**1. Introduction**

*Candida glabrata* is one of the most common causes of systemic fungal infection (candidemia), surpassed only by *Candida albicans* [1–3]. It is the second most common isolated yeas<sup>t</sup> in the United States of America and the third in Europe, after *Candida parapsilosis*, accounting for 20% of candidemia [2,4]. As a commensal yeast, *C. glabrata* colonizes and adapts to many different niches in the human body and can be isolated from the mucosae of healthy individuals [2,5]. Yet, as an opportunistic pathogen, this fungus can also be the point of origin for mucosal infections and severe candidemia. Its biofilm-forming ability and the ability to rapidly acquire resistance to antifungals (especially to azoles) [2,5,6], which in many cases can be further increased by genetic and genomic

mutations (e.g., polymorphisms, the formation of new chromosomes, karyotype variations) [7–9], may contribute to increased virulence.

Risk factors for the development of invasive *C. glabrata* infections in human patients comprise immunosuppression (e.g., cancer chemotherapy, human immunodeficiency virus (HIV) infection, diabetes mellitus, neutropenia), mucosal colonization by *Candida* spp., the use of indwelling medical devices (e.g., vascular catheters), and gastrointestinal surgery [10–12].

During infection, *C. glabrata* virtually colonizes all sites and organs, which reveals a high capacity to adapt to the many different niches inside the human host [1]. Oral and systemic *C. glabrata* infections have high associated morbidity and mortality [13–15] and the rise in incidence infections caused by this yeas<sup>t</sup> is to some extent attributable to its ability to tolerate or resist many antifungals commonly used in clinical practice [2,16,17]. The occurrence of oral candidiasis related to *C. glabrata* is increasing [15,18]. Although *C. glabrata* colonization does not always lead to infection, it is a foreword to infection when the risk of systemic infection is elevated, or the host immunity is compromised. *C. glabrata* infections are a major challenge [15,19,20]. The good biofilm-forming ability and raised enzymatic activity of *C. glabrata* are two of the most important features favoring oral and systemic candidiasis. In fact, biofilms can be formed on both biotic (e.g., gastrointestinal or mouth mucosae) and abiotic surfaces (e.g., indwelling medical devices) [21,22] and biofilm cells are recognized to be more resistant to antifungal treatment than planktonic cells, as well as responsible for more severe infections [2,23–25]. Systemic candidiases are the most prevalent invasive mycoses worldwide with mortality rates close to 40% and *C. glabrata* is frequently recognized as a causative agen<sup>t</sup> [26]. In nearly all these cases, the infections are related to the use of a medical device and biofilm formation on its surface [20]. The contamination of medical devices (mostly catheters) or infusion fluids can occur from the skin of the patient, the hands of health professionals [27], or by migration into medical devices from a previous lesion. Less commonly, *Candida* spp. that commensally colonize the gastrointestinal tract switch to having a pathogenic behavior, being able to infiltrate the intestinal mucosa, disseminate through the bloodstream, and colonize medical devices endogenously (this is more common in cancer patients, since chemotherapy harms the mucosa) [28]. Depending on the clinical situation, the removal of medical devices can be recommended in patients with disseminated *Candida* spp. infection to enable pathogen eradication and to improve the prognosis [29,30]. In contrast, experimental intravenous infection of laboratory animals with *C. glabrata* does not usually cause mortality, since it appears that this species has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts [1,31].

Because of the high probability of innate resistance to azoles, echinocandins are recommended as first-line therapy against *C. glabrata* candidemia [32]. Nonetheless, and worryingly, *C. glabrata* is the first *Candida* spp. for which resistance to echinocandins has been identified and described [33,34]. Recently, case reports of echinocandin-resistant *C. glabrata* subsequent to different echinocandin therapies are becoming more common [35–41]. Indeed, one third of those isolates may be multidrug resistant [42] and have specific mutations in one of two "hot spot" regions of the *FKS1* or *FKS2* (1,3-β-glucan synthase) genes, which encode a subunit of the β-1,3- D glucan synthase protein, a target of echinocandins [35,43–45].

Therefore, in this work, a simulation of a hematogenously disseminated *C. glabrata* infection derived exclusively from biofilm cells (as occurs in catheter infections) was performed. CD1 mice were infected with 48 h-biofilm cells of the wild type *C. glabrata* strain ATCC2001, and then treated with the echinocandins caspofungin (Csf) and micafungin (Mcf) in order to evaluate organ fungal burdens after 72 h, the efficacy of each drug after two administrations, and the associated inflammatory response.

## **2. Experimental Section**

## *2.1. Ethics Statement*

This study was performed in strict accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific

Purposes (ETS 123), the 86/609/EEC directive, and Portuguese rules (DL 129/92). All experimental protocols were approved by the competent national authority (Direcção-Geral de Veterinária), document 0420/000/000/2010. Female CD1 mice, 8–12 weeks old, were purchased from Charles River (Barcelona, Spain) and kept under specific pathogen-free conditions at the Animal Facility of the Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal. Mice were maintained in individually ventilated cages (five animals per cage) with corncob bedding, and under controlled conditions of temperature (21 ± 1 ◦C), relative humidity (between 45 and 65%), and light (12 h light/dark cycle). Mice had ad libitum access to food and water. Hiding and nesting materials were provided for enrichment. All procedures such as cage changing, water and food supply, as well as intravenous and intraperitoneal injections were always performed during the day cycle (between 7 a.m. and 7 p.m.).

#### *2.2. Organisms and Growth Conditions*

One strain of the American Type Culture Collection (ATCC), *C. glabrata* ATCC2001, was subcultured on Sabouraud dextrose agar (SDA) (Merck, Darmstadt, Germany) for 24 h at 37 ◦C. Cells were then inoculated in Sabouraud dextrose broth (SDB) (Merck, Darmstadt, Germany) and incubated for 18 h at 37 ◦C under agitation at 120 rpm. Biofilms were formed in 24-well polystyrene microtiter plates (Orange Scientific, Braine-l'Alleud, Belgium) [46]. For this, 1000 μL of the yeas<sup>t</sup> cell suspension (1 × 10<sup>5</sup> cells/mL) was added to each well and incubated for 24 h. After 24 h, 500 μL of RPMI 1640 was removed and an equal volume of fresh medium was carefully added. Biofilms allowed to grow, under the same temperature and agitation conditions, for an additional 24 h. After this time (total 48 h), all media were removed and the biofilms carefully washed to remove non-adhered cells. Biofilms were scraped from the 24-well plates, resuspended in ultra-pure water, sonicated (Ultrasonic Processor, Cole-Parmer, IL, USA) for 30 s at 30 W, and then suspension vortexed for 2 min. The suspension was centrifuged at 5000 *g* for 5 min at 4 ◦C, as previously optimized [46,47]. The pellets of the biofilm cells were then suspended in RPMI 1640 and the cellular density was adjusted to 5 × 10<sup>8</sup> cells/mL using a Neubauer counting chamber.

## *2.3. Antifungal Drugs*

Csf and Mcf were kindly provided by MSD® and Astellas®, respectively. Aliquots of 5000 mg/L were prepared using dimethyl-sulfoxide (DMSO). The final concentrations used were prepared with pyrogen-free phosphate buffer saline (PBS) for both drugs.

#### *2.4. Murine Model of Hematogenously Disseminated Infection*

*Candida glabrata* inoculum was prepared following previously described procedures [47,48]. The number of cultivable cells was assessed by colony forming units (CFU) counting and were injected intravenously in the lateral tail vein, with the support of a restrainer. Sample size was determined based on the results of preliminary experiments. On day 0, adult CD1 mice, randomly allocated to each experimental group, received 200 μL of *C. glabrata* biofilm cell suspensions containing 5 × 10<sup>8</sup> CFU i.v. via the tail vein. Control mice were injected intravenously with 200 μL of pyrogen-free PBS. Treatment with the echinocandins started 24 h post-inoculation and was administered intraperitoneally (i.p.) with a volume of 0.5 mL at 24 and 48 h post-inoculation. Doses were as follows: caspofungin 6 mg/kg and micafungin 12 mg/kg. This experimental scheme (days and dosages) were chosen on the basis of previous pharmacodynamic studies of echinocandins against *C. glabrata* and a need to reach drug exposures in mice that were comparable to those in humans receiving currently licensed echinocandin regimens [32,49,50]. Liver and kidneys were aseptically removed, weighed, homogenized, and quantitatively cultured on Sabouraud dextrose agar (Difco) at 37 ◦C. Values are expressed as log CFU per gram of liver. Two independent experiments were performed, with at least five animals per infected group.
