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. 2007 May;45(5):1455-62.
doi: 10.1128/JCM.00243-07. Epub 2007 Feb 28.

Genotyping of Candida orthopsilosis clinical isolates by amplification fragment length polymorphism reveals genetic diversity among independent isolates and strain maintenance within patients

Arianna Tavanti  1 , Lambert A M Hensgens, Emilia Ghelardi, Mario Campa, Sonia Senesi
Affiliations

Genotyping of Candida orthopsilosis clinical isolates by amplification fragment length polymorphism reveals genetic diversity among independent isolates and strain maintenance within patients

Arianna Tavanti et al. J Clin Microbiol. 2007 May.

Abstract

Candida parapsilosis former groups II and III have recently been established as independent species named C. orthopsilosis and C. metapsilosis, respectively. In this report, 400 isolates (290 patients) previously classified as C. parapsilosis by conventional laboratory tests were screened by BanI digestion profile analysis of the secondary alcohol dehydrogenase gene fragment and by amplification fragment length polymorphism (AFLP). Thirty-three strains collected from 13 patients were identified as C. orthopsilosis, thus giving the first retrospective evidence that C. orthopsilosis was responsible for 4.5% of the infections/colonization attributed to C. parapsilosis. AFLP was proven to unambiguously identify C. orthopsilosis at the species level and efficiently delineate intraspecific genetic relatedness. A high percentage of polymorphic AFLP bands was observed for independent isolates collected from each patient. Statistical analysis of the pairwise genetic distances and bootstrapping revealed that clonal reproduction and recombination both contribute to C. orthopsilosis genetic population structure. AFLP patterns of sequential isolates obtained from two patients demonstrated that a successful strain colonization within the same patient occurred, as revealed by strain maintenance in various body sites. No association between AFLP markers and drug resistance was observed, and none of the clinical C. orthopsilosis isolates were found to produce biofilm in vitro.

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Figures

FIG. 1.
FIG. 1.
(A) Representative gel of BanI restriction analysis of SADH PCR products in 12 clinical isolates. Lanes: M, 100-bp DNA ladder molecular weight marker; 2, C. metapsilosis; 1, 3 to 9, 11, and 12, C. parapsilosis; 10, C. orthopsilosis (Cp 25). (B) Positions of BanI sites within the 716-bp SADH amplicon for the three species.
FIG. 2.
FIG. 2.
(A) AFLP profiles obtained from strains of 16 different fungal species. Lanes: 1 and 2, Candida orthopsilosis (ATCC 96139 and ATCC 96140); 3 and 4, C. metapsilosis (ATCC 96144 and CP5); 5 and 6, C. parapsilosis (ATCC 22019 and CBS 604); lanes 7 and 8, C. albicans (SC5314 and DSM 1386); 9, C. tropicalis (DSM 11953); 10 and 11, C. dubliniensis (CPx and CP142); 12 and 13, C. lusitaniae (DSM 70102 and CP153); 14 and 15, C. rugosa (DSM 70761 and CP301C); 16, C. krusei (DSM 6128); 17, S. cerevisiae (DSM 70449); 18, C. glabrata (DSM 11226); 19, C. famata (DSM 70590); 20, C. pulcherrima (DSM 70336); 21, Cryptococcus neoformans (DSM 11959); 22, Aspergillus fumigatus (DSM 819); 23, A. terreus (DSM 826); M, molecular weight marker (50- to 500-base ladder). (B) UPGMA dendrogram indicating genetic relatedness among the different fungal species. SAB values of 1 indicate undistinguishable strains.
FIG. 3.
FIG. 3.
(A) AFLP profiles obtained from 33 clinical isolates of Candida orthopsilosis (lanes 3 to 35) and two C. orthopsilosis reference strains tested in duplicate. The loading order for the indicated lanes is as follows: 1, ATCC 96140; 2, ATCC 96139; 3 to 21, sequential isolates from patient P12; 22, CP25 (patient P1); 23, CP47 (patient P2); 24, CP85 (patient P3); 25, CP125 (patient P4); 26, CP185 (patient P5); 27 to 29, sequential isolates from patient P13; 30, CP296 (patient P9); 31, CP331 (patient P10); 32, CP344 (patient P11); 33, CP287 (patient P6); 34, CP288 (patient P7); 35, CP289 (patient P8); 36, ATCC 96139; 37, ATCC 69140; M, molecular weight marker (50- to 500-base ladder). (B) UPGMA dendrogram indicating genetic relatedness among the different C. orthopsilosis isolates. SAB values of 1 indicate undistinguishable strains.
FIG. 4.
FIG. 4.
Distribution of pairwise comparisons of AFLP genotypes for 15 independent C. orthopsilosis isolates. (A) Bar graph of the frequency distribution of pairwise distances among AFLP genotypes. Recombining organisms show a normal distribution, while the high number of close relatives (n = 28) suggests that clonality also occurs (30). (B) Bar graph of the Ia values obtained for our data set.
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