Novel 5-flucytosine-resistant clade of Candida dubliniensis from Saudi Arabia and Egypt identified by Cd25 fingerprinting

Abstract

DNA fingerprinting of Candida dubliniensis isolates using the species-specific probe Cd25 previously showed that this species consists of two distinct groups, termed Cd25 group I and Cd25 group II. The present study investigated the population structure of 30 C. dubliniensis oral isolates from Saudi Arabia and Egypt using Cd25 fingerprinting and rRNA gene internal transcribed spacer region-based genotyping. Cd25 fingerprinting analysis of these isolates revealed two distinct populations, the first of which consisted of 10 closely related genotype 1 isolates (average similarity coefficient [S(AB)] value, 0.86). The second population of 20 isolates was much more heterogeneous (average S(AB) value, 0.35) and consisted of two distinct subpopulations, one of which consisted of genotype 3 isolates (n = 13) and the other of genotype 4 isolates (n = 7). A mixed dendrogram generated from the fingerprint data from the 30 Saudi Arabian and Egyptian isolates, 5 Israeli isolates, and 51 previously characterized international isolates (32 of Cd25 group I and 19 of Cd25 group II) revealed the presence of three distinct main clades. The first corresponded to the previously described Cd25 group I and contained all the Saudi Arabian, Egyptian, and Israeli genotype 1 isolates mixed with international isolates. The second clade corresponded to the previously described Cd25 group II and contained three Israeli isolates, one genotype 2 isolate, one genotype 3 isolate, and a genotype 4 variant isolate, which were mixed with international isolates. The third clade has not been described before and consisted solely of the 20 Saudi Arabian and Egyptian genotype 3 and 4 isolates identified in this study and a previously described genotype 4 Israeli isolate. All 20 Cd25 group III isolates exhibited high-level resistance to 5-flucytosine (MIC > or = 128 microg/ml), whereas all Cd25 group I and Cd25 group II isolates tested (10 Saudi Arabian and Egyptian, 16 Israeli, and 24 international) were susceptible to 5-flucytosine (MIC < or = 0.125 microg/ml). The results of this study show for the first time the presence of a novel 5-flucytosine-resistant clade of C. dubliniensis (Cd25 group III) that is predominant among isolates from Saudi Arabia and Egypt and absent from a previously characterized international collection of 98 isolates from 15 countries.

FIG. 1.

Dendrogram generated from the S_ABs computed for every possible pairwise combination of 30 C. dubliniensis isolates recovered from individual patients in Saudi Arabia and Egypt fingerprinted with Cd25. The provenances of the isolates are shown in Tables 1 and 3. At an S_AB node of 0.05, the isolates are divided into two main populations. The first of these populations consists solely of genotype 1 isolates and are closely related, with an average S_AB value of 0.86 ± 0.27. The second population consists of genotype 3 and genotype 4 isolates that are less closely related to each other than are genotype 1 isolates and have an average S_AB value of 0.35 ± 0.19. At an S_AB node of 0.22, the second population is divided into two subpopulations consisting of genotype 3 and genotype 4 isolates, respectively.

FIG.2.

Dendrogram generated from the S_ABs computed for every possible pairwise combination of independent C. dubliniensis isolates from Saudi Arabia and Egypt (n = 30), Israel (n = 5), and 13 other countries (n = 51) from the study of Gee et al. (18) fingerprinted with Cd25. At an S_AB node of 0.05 (short dashed vertical line), the isolates are divided into three main populations, the first and second of which correspond to the major clades Cd25 group I and Cd25 group II described previously (18, 22). The third main population (Cd25 group III) corresponds to a third major clade identified in this study and contains 20/30 of the Saudi Arabian and Egyptian isolates investigated. The Cd25 group I major clade consists solely of closely related genotype 1 isolates with an average S_AB value of 0.63 ± 0.12. At an S_AB node of 0.13 (short dashed vertical line), the Cd25 group II major clade can be divided into two minor clades, the first of which consists solely of genotype 2 isolates. The second minor clade consists of genotype 3 isolates and the genotype 4B variant isolate Is35. At an S_AB node of 0.22 (short dashed vertical line), the Cd25 group III major clade can also be divided into two minor clades, the first of which consists solely of genotype 3 isolates and the second solely of genotype 4 isolates, including the genotype 4A variant isolate Eg207. Within the Cd25 group III major clade, all of the isolates were from Saudi Arabia or Egypt and were resistant to 5FC (MIC₅₀ ≥ 128 μg/ml), apart from the Israeli isolate, p7718 (18), which was susceptible to 5FC (MIC₅₀ ≤ 0.125 μg/ml). All of the other Saudi Arabian, Egyptian, and Israeli isolates investigated in this study and a selection of 24 independent isolates from the study of Gee et al. (18) (10 Cd25 group I isolates and 14 Cd25 group II isolates) were susceptible to 5FC (MIC₅₀ ≤ 0.125 μg/ml). The Saudi Arabian, Egyptian, and Israeli isolates investigated in this study are highlighted in boldface. The Cd25 groups (a) and the ITS genotypes (b) of the isolates are shown to the right of the dendrogram.

FIG. 3.

Model generated using the DENDRON software package showing band positions and intensities of Cd25-generated hybridization fingerprint patterns of EcoRI-digested genomic DNA of C. dubliniensis isolates belonging to the major clades Cd25 group I, Cd25 group II, and the novel 5FC-resistant Saudi Arabian and Egyptian clade Cd25 group III identified in this study. Molecular sizes in kilobases are shown on the left. The C. dubliniensis isolates from which the corresponding patterns in the lanes were obtained are as follows (genotypes are shown in parentheses): lane 1, CM6 (1); lane 2, CD518 (1); lane 3, SA102 (1); lane 4, Eg204 (1); lane 5, Can9 (2); lane 6, CD514 (2); lane 7, Is49 (2); lane 8, p6265 (3); lane 9, SA103 (3); lane 10, SA121 (4); lane 11, SA119 (3); lane 12, Eg201 (4).