Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical <i>Candida dubliniensis</i> isolates from Kuwait

<div><p><i>Candida dubliniensis</i> and <i>Candida albicans</i> are two closely related species. Although <i>C</i>. <i>dubliniensis</i> is less pathogenic, it has a higher propensity to develop resistance to fluconazole and some strains exhibit intrinsic resistance to 5-flucytosine (5-FC). All 5-FC-resistant isolates from Kuwait were previously shown to belong to one of seven internal transcribed spacer (ITS) region of rDNA-based haplotypes. This study performed fingerprinting of <i>C</i>. <i>dubliniensis</i> isolates by multilocus sequence typing (MLST) to determine population structure of 5-FC-resistant and -susceptible strains and compared the results with data from a global collection of isolates. Fifty-two <i>C</i>. <i>dubliniensis</i> isolates previously analyzed and 58 additional isolates mostly collected during 2010–2013 and characterized by phenotypic and molecular methods were used. ITS-based haplotypes were identified by haplotype-specific PCR and/or by PCR-DNA sequencing of rDNA. Population structure was determined by 8-loci-based MLST. E-test was used to determine susceptibility to 5-FC, fluconazole, voriconazole and amphotericin B. Five ITS haplotypes (ITSH) were detected among 110 <i>C</i>. <i>dubliniensis</i> isolates. The ITSH1 was most common (n = 80 isolates) followed by ITSH4 (n = 25 isolates). Two isolates each belonged to ITSH5 and ITSH8 while one isolate belonged to ITSH7. MLST identified 16 diploid sequence types (DSTs) including six new DSTs. DST11 (n = 52) and DST14 (n = 25) were dominant genotypes and were confined (together with DST21) to Middle-Eastern countries. Other DSTs (excluding some new DSTs) had a wider global distribution as they were identified from various other countries. Only ITSH4 isolates (n = 25) belonged to DST14, were resistant to 5-FC and contained S29L mutation in Cd<i>FCA</i>1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical <i>C</i>. <i>dubliniensis</i> isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant <i>C</i>. <i>dubliniensis</i> isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.</p></div

Common underlying conditions among patients yielding <i>C</i>. <i>tropicalis</i> isolates.

<p>Common underlying conditions among patients yielding <i>C</i>. <i>tropicalis</i> isolates.</p

Minimum spanning tree showing relationship of 63 <i>C</i>. <i>tropicalis</i> isolates from Kuwait with 804 isolates from other countries available from the MLST website as of January 2017.

<p>Each circle corresponds to a unique genotype, and lines between circles represent relative distance between isolates. The sizes of the circles correspond to the number of isolates of the same genotype (DST). Connecting lines correspond to the number of allele differences between genotypes, with a solid thick line connecting genotypes that differ in one locus, a solid thin line connecting genotypes that differ in two-three loci, a dashed line connecting genotypes that differ in four loci, and a dotted line connecting genotypes that differ in more than four loci.</p

An UPGMA-derived dendrogram based on locus-specific Diploid Sequence Type (DST) of 8 housekeeping gene fragments from one representative <i>C</i>. <i>dubliniensis</i> isolate from Kuwait for each genotype together with the data from a global collection of 50 epidemiologically distinct <i>C</i>. <i>dubliniensis</i> isolates analyzed previously.

<p>Similarity is presented in percentages using the scale bar in the upper left corner and the country of isolation of the isolates are color coded. The columns from left to right include, isolate number, country of isolation and MLST-based DST. The 5-FC-resistant isolates are indicated by an asterisk (*) before the isolate number and the numbers in parenthesis indicate the number of isolates from Kuwait with the identical DST.</p

Summary of fingerprinting data for 110 <i>C</i>. <i>dubliniensis</i> isolates using ITS-region sequencing, MLST and susceptibility to 5-flucytosine.

<p>Summary of fingerprinting data for 110 <i>C</i>. <i>dubliniensis</i> isolates using ITS-region sequencing, MLST and susceptibility to 5-flucytosine.</p

Population structure and molecular genetic characterization of clinical <i>Candida tropicalis</i> isolates from a tertiary-care hospital in Kuwait reveal infections with unique strains

<div><p><i>Candida tropicalis</i> is a frequently isolated yeast species causing bloodstream, urinary tract and other infections particularly in patients admitted to intensive care units (ICUs) and those requiring prolonged urinary catheterization (UC) or receiving broad-spectrum antibiotics (BSA). This study investigated clinical characteristics and genetic relatedness among <i>C</i>. <i>tropicalis</i> strains isolated from patients at Al-Amiri Hospital in Kuwait. <i>C</i>. <i>tropicalis</i> strains (n = 63) isolated from blood, genito-urinary, respiratory (RT) and digestive (GIT) tracts and wound sites from 54 patients were used. All isolates were phenotypically identified and tested against six antifungal drugs by using Vitek 2 system. Molecular identification was performed by PCR amplification of rDNA. Fingerprinting was achieved by 6-loci-based multilocus sequence typing (MLST) and data were analyzed by BioNumerics software for phylogenetic relationships. Patients mean age was >65 years and >20% patients were hospitalized in ICUs. Most patients had underlying conditions that included UC, BSA, diabetes and RT/GIT abnormalities. Most candiduria cases had UC, ureteric stent or suprapubic catheters. All isolates were identified as <i>C</i>. <i>tropicalis</i> by Vitek 2 and by species-specific PCR. Sixty-two isolates were susceptible to all tested antifungal drugs. MLST identified 59 diploid sequence types (DSTs) including 54 newly-identified DSTs. <i>C</i>. <i>tropicalis</i> isolates from multiple sites of same patient usually belonged to different DSTs. Interestingly, 56 of 57 isolates from 48 patients belonged to unique genotypes. Only six isolates from six patients belonged to three DSTs (clusters), however, <i>C</i>. <i>tropicalis</i> strains in each cluster were isolated >3 months apart. Our data show diverse origins of <i>C</i>. <i>tropicalis</i> infections in Kuwait as most isolates were unique strains. There was no obvious correlation between cluster isolates with time of isolation and/or hospital ward of their origin. This study presents the first MLST analysis of <i>C</i>. <i>tropicalis</i> isolates from Middle East and may be useful for studying genetic relationships among global <i>C</i>. <i>tropicalis</i> strains.</p></div

MLST data for multiple <i>C</i>. <i>tropicalis</i> isolates from different anatomic sites of seven patients.

<p>MLST data for multiple <i>C</i>. <i>tropicalis</i> isolates from different anatomic sites of seven patients.</p

An UPGMA-derived dendrogram based on allelic profile of 6 gene fragments from 63 <i>C</i>. <i>tropicalis</i> isolate from Kuwait.

<p>Similarity is presented in percentages using the scale bar in the upper left corner. The columns from left to right include, isolate number, MLST-based diploid sequence type (DST), clinical specimen yielding the isolate, data of isolation, hospital ward/unit where the patients were housed and eBURST group. Repeat isolates from the same patient are indicated by alphabets within brackets and DSTs for cluster isolates are shown by an asterisk (*) before the DST.</p

Clinical and epidemiological characteristics of patients yielding <i>C</i>. <i>tropicalis</i> isolates belonging to the three MLST-based clusters.

<p>Clinical and epidemiological characteristics of patients yielding <i>C</i>. <i>tropicalis</i> isolates belonging to the three MLST-based clusters.</p

Minimum Inhibitory Concentration (MIC) ranges and susceptibility data for 110 <i>Candida dubliniensis</i> isolates by E-test.

<p>Minimum Inhibitory Concentration (MIC) ranges and susceptibility data for 110 <i>Candida dubliniensis</i> isolates by E-test.</p