Biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface. Nearly all device-associated infections involve growth in the biofilm life style. Biofilm communities have characteristic architecture and distinct phenotypic properties. The most clinically important phenotype involves extraordinary resistance to antimicrobial therapy, making biofilm infections very difficulty to cure without device removal. The current studies examine drug resistance in Candida albicans biofilms. Similar to previous reports, we observed marked fluconazole and amphotericin B resistance in a C. albicans biofilm both in vitro and in vivo. We identified biofilm-associated cell wall architectural changes and increased ␤-1,3 glucan content in C. albicans cell walls from a biofilm compared to planktonic organisms. Elevated ␤-1,3 glucan levels were also found in the surrounding biofilm milieu and as part of the matrix both from in vitro and in vivo biofilm models. We thus investigated the possible contribution of ␤-glucans to antimicrobial resistance in Candida albicans biofilms. Initial studies examined the ability of cell wall and cell supernatant from biofilm and planktonic C. albicans to bind fluconazole. The cell walls from both environmental conditions bound fluconazole; however, four-to fivefold more compound was bound to the biofilm cell walls. Culture supernatant from the biofilm, but not planktonic cells, bound a measurable amount of this antifungal agent. We next investigated the effect of enzymatic modification of ␤-1,3 glucans on biofilm cell viability and the susceptibility of biofilm cells to fluconazole and amphotericin B. We observed a dose-dependent killing of in vitro biofilm cells in the presence of three different ␤-glucanase preparations. These same concentrations had no impact on planktonic cell viability. ␤-1,3 Glucanase markedly enhanced the activity of both fluconazole and amphotericin B. These observations were corroborated with an in vivo biofilm model. Exogenous biofilm matrix and commercial ␤-1,3 glucan reduced the activity of fluconazole against planktonic C. albicans in vitro. In sum, the current investigation identified glucan changes associated with C. albicans biofilm cells, demonstrated preferential binding of these biofilm cell components to antifungals, and showed a positive impact of the modification of biofilm ␤-1,3 glucans on drug susceptibility. These results provide indirect evidence suggesting a role for glucans in biofilm resistance and present a strong rationale for further molecular dissection of this resistance mechanism to identify new drug targets to treat biofilm infections.

Numerous factors have been theorized to affect the development of antimicrobial resistance, including those specific to the host, the organism, the environment, the drug, and the drug prescriber. One variable under the control of the prescriber is the drug dosing regimen. Dosing regimens can vary in dose level, dosing interval, and treatment duration. The current studies examined the relationships between antimicrobial dosing regimens and resistance development by use of an in vivo model. A murine model of systemic Candida albicans infection was used to examine resistance emergence during exposure to the triazole antifungal fluconazole.Data from this experimental model demonstrated that the more frequently administered dosing prevented selection of the isogenic resistant cell populations. Conversely, dosing regimens producing prolonged sub-MIC effects appeared to contribute to the outgrowth of isogenic resistant strains. The association between dosing and resistance emergence observed in the current investigation is disparate from that described for antimicrobial compounds with cidal killing characteristics. The inhibitory or static antimicrobial activity of the triazole compounds may explain these differences.The rapid development and spread of antimicrobial resistance has become an increasingly serious public health problem in a wide range of infectious diseases (3,28,29,37,38,44,45,53,58). New drugs for the treatment of these resistant infections are unlikely to appear soon enough and in sufficient numbers to solve many of the resistance problems. Thus, it is imperative to understand the factors that lead to the evolution of resistance and to design strategies to prevent or delay the emergence of antimicrobial-resistant pathogens.The need to stem the growing problem of antimicrobial resistance has prompted multiple calls for change in the use of antimicrobial agents to maximize the life spans of these drugs (6,8,10,16,21,22,30,31,38,42,53,55). However, the relationship in the context of use pattern and resistance development is complex and remains, for the most part, undefined. One dosing regimen approach that has been shown to reduce the amplification of resistant strains involves the use of large, infrequent doses of antimicrobials to eliminate not only the susceptible populations but also any resistant mutants (7,13,15,18,25,26,28). The success of this dosing strategy has been demonstrated with a few antimicrobial drug class-organism combinations in which antimicrobial drugs exhibit extensive organism killing or "cidal" activity. For example, Jumbe et al. used an in vivo model system to define the fluoroquinolone concentration likely to select for resistant cell populations (25). However, studies have not considered the large group of antimicrobials that exhibit only inhibitory or "static" effects. These drugs are unable to effectively eliminate pathogen populations. Drug effectiveness is primarily the result of limiting additional organism generations. It is possible that alternative dosing strategies wil...

Biofilms are microbial communities that are associated with solid surfaces such as intravascular catheters. Candida species are a major cause of medical device-associated infections. Twenty percent to 70% of all candidemias are associated with this biofilm process. Diagnosis and effective treatment of Candida device-associated infections requires removal of the involved device. The ability to identify a biofilm device infection before catheter removal may obviate removal of a substantial number of devices. Prior studies in our laboratory identified cell wall changes (specifically, increased beta -1,3 glucan) associated with biofilm, compared with planktonic C. albicans. Both in vitro and in vivo (catheter) biofilm models were used to determine whether biofilm cells secreted more beta -1,3 glucan and whether these differences could be used to discern the presence of a Candida biofilm infection with 3 species (C. albicans, C. glabrata, and C. parapsilosis). A limulus lysate assay was used to quantify beta -1,3 glucan in supernatants from planktonic or biofilm cultures and in the serum of rats with an intravascular catheter biofilm infection or disseminated candidiasis. beta -1,3 glucan was detected from both in vitro and in vivo models from each condition. However, the concentrations of beta -1,3 glucan from the biofilm conditions were 4-10-fold greater in vitro (P<.001) and were 10-fold greater in vivo (P<.001), despite equal or fewer numbers of cells in the biofilm conditions. These results suggest the secreted polysaccharide beta -1,3 glucan may serve as a useful tool for the diagnosis of Candida biofilm and device-associated infections.

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Pharmacodynamics (PD) considers the relationship between drug exposure and effect. The two factors that have been used to distinguish the PD behaviors of antimicrobials are the impact of concentration on the extent of organism killing and the duration of persistent microbiologic suppression (postantibiotic effect). The goals of these studies were (i) to examine the relationship between antimicrobial PD and gene expression and (ii) to gain insight into the mechanism of fluconazole effects persisting following exposure. Microarrays were used to estimate the transcriptional response of Candida albicans to a supra-MIC F exposure over time in vitro. Fluconazole at four times the MIC was added to a log-phase C. albicans culture, and cells were collected to determine viable growth and for microarray analyses. We identified differential expression of 18% of all genes for at least one of the time points. More genes were upregulated (n ‫؍‬ 1,053 [16%]) than downregulated (174 [3%]). Of genes with known function that were upregulated during exposure, most were related to plasma membrane/cell wall synthesis (18%), stress responses (7%), and metabolism (6%). The categories of downregulated genes during exposure included protein synthesis (15%), DNA synthesis/repair (7%), and transport (7%) genes. The majority of genes identified at the postexposure time points were from the protein (17%) and DNA (7%) synthesis categories. In subsequent studies, three genes (CDR1, CDR2, and ERG11) were examined in greater detail (more concentration and time points) following fluconazole exposure in vitro and in vivo. Expression levels from the in vitro and in vivo studies were congruent. CDR1 and CDR2 transcripts were reduced during in vitro fluconazole exposure and during supra-MIC exposure in vivo. However, in the postexposure period, the mRNA abundance of both pumps increased. ERG11 expression increased during exposure and fell in the postexposure period. The expression of the three genes responded in a dose-dependent manner. In sum, the microarray data obtained during and following fluconazole exposure identified genes both known and unknown to be affected by this drug class. The expanded in vitro and in vivo expression data set underscores the importance of considering the time course of exposure in pharmacogenomic investigations.The time course of antimicrobial activity is dependent on a drug's pharmacokinetics and two major pharmacodynamic characteristics (2, 13). The first is the rate of organism killing and whether increasing drug concentrations enhance the rate and extent of killing. The second is the presence or absence of inhibitory effects on organism growth which persist after drug levels have fallen below the MIC. These persistent effects are termed postantibiotic effects (PAE) (3,4,5,9,14,16,29,38,39,40). The PAE pharmacodynamic phenomenon underscores the importance of investigating the effects of antimicrobial exposure over time. Drugs from the triazole class demonstrate time-dependent killing but prolonged persistent effects...