The Fourier-transform infrared spectroscopy-based method as a new typing tool for Candida parapsilosis clinical isolates

Abstract

The Fourier-transform infrared spectroscopy-based IR Biotyper is a straightforward typing tool for bacterial species, but its use with Candida species is limited. We applied IR Biotyper to Candida parapsilosis, a common cause of nosocomial bloodstream infection (BSI), which is aggravated by the intra-hospital spread of fluconazole-resistant isolates. Of 59 C. parapsilosis isolates studied, n = 56 (48 fluconazole-resistant and 8 fluconazole-susceptible) and n = 3 (2 fluconazole-resistant and 1 fluconazole-susceptible) isolates, respectively, had been recovered from BSI episodes in 2 spatially distant Italian hospitals. The latter isolates served as an outgroup. Of fluconazole-resistant isolates, n = 40 (including one outgroup) harbored the Y132F mutation alone and n = 10 (including one outgroup) harbored both Y132F and R398I mutations in the ERG11-encoded azole-target enzyme. Using a microsatellite typing method, which relies on the amplification of genomic short tandem repeats (STR), two major clusters were obtained based on the mutation(s) (Y132F or Y132F/R398I) present in the isolates. Regarding IR Biotyper, each isolate was analyzed in quintuplicate using an automatic (i.e., proposed by the manufacturer's software) or tentative (i.e., proposed by us) cutoff value. In the first case, four clusters were identified, with clusters I and II formed by Y132F or Y132F/R398I isolates, respectively. In the second case, six subclusters (derived by the split of clusters I and II) were identified. This allowed to separate the outgroup isolates from other isolates and to increase the IR Biotyper typeability. The agreement of IR Biotyper with STR ranged from 47% to 74%, depending on type of cutoff value used in the analysis. IMPORTANCE Establishing relatedness between clinical isolates of Candida parapsilosis is important for implementing rapid measures to control and prevent nosocomial transmission of this Candida species. We evaluated the FTIR-based IR Biotyper, a new typing method in the Candida field, using a collection of fluconazole-resistant C. parapsilosis isolates supposed to be genetically related due to the presence of the Y132F mutation. We showed that IR Biotyper was discriminatory but not as much as the STR method, which is still considered the method of choice. Further studies on larger series of C. parapsilosis isolates or closely related Candida species will be necessary to confirm and/or extend the results from this study.

Keywords: C. parapsilosis; Erg11p mutation; FTIR spectroscopy; IR Biotyper; STR; fluconazole resistance; microsatellite genotyping.

Fig 1

STR typing of C. parapsilosis isolates with (n = 50) or without (n = 9) azole-resistance associated Erg11p mutations. Dendrogram was based on the analysis of STR markers, three trinucleotide (M3) and three hexanucleotide (M6), which generated two series of microsatellite genotypes. The scale bar (visible in the upper left corner) indicates the similarity percentages. Two major clusters, namely, STR I and STR II, were identified.

Fig 2

FTIR typing of C. parapsilosis isolates with (n = 50) or without (n = 9) azole-resistance associated Erg11p mutations. Dendrogram was based on the IR Biotyper analysis of spectra obtained from replicate measurements for each isolate. The vertical line represents the automatic cutoff value (0.967). Four clusters (two major and two minor), namely, IR I to IR IV, were identified.

Fig 3

FTIR typing of C. parapsilosis isolates with (n = 50) or without (n = 9) azole-resistance-associated Erg11p mutations. Dendrogram was based on the IR Biotyper analysis of spectra obtained from replicate measurements for each isolate. The vertical line represents the tentative cutoff value (0.995). Six subclusters (derived, respectively, from clusters IR I and IR II; see Fig. 2), namely, IR Ia to IR Id and IR IIa to IR IIb, were identified.

Fig 4

FTIR spectral profiles of fluconazole-resistant (Y132F or Y132F/R398I mutation(s) harboring) or fluconazole-susceptible C. parapsilosis isolates. Different colors (green, pink, or red) indicate the three isolates, respectively. Profiles overlap and only subtle differences in absorbance are visible. Spectra were acquired in the mid-IR (4,000–400 cm⁻¹), which includes spectral windows corresponding to the absorption of lipids (3,000–2,800 cm⁻¹, dominated by fatty acids), proteins and peptides (1,800–1,500 cm⁻¹, dominated by amides), or polysaccharides (1,200–900 cm⁻¹, dominated by carbohydrates).