Time-Kill Assay and Etest Evaluation for Synergy with Polymyxin B and Fluconazole against Candida glabrata

Abstract

Fluconazole-resistant Candida glabrata is an emerging pathogen that causes fungemia. Polymyxin B, a last-resort antibiotic used to treat multidrug-resistant Gram-negative bacterial infections, has been found to possess in vitro fungicidal activity and showed synergy with fluconazole against a single strain of C. glabrata. Since both agents may be used simultaneously in intensive care unit (ICU) patients, this study was performed to test for possible synergy of this combination against 35 C. glabrata blood isolates, using 2 methods: a time-kill assay and an experimental MIC-MIC Etest method. Thirty-five genetically unique C. glabrata bloodstream isolates were collected from 2009 to 2011, identified using an API 20C system, and genotyped by repetitive sequence-based PCR (rep-PCR). MICs were determined by Etest and broth microdilution methods. Synergy testing was performed using a modified bacterial Etest synergy method and time-kill assay, with final results read at 24 h. The Etest method showed synergy against 19/35 (54%) isolates; the time-kill assay showed synergy against 21/35 (60%) isolates. Isolates not showing drug synergy had an indifferent status. Concordance between methods was 60%. In vitro synergy of polymyxin B and fluconazole against the majority of C. glabrata isolates was demonstrated by both methods. The bacterial Etest synergy method adapted well when used with C. glabrata. Etest was easier to perform than time-kill assay and may be found to be an acceptable alternative to time-kill assay with antifungals. Candida glabrata has been a rising cause of candidemia in the past few years, second only to Candida albicans (1). According to the Centers for Disease Control and Prevention (CDC), candidemia is the fourth most common hospital-acquired infection in the United States (2). C. glabrata has been known to develop resistance to the azole family of antifungals, the most inexpensive and easily accessible medications to treat candidemia. Further data from the CDC have shown that 7% of Candida species have become fluconazole resistant (2). This development of resistance is most likely due to the routine use of azoles in treatment or prophylaxis. Other treatment options for Candida infections include amphotericin B and the echinocandin family (to which resistance by C. glabrata has occurred) (1). A U.S. surveillance study evaluating Candida isolates from San Francisco, CA, Atlanta, GA, Baltimore, MD, and Connecticut (from 1998 to 2010) determined that C. glabrata isolates are able to adapt to new environments to increase their fitness and antifungal resistance (3). Another surveillance study, evaluating developing resistance in fluconazolesusceptible isolates collected from 2008 to 2011, showed that among 2,329 Candida isolates, the prevalence of azole resistance was unchanged (7%), 32 of the isolates were echinocandin resistant, and 8 C. glabrata isolates were resistant to both fluconazole and echinocandins (4).Polymyxin B is a last-resort antibiotic used to treat multidrug-resistant Gram-negative bacte...

Antifungal Susceptibly Testing by Concentration Gradient Strip Etest Method for Fungal Isolates: A Review

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“…13 Recent scientific studies have reevaluated old antibiotics, such as chloramphenicol, tetracyclines, and polymyxins, traditionally used for bacterial infections, for their potential antifungal activity. [14][15][16][17] Based on literature and clinical experience, in this study, we evaluated the antifungal activity of a widely used antibacterial ophthalmic combination (Colbiocin; SIFI SpA, Catania, Italy) containing tetracycline (TET), chloramphenicol (CAF), and colistimethate sodium (CS), using in vitro tests and an ex vivo mycotic keratitis model. Specific assays were also carried out to understand the mechanism of action.…”