Biofilm Formation and Effect of Caspofungin on Biofilm Structure of Candida Species Bloodstream Isolates

Candida biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different Candida species, particularly Candida albicans, C. tropicalis, and C. parapsilosis, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against C. albicans and C. tropicalis cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed Candida species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in C. albicans and C. tropicalis biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in Candida species biofilm populations. The clinical implications of these findings are still unknown.Candida species are opportunistic pathogens that cause superficial and systemic diseases in critically ill patients (8, 22, 44) and are associated with high mortality rates (35%) and costly treatments (8, 19). They rank among the four most common causes of bloodstream infection in U.S. hospitals, surpassing gram-negative rods in incidence (6, 17).Recent studies suggest that the majority of disease produced by this pathogen is associated with a biofilm growth style (7, 16, 28, 48). Biofilms are self-organized communities of microorganisms that grow on an abiotic or biotic surface, are embedded in a self-produced matrix consisting of an extracellular polymeric substance (14, 15, 55), and when associated with implanted medical devices are commonly refractive to antimicrobial therapy.As opportunistic pathogens, Candida species are able to attach to polymeric surfaces and generate a biofilm structure, protecting the organisms from the host defenses and antifungal drugs (11, 16, 45, 48). Candida biofilms are more resistant than their planktonic counterparts to various antifungal agents, including amphotericin B (AMB), fluconazole, itraconazole, and ketoconazole (20, 38, 50). However, the molecular basis for the antifungal resistance of biofilm-related organisms is not completely understood.The complex architecture of Candida biofilms observed both in vitro and in vivo suggests that morphological differentiation to produce hyphae plays an important role in biofilm formation and maturation (7, 32, 33). Baillie and Douglas demonstrated that although mutant cells fixed in either a hyphal or a yeast form can develop into biofilms, the hyphal structure is the essential element for providing the integrity and multilayered architecture of a biofilm (4). It has been reported that Candida parapsilosis, C. glabrata, and C. tropicalis biofilms are not as large as those generated by C. albicans; however, further structural analysis studies are needed to describe biofilm formation by these organisms (30, 31).The mechanisms responsible for the resistance characteristics displayed by Candida biofilms are unclear. Possible mechanisms include a decreased growth rate; nutrient limitation of cells in the biofilm; expression of resistance genes, particularly those encoding efflux pumps; increased cell density; cell aging; or the presence of “persister” cells in the biofilm (1, 3, 5, 29, 34, 36, 38, 43, 46, 48, 50, 51).The echinocandins are a novel class of semisynthetic amphiphilic lipopeptides that display important antifungal activity. The echinocandins that are presently marketed are caspofungin (CAS), micafungin, and anidulafungin. The echinocandins show considerable efficacy in vitro and in vivo in the treatment of candidemia and invasive candidiasis (25, 27, 42). CAS is the first antifungal agent to be licensed that inhibits the synthesis of β-1,3-glucan, the major structural component of Candida cell walls; glucan synthesis might prove to be a particularly effective target for biofilms (29, 31, 38, 48, 50). The paradoxical attenuation of antifungal activity at high echinocandin concentrations is a phenomenon that usually occurs with C. albicans isolates and appears to be specific to CAS among echinocandins. The cells surviving at high concentrations appear to be subject to some drug effect, showing evidence of slowed growth in the presence of CAS (53, 54). Recent studies have described this effect in Candida species biofilms (24, 37, 47); however, we are not aware of studies that have elucidated the effect of CAS on Candida biofilm structure. The present study was designed to (i) characterize the in vitro biofilm growth of Candida species bloodstream isolates and (ii) use scanning electron microscopy (SEM) to obtain visual evidence of the effect of CAS on biofilm morphology changes associated with paradoxical growth (PG).     

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.     

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

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

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.     

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

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

Borrelia burgdorferi Resistance to a Major Skin Antimicrobial Peptide Is Independent of Outer Surface Lipoprotein Content

We hypothesize a potential role for Borrelia burgdorferi OspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferi is resistant to high levels (>200 μg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.Borrelia burgdorferi, a spirochete and the causal organism of Lyme disease, is naturally transmitted to mammals through the bite of infected Ixodes ticks (5, 8). A significant change in B. burgdorferi gene expression accompanies transmission between these diverse environments. This was first described for the inverse relationship between two abundant outer surface proteins of B. burgdorferi, in which synthesis of OspA declines and that of OspC increases during tick feeding (41). We and others have demonstrated the essential nature of OspC for colonization of the murine host (23, 35, 42, 45, 47, 49). These findings suggest a critical role for OspC in evasion of host innate immunity immediately after transmission (47). However, the essential contribution of OspC to early mammalian infection by B. burgdorferi remains undefined.Microorganisms induce a variety of responses from the skin epithelial cells of their hosts, including the production of antimicrobial peptides, which are recognized as integral components of the innate immune system (20, 22). Defensins and cathelicidins comprise two major families of cationic antimicrobial peptides secreted by human and other mammalian skin neutrophils (20). Mouse neutrophils lack α defensins (14, 24), but about 30 cathelicidin members have been identified in various mammalian species, including mice (21, 50). These small, cationic, amphipathic molecules are primarily stored as inactive propeptides in the secretory granules of skin neutrophils. The mature bioactive peptides assume an α-helical structure in solution and preferentially interact with negatively charged cell surface components of a broad spectrum of bacteria and fungi, in which they disrupt cell membrane integrity (6, 9, 12, 20, 34). The importance of the sole murine cathelicidin, known as mCRAMP (mouse cathelin-related antimicrobial peptide) (19, 36), to innate host defense is well established, and mCRAMP has been shown to provide protection against bacterial skin infections in mice (33).     

Intra- and Extracellular Activities of Dicloxacillin against Staphylococcus aureus In Vivo and In Vitro

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT_MIC]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT_MIC is the index that is the most predictive of the outcome of infection both intra- and extracellularly.Staphylococcus aureus is a major cause of both community- and hospital-acquired infections (28, 30), which range from simple and uncomplicated skin and wound infections (2, 24) to more serious and life-threatening situations such as pneumonia (15, 36), endocarditis (16, 37), osteomyelitis (13, 25), and meningitis (34). S. aureus infections often show poor and slow responses to therapy, with recurrences and ensuing mortality (8, 9, 27, 37, 38, 46). These responses could be caused by the ability of the bacteria to invade and survive inside cells (5, 10, 21, 22, 31, 32). Intracellular antimicrobial activity depends on both drug- and bacterium-related factors (penetration, accumulation, subcellular bioavailability, expression of activity in the local environment, and the state of responsiveness of the organisms [42, 44]). In general, intracellular antimicrobial activity is markedly impaired compared to the activity seen in broth or the extracellular milieu (3, 39, 45), although we know about situations in which the opposite is true (7). Thus, the direct assessment of antibiotic activity in the pertinent models is warranted. Several in vitro models with either human or animal cells have been developed to study the intracellular activities of antibiotics (3, 6, 14, 21, 35, 41), and a corresponding in vivo model (a modified version of a murine peritonitis model) has recently been described (39). We have now combined these models and report here our results obtained by using dicloxacillin (DCX) as a prototype of antistaphylococcal β-lactam antibiotics. Isoxazolyl penicillins have usually been preferred for the treatment of methicillin-susceptible S. aureus (MSSA) infections (2, 20, 26, 30). DCX has been the main choice in Denmark and many other countries due to its stability against penicillinases, low level of toxicity, and availability for both oral and intravenous administration (19). We examined the intra- and extracellular time- and concentration-kill relationships for two MSSA strains in vitro using macrophages and performed corresponding intra- and extracellular dose-kill studies with the murine peritonitis model. In combination with pharmacokinetic (PK) analysis and measurement of the amount of free drug (f) versus protein-bound drug, this allowed us to estimate which PK/pharmacodynamic (PD) index best predicts the efficacy of DCX intra- and extracellularly.(Part of this study was presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 2006, San Francisco, CA.)     

Risk Factors for Fluconazole-Resistant Candidemia

Previous studies have sought to determine the risk factors associated with candidemia caused by non-albicans Candida spp. or with potentially fluconazole-resistant Candida spp. (C. glabrata and C. krusei). Non-albicans Candida strains are a heterogeneous group that includes species with different levels of virulence, and only a limited number of C. glabrata isolates are resistant to fluconazole. We set out to identify the risk factors associated with microbiologically proven fluconazole-resistant candidemia. A prospective study including adult patients with candidemia was performed. Data were collected on patient demographics; underlying diseases; exposure to corticosteroids, antibiotics, or fluconazole; and invasive procedures. Risk factors associated either with non-albicans Candida spp. or potentially fluconazole-resistant Candida spp. (C. glabrata or C. krusei) or with Candida spp. with microbiologically confirmed fluconazole resistance were assessed using logistic regressions. We included 226 candidemia episodes. Non-albicans Candida isolates accounted for 53.1% of the fungal isolates, but only 18.2% of the cases were caused by potentially fluconazole-resistant organisms. Thirty isolates exhibited microbiologically confirmed fluconazole resistance. The multivariate analysis revealed that independent predictors associated with fluconazole-resistant Candida spp. were neutropenia (odds ratio [OR] = 4.94; 95% confidence interval [CI] = 1.50 to 16.20; P = 0.008), chronic renal disease (OR = 4.82; 95% CI = 1.47 to 15.88; P = 0.01), and previous fluconazole exposure (OR = 5.09; 95% CI = 1.66 to 15.6; P = 0.004). Independently significant variables associated with non-albicans Candida bloodstream infection or with potentially fluconazole-resistant Candida spp. did not include previous fluconazole exposure. We concluded that prior fluconazole treatment is an independent risk factor only for candidemia caused by microbiologically confirmed fluconazole resistant species. Our findings may be of value for selecting empirical antifungal therapy.Bloodstream infections (BSIs) due to Candida species are a serious complication in hospitalized patients. Candidemia is associated with a high mortality rate and has obvious effects on resource use (11). Over the past few decades, the incidence of candidemia has increased markedly (2, 3). Moreover, the increment of candidemia caused by Candida species other than Candida albicans is a common finding in recent series (3, 12). These species include C. glabrata and C. krusei, which tend to be more resistant to fluconazole and therefore present a particular challenge for clinical management.Diverse studies highlight the importance of early and appropriate empirical therapy in invasive Candida infection (14, 21). Fluconazole is currently considered the initial therapy for most adult patients with candidemia (20). As the selection of empirical therapy is driven in large part by the likely epidemiology, there is a need to identify patients at risk of candidemia caused by species resistant to fluconazole in order to begin with adequate empirical antifungal therapy.In order to provide guidance to clinicians to initiate empirical treatment for candidemia, prior studies have addressed risk factors for candidemia caused by non-albicans Candida spp. (1, 9, 10, 23) or by potentially fluconazole-resistant organisms (Candida glabrata and Candida krusei) (16, 23, 28) but have not taken into consideration in vitro resistance to fluconazole. In these studies, prior fluconazole exposure was an independent risk factor for candidemia caused by non-albicans Candida species (1) or by potentially fluconazole-resistant species (23), whereas other studies did not find this association (16, 28).Therefore, to the best of our knowledge, the risk factors for microbiologically proven fluconazole-resistant Candida spp. BSIs have not been comprehensively studied. We conducted the prospective single-center study described here to identify the variables associated with the diagnosis of fluconazole-resistant candidemia. Our working hypothesis was that fluconazole administration is an independent risk factor only in the emergence of BSIs caused by fluconazole-resistant Candida.     

Imcroporin,a New Cationic Antimicrobial Peptide from the Venom of the Scorpion Isometrus maculates

The pace of resistance against antibiotics almost exceeds that of the development of new drugs. As many bacteria have become resistant to conventional antibiotics, new drugs or drug resources are badly needed to combat antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial peptides, rich sources existing in nature, are able to effectively kill multidrug-resistant pathogens. Here, imcroporin, a new antimicrobial peptide, was screened and isolated from the cDNA library of the venomous gland of Isometrus maculates. The MIC of imcroporin against MRSA was 50 μg/ml, 8-fold lower than that of cefotaxime and 40-fold lower than that of penicillin. Imcroporin killed bacteria rapidly in vitro, inhibited bacterial growth, and cured infected mice. These results revealed that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.The war between human beings and bacteria is still going on. On one hand, conventional antibiotics defend against bacterial infection. On the other hand, antibiotic resistance fights back. Among the pathogens of concern, methicillin-resistant Staphylococcus aureus (MRSA) takes priority. From 1999 to 2005, the estimated number of MRSA-related hospitalizations in the United States more than doubled, from 127,036 to 278,203, and MRSA-related deaths averaged about 5,500 per year (range, 3,809 to 7,372) (27). Vancomycin was most commonly used in the past 2 decades to treat MRSA infections. However, vancomycin-intermediate S. aureus has emerged (1, 3, 34). Therefore, new kinds of antimicrobial agents are badly needed.Cationic host defense peptides play an important role in the innate immune response. These peptides have potent antimicrobial activity against gram-positive and gram-negative bacteria, fungi, parasites, and some viruses (19). Such peptides can be constitutively expressed or induced by bacteria in many organisms (20, 23, 29, 38). They are widely distributed in nature, from insects and plants to highly evolved animal species with more complex immune systems (6, 7). More than 2 decades ago, these defense molecules were initially isolated from insect lymph, the skin of frogs, and mammalian neutrophil granules and were demonstrated to have antibacterial properties. Since then, interest in the distribution and application of these peptides has been escalating, leading to the discovery of more than 1,300 cationic peptides from numerous species (16, 31, 37).Cationic antimicrobial peptides are defined as peptides of 12 to 50 amino acids with a net positive charge of 2 to 9 (5, 7, 20). Despite their small size and common physicochemical features, cationic antimicrobial peptides exhibit a range of structures. The peptides can be divided into four groups according to their secondary structures: amphipathic α-helices, amphiphilic peptides with two to four β-strands, loop structures, and extended structures (7, 17, 26). Moreover, many cationic antimicrobial peptides have activity against antibiotic-resistant bacteria. As antibiotic-resistant pathogens have become a threat to human health (2, 4, 25, 36), the cationic antimicrobial peptides are one of the new strategies against infective diseases (21, 22, 31, 33).Several cationic antimicrobial peptides have been found in scorpion hemolymph and venom (9, 15), including hadrurin (35), scorpine (10), opistoporins (30), parabutoporin (30), IsCTs (13, 28), pandinins (11), and mucroporin (12). In the present study, we isolated a cationic antimicrobial peptide, termed imcroporin, from the cDNA library of the venomous gland of the scorpion Isometrus maculates. Imcroporin showed potent growth-inhibitory activity against antibiotic-resistant gram-positive pathogens, but not gram-negative bacteria, and relatively low hemolytic activity against human erythrocytes. What is more, imcroporin killed the bacteria rapidly and cured infected mice. These results indicate that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.     

Defining the Role of Mutations in Plasmodium vivax Dihydrofolate Reductase-Thymidylate Synthase Gene Using an Episomal Plasmodium falciparum Transfection System

Plasmodium vivax resistance to antifolates is prevalent throughout Australasia and is caused by point mutations within the parasite dihydrofolate reductase (DHFR)-thymidylate synthase. Several unique mutations have been reported in P. vivax DHFR, and their roles in resistance to classic and novel antifolates are not entirely clear due, in part, to the inability to culture P. vivax in vitro. In this study, we use a homologous system to episomally express both wild-type and various mutant P. vivax dhfr (pvdhfr) alleles in an antifolate-sensitive line of P. falciparum and to assess their influences on the susceptibility of the recipient P. falciparum line to commonly used and new antifolate drugs. Although the wild-type pvdhfr-transfected P. falciparum line was as susceptible to antifolate drugs as the P. falciparum parent line, the single (117N), double (57L/117T and 58R/117T), and quadruple (57L/58R/61M/117T) mutant pvdhfr alleles conferred a marked reduction in their susceptibilities to antifolates. The resistance index increased with the number of mutations in these alleles, indicating that these mutations contribute to antifolate resistance directly. In contrast, the triple mutant allele (58R/61M/117T) significantly reversed the resistance to all antifolates, indicating that 61M may be a compensatory mutation. These findings help elucidate the mechanism of antifolate resistance and the effect of existing mutations in the parasite population on the current and new generation of antifolate drugs. It also demonstrates that the episomal transfection system has the potential to provide a rapid screening system for drug development and for studying drug resistance mechanisms in P. vivax.Of the four species of Plasmodium that commonly cause malaria in humans, Plasmodium vivax is the most widely distributed and can account for up to 80 million cases annually (25). Although P. vivax infections cause less mortality than P. falciparum, they do cause a debilitating disease that contributes to significant morbidity and economic loss in many regions where P. vivax is endemic (25). This is compounded by frequent relapses that can occur many times and for many months after the initial infection (6, 16, 22).Plasmodium vivax parasites are susceptible to most antimalarial drugs. However, over the last 20 years there have been many reports that highlight the significant increase in resistance of P. vivax malaria to chloroquine, the recommended first-line treatment of P. vivax, and/or sulfadoxine-pyrimethamine (SP) (1, 7, 9, 13, 29, 31, 41, 42). Although the determinant(s) for chloroquine resistance in P. vivax remains elusive, a genetic basis for antifolate resistance in P. vivax has been identified as polymorphisms in the P. vivax dihydrofolate reductase (PvDHFR) active site, the same mechanism observed in antifolate resistance in P. falciparum (2, 11, 12, 17, 18, 20, 21, 24, 39).In P. falciparum, point mutations within DHFR have been determined as the cause for antifolate resistance, such as pyrimethamine and cycloguanil resistance (4, 5, 8, 30, 32-36, 44, 45). It has been shown that resistance to antifolates results from the accumulation of mutations in the P. falciparum DHFR, principally A16V, N51I, C59R, S108N or S108T, and I164L.A large number of point mutations have been identified in PvDHFR, and some were reported to be prevalent in many areas of P. vivax endemicity (2, 12, 18, 20, 21, 39). A particular set of mutations (F57L + S58R + T61M + S117T) within the P. vivax DHFR was shown to correlate with SP treatment failures (18, 39) and to confer significant antifolate resistance when transfected into antifolate-sensitive P. falciparum (28). Resistance is due to alterations to the pyrimethamine binding site of PvDHFR that reduces parasite-drug interactions (23, 28). However, the contribution of these PvDHFR mutations to resistance to a variety of antifolates drugs is not clear.Due to the difficulty in maintaining P. vivax in in vitro cultures, most studies of the mutations within PvDHFR have been limited to surrogate biological systems such as yeast and Escherichia coli (17, 19, 24, 38). Although these systems have obvious experimental utility, they are different from Plasmodium in many biological aspects, particularly in membrane structures and transporters that can potentially affect the susceptibility to drugs. A recent publication (28) showed that the pvdhfr-ts quadruple mutant allele (57L + 58R + 61M + 117T) that is episomally expressed in P. falciparum provides significant protection against antifolates. This demonstrated an excellent potential of using P. falciparum as a biological system for the transgenic expression of pvdhfr-ts alleles to assess DHFR-TS interactions with antifolates.We report here the use of this P. falciparum expression system to assess the effect that specific mutations within the P. vivax DHFR have on conventional and new-generation antifolate drugs. The findings improve our understanding of the effect of various mutant pvdhfr alleles observed in the field on the parasite responses to current and new generations of antifolates, improve the prediction of malaria drug treatment outcome, and provide a useful tool for drug development.     

Human Antimicrobial Peptide LL-37 Induces MefE/Mel-Mediated Macrolide Resistance in Streptococcus pneumoniae

Macrolide resistance is a major concern in the treatment of Streptococcus pneumoniae. Inducible macrolide resistance in this pneumococcus is mediated by the efflux pump MefE/Mel. We show here that the human antimicrobial peptide LL-37 induces the mefE promoter and confers resistance to erythromycin and LL-37. Such induction may impact the efficacy of host defenses and of macrolide-based treatment of pneumococcal disease.Macrolides are in widespread use and are recommended as the first line of treatment for community-acquired pneumonia (CAP), except in areas compromised by high rates of macrolide resistance (25). Macrolide resistance is therefore a major concern in the treatment of Streptococcus pneumoniae, a major causative agent of CAP, sinusitis, otitis media, and meningitis. In S. pneumoniae, macrolide resistance is conferred primarily by the methylase ErmB (MIC ≥ 64 μg/ml) or the macrolide efflux pump Mef/Mel (1, 20, 30). Mef/Mel-mediated efflux has been shown to be specific for 14- and 15-membered macrolides (2, 32). Mef is encoded by two variants, either mefE or mefA, with mefE being more prevalent in the United States (2). MefE/Mel is encoded on the mobile genetic element mega (8) and has been shown to be inducible by 14- and 15-membered macrolides (1, 2, 32). MefE/Mel-mediated MICs for erythromycin range from 1 to 32 μg/ml and increase by 4-fold upon macrolide induction, on average (37). The clinical implications of low-level macrolide resistance (1 to 8 μg/ml) are controversial (3, 14, 17, 26). Recent studies provide evidence that infection with low-level macrolide-resistant pneumococci constitutes a risk factor for treatment failure (5, 4, 13, 22, 23). About 35% of all pneumococcal isolates from community-acquired respiratory tract infections in the Unites States are macrolide resistant, and Mef/Mel is present in more than 50% of the resistant isolates (15).Cationic antimicrobial peptides (CAMPs) are small cationic, amphiphilic peptides. The major mammalian CAMPs are cathelicidins and defensins, which are constitutively expressed in macrophages and neutrophils and inducibly produced by epithelial cells and at mucosal surfaces. They play an important role in host defense against bacterial infections and are a major component of the innate immune response (11, 19). Sublethal concentrations of CAMPs are known to alter the levels of bacterial gene expression (9). In this respect, we determined that the sole human cathelicidin, LL-37, could alter mefE and mel expression and that this alters pneumococcal susceptibility to macrolides and LL-37.     

Correlation between Azole Susceptibilities,Genotypes, and ERG11 Mutations in Candida albicans Isolates Associated with Vulvovaginal Candidiasis in China

The relationship between susceptibilities to fluconazole and itraconazole and microsatellite CAI genotypes were examined from a total of 154 Candida albicans isolates (97 isolates causing vulvovaginitis in Chinese women and 6 vaginal isolates and 51 oral cavity isolates from asymptomatic carriers). The two dominant genotypes, CAI 30-45 (45 isolates) and CAI 32-46 (33 isolates), associated with vulvovaginitis showed significantly different azole susceptibility patterns with strong statistical support. CAI 32-46 isolates were usually less susceptible to both fluconazole and itraconazole than CAI 30-45 isolates and than the oral isolates with other diversified CAI genotypes. Remarkably different mutation patterns in the azole target gene ERG11 were correspondingly observed among C. albicans isolates representing different genotypes and sources. Isolates with the same or similar CAI genotypes usually possessed identical or phylogenetically closely related ERG11 sequences. Loss of heterozygosity in ERG11 was observed in all the CAI 32-46 isolates but not in the CAI 30-45 isolates and most of the oral isolates sequenced. Compared with the ERG11 sequence of strain SC5314 ({"type":"entrez-nucleotide","attrs":{"text":"X13296","term_id":"2503","term_text":"X13296"}}X13296), two homozygous missense mutations (G487T and T916C) leading to two amino acid changes (A114S and Y257H) in Erg11p were found in CAI 32-46 isolates. The correlation between azole susceptibility and C. albicans genotype may be of potential therapeutic significance.Vulvovaginal candidiasis (VVC) is a common vaginal infection, affecting up to 75% of women of child-bearing age at least once in their lifetime (7, 21, 22). The most frequent cause of VVC is Candida albicans, which is responsible for 70 to 90% of vulvovaginitis cases. Non-C. albicans species of Candida, predominantly Candida glabrata, are responsible for the remainder of cases (21). On the basis of the severity of symptoms, frequency, and causative agents, VVC is usually classified as either uncomplicated (mild and sporadic) or complicated (recurrent, severe, or caused by non-C. albicans species) (7, 21). Ten to 20% of women suffer complicated VVC in their lifetime (21). When properly diagnosed, uncomplicated VVC may be treated easily and reliably. However, complicated VVC often causes long-term physical and mental discomfort, significant economic burden from treatments, and considerable negative effect on sexual relations (21-23).At present, prolonged suppressive therapy using fluconazole is recommended as the standard management for chronic, recurrent Candida vulvovaginitis (23). Therefore, there is a great concern about the emergence and spread of azole resistance of C. albicans isolates associated with VVC. Indeed, susceptibility testing of VVC-causing isolates has been performed in different countries and regions of the world (1, 2, 4, 5, 6, 13-15, 17, 18, 20, 24). Although relatively high frequencies of fluconazole- and/or itraconazole-resistant C. albicans isolates causing VVC have been observed in a few reports (13, 20, 24), most studies failed to identify any clear correlation between azole susceptibility and VVC association among C. albicans isolates (1, 2, 4, 5, 6, 14, 15, 17, 18).Recently, we compared the genotype distribution patterns among independent C. albicans isolates associated with VVC in Chinese women and those from various extragenital sites by using the polymorphic microsatellite locus CAI (8, 11). The results showed that the CAI genotypes of C. albicans isolates from extragenital sites were highly diversified. In contrast, isolates associated with VVC from unrelated patients were more homogeneous and belonged to only a few genotypes, with two genotypes, CAI 30-45 and CAI 32-46, being the most common. These two dominant genotypes were rarely found among isolates from extragenital sites (11). In addition, the distribution of the dominant genotypes correlated positively with the severity of VVC (8, 11). These results suggested that C. albicans isolates with genotypes CAI 30-45 and CAI 32-46 might be more virulent and/or more resistant to the commonly used azole drugs than those with other genotypes as causative agents of vaginal infection.Antifungal susceptibility testing using the Etest method revealed that the C. albicans isolates causing VVC in Chinese women were generally susceptible to fluconazole, amphotericin B, ketoconazole, and flucytosine; however, 19.1% of the isolates could be interpreted as being resistant to itraconazole in vitro. Interestingly, most of the itraconazole-resistant isolates belonged to a specific genotype (13). Contrary to the report described above, recent susceptibility testing and microsatellite typing of vulvovaginitis-causing Candida isolates from Europe did not find an association between azole resistance and any particular genotype cluster among C. albicans isolates (1). In the present study, fluconazole and itraconazole susceptibilities of the C. albicans isolates with the dominant genotypes CAI 30-45 and CAI 32-46 from VVC patients were compared with those of isolates possessing other minor genotypes and of isolates from the oral cavity by using the standard broth microdilution method. Furthermore, ERG11 (encoding lanosterol-14-α-demethylase, the target of azoles) gene sequences of C. albicans isolates representing different genotypes and sources were determined. The correlation between azole susceptibilities, genotypes, and ERG11 mutations was examined.