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DNA macrorestriction profiles and antifungal susceptibility of Candida (Torulopsis) glabrata
M G Cormican1, R J Hollis, M A Pfaller
1Department of Pathology, University of Iowa College of Medicine, Iowa City 52242, USA.
Abstract:
Candida (Torulopsis) glabrata is an emerging nosocomial pathogen that may be relatively resistant to fluconazole. A series of 75 isolates (blood, urine, tissue, and other sites) from 16 patients (1 to 12 isolates per patient) at a single university medical center were analyzed by pulsed field gel electrophoresis (PFGE) of restriction endonuclease digests of chromosomal DNA. The MICs of the isolates for amphotericin B, flucytosine, fluconazole, and itraconazole, were determined by a microdilution broth method. A preliminary study of seven restriction enzymes, three producing small fragments (Hinf I, Hind III, Eco RI) and four producing large fragments (Eag I, BssH II, Sfi I, Not I) identified Not I as giving interpretable banding patterns. Isolates were considered of different types if they differed by two or more bands. Sixteen distinct DNA types (A to P) were identified. Karyotyping was used an an additional technique to compare strains with a common PFGE type. Most patients were colonized or infected with a single type at multiple body sites and over time. One PFGE type was shared by four patients housed in different areas of the institution at different times. For two of these four patients, the karyotype was also indistintuishable. Five patients were each colonized with two distinct types. The MIC of the strains studied were amphotericin B 0.5-1.0 microgram/ml (MIC90 = 1.0 microgram/ ml), 5-fluorocytosine 0.25-->256 micrograms/ml (MIC90 = 2 micrograms/ml), fluconazole 0.25-->128 micrograms/ml (MIC90 = 32 micrograms/ml), and itraconazole 0.06-8.0 micrograms/ml). Molecular typing by PFGE using Not I digestion is a useful technique for epidemiological investigation as epidemiologically related isolates are generally identical and epidemiologically unrelated isolates are different by this method.
Insights
Candida glabrata, a growing hospital pathogen, shows potential resistance to fluconazole. Pulsed field gel electrophoresis (PFGE) effectively distinguished DNA types, aiding in tracking its spread and understanding antifungal resistance patterns.
Area of Science:
- Clinical Microbiology
- Infectious Diseases
- Molecular Epidemiology
Background:
- Candida glabrata is an emerging opportunistic pathogen frequently associated with hospital-acquired infections.
- This yeast species exhibits intrinsic or acquired resistance to azole antifungal agents, particularly fluconazole, complicating treatment strategies.
- Understanding the genetic diversity and transmission patterns of C. glabrata is crucial for effective infection control in healthcare settings.
Purpose of the Study:
- To investigate the genetic relatedness and molecular epidemiology of Candida glabrata isolates from a university medical center.
- To correlate genotypic profiles with antifungal susceptibility patterns, focusing on resistance to fluconazole.
- To evaluate the utility of pulsed field gel electrophoresis (PFGE) as a tool for tracking C. glabrata infections and colonization.
Main Methods:
- Seventy-five C. glabrata isolates from 16 patients were analyzed using pulsed field gel electrophoresis (PFGE) after Not I restriction endonuclease digestion of chromosomal DNA.
- Minimum Inhibitory Concentrations (MICs) for amphotericin B, flucytosine, fluconazole, and itraconazole were determined using a microdilution broth method.
- Karyotyping was employed as a secondary method for strain comparison within common PFGE types.
Main Results:
- Sixteen distinct DNA types (A-P) were identified among the C. glabrata isolates.
- Most patients harbored a single C. glabrata type across multiple body sites and over time; however, five patients were colonized with two distinct types.
- Antifungal susceptibility testing revealed a wide range of MICs, with significant variability for fluconazole (MIC90 = 32 µg/mL) and flucytosine (MIC90 = 2 µg/mL), indicating potential resistance.
- A single PFGE type was found to be shared among four patients in different hospital areas, suggesting potential transmission.
Conclusions:
- Pulsed field gel electrophoresis (PFGE) using Not I digestion is a highly discriminatory and valuable technique for the molecular typing and epidemiological investigation of Candida glabrata.
- The study highlights the potential for C. glabrata to colonize patients with multiple strains and the possibility of inter-patient transmission within a healthcare facility.
- The observed variability in antifungal susceptibility underscores the importance of molecular surveillance and susceptibility testing for guiding therapeutic decisions in C. glabrata infections.