided the C. gattii isolates into groups according to their serotype or genotype (15, 29).Therefore, we studied the in vitro susceptibilities of each of the C. gattii genotypes from a large worldwide collection, subdivided by AFLP genotyping, to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and the new experimental broad-spectrum antifungal triazole isavuconazole.     

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.     

In Vitro Antifungal Activities of Bis(Alkylpyridinium)Alkane Compounds against Pathogenic Yeasts and Molds

Ten bis(alkylpyridinium)alkane compounds were tested for antifungal activity against 19 species (26 isolates) of yeasts and molds. We then determined the MICs and minimum fungicidal concentrations (MFCs) of four of the most active compounds (compounds 1, 4, 5, and 8) against 80 Candida and 20 cryptococcal isolates, in comparison with the MICs of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin, using Clinical Laboratory and Standards Institutes broth microdulition M27-A3 (yeasts) or M38-A2 (filamentous fungi) susceptibility protocols. The compounds were more potent against Candida and Cryptococcus spp. (MIC range, 0.74 to 27.9 μg/ml) than molds (0.74 to 59.7 μg/ml). MICs against Exophiala were 0.37 to 5.9 μg/ml and as low as 1.48 μg/ml for Scedosporium but ≥25 μg/ml for zygomycetes, Aspergillus, and Fusarium spp. Compounds 1, 4, 5, and 8 exhibited good fungicidal activity against Candida and Cryptococcus, except for Candida parapsilosis (MICs of >44 μg/ml). Geometric mean (GM) MICs were similar to those of amphotericin B and lower than or comparable to fluconazole GM MICs but 10- to 100-fold greater than those for the other azoles. GM MICs against Candida glabrata were <1 μg/ml, significantly lower than fluconazole GM MICs (P < 0.001) and similar to those of itraconazole, posaconazole, and voriconazole (GM MIC range of 0.4 to 1.23 μg/ml). The GM MIC of compound 4 against Candida guilliermondii was lower than that of fluconazole (1.69 μg/ml versus 7.48 μg/ml; P = 0.012). MICs against Cryptococcus neoformans and Cryptococcus gattii were similar to those of fluconazole. The GM MIC of compound 4 was significantly higher for C. neoformans (3.83 μg/ml versus 1.81 μg/ml for C. gattii; P = 0.015). This study has identified clinically relevant in vitro antifungal activities of novel bisalkypyridinium alkane compounds.Invasive fungal disease is a significant cause of morbidity and mortality in seriously ill and immunocompromised patients (16, 26, 35). Despite the recent addition of a new class of antifungal agent (the echinocandins) (20) and more potent, broader-spectrum triazoles such as voriconazole (VRC) and posaconazole (POS) (23, 25), the number of available drugs for treatment of fungal infections remains limited. Many are fungistatic rather than fungicidal, and others are associated with substantial toxicity (4). Furthermore, clinical efficacy may be compromised by intrinsic or acquired drug resistance (29, 34). There is therefore a continuing need to develop and test novel antifungal agents with different modes of action.Targeting of fungal virulence determinants, such as, for example, phospholipase B (PLB), is a potentially fruitful approach to new drug development. PLB is a proven virulence determinant of Candida albicans and Cryptococcus neoformans and is secreted by other pathogenic fungi, including Aspergillus spp. (6, 7, 13). Cryptococcal PLB (PLB1) in particular, has been well characterized (8, 13). As part of a study seeking inhibitors of cryptococcal PLB1, Ganendren et al. identified a novel class of phospholipase inhibitors and observed that the bis(quaternary phosphonium)-alkane 1,12-bis(tributylphosphonium) dodecane dibromide not only inhibited cryptococcal PLB1 but also exhibited in vitro antifungal activity (18).Properties of an “ideal” antifungal agent include ease of manufacture, potent antifungal activity, an excellent safety profile, and low cost. Bis-quaternary ammonium salts, which fulfill the above conditions, have long been recognized as potential antimicrobial agents (21, 32). Other than bisphosphonium salts (as described above) (18), we have previously determined that bisammonium-alkanes with a 12-carbon spacer between the positively charged bisammonium head groups exhibit antifungal activity with MICs of ∼1 to 2.5 μg/ml against C. neoformans and C. albicans and that antifungal activity correlated with inhibition of cryptococcal PLB1 activity (27). Subsequent work on bis(aminopyridinium)alkane molecules indicated that these were also strongly antifungal, but they did not inhibit cryptococcal PLB1. This second class of compounds was significantly less toxic to human erythrocytes than the bisammonium-alkanes (28). Most recently, Obando et al. designed a third novel class of antifungal compound—the bis(alkylpyridinium)alkanes—with combined structural features of the bis(quaternary ammonium)alkanes and bis(aminopyridinium)alkanes (30). The compounds differ from previously described antimicrobial bispyridinium compounds (21, 28) as the pyridinium rings are attached to each other through the ring nitrogen atoms, with alkyl substituents appended directly to the pyridinium rings at the 2-, 3-, or 4-positions; preliminary testing of two of these compounds (compounds 1 and 9 in the present study) against 11 unique fungal strains indicated that they may have useful antifungal activities (30).Given the promising antifungal activity of this class of compounds as observed by Obando et al. (30), we evaluated the in vitro antifungal activities of 10 novel bisalkylpyridinium compounds, including compounds designated in the present study as 1 and 9 (described above); the in vitro hemolytic and cytotoxic activities of these compounds have previously been determined (30). Initially, the 10 compounds were screened for antifungal activity against a panel of key fungal pathogens. The MICs and minimum fungicidal concentrations (MFCs) of four of the most active compounds and MICs of marketed triazoles, amphotericin B (AMB), and caspofungin (CAS) were then determined against a large number of Candida (representing eight species) and cryptococcal isolates.     

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.