Locus CauMT1 Provides a Higher-Resolution Alternative to Ribosomal Gene Sequencing for Initial Candida auris Genotyping.
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ABSTRACT:
SUBMITTER: Katiyar S
PROVIDER: S-EPMC7448618 | biostudies-literature | 2020 Aug
REPOSITORIES: biostudies-literature
Publications
Locus CauMT1 Provides a Higher-Resolution Alternative to Ribosomal Gene Sequencing for Initial Candida auris Genotyping.
Katiyar Santosh S Edlind Tom T
Journal of clinical microbiology 20200824 9
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Project description:Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.
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Project description:The most widely used DNA-based method for bacterial strain typing, multi-locus sequence typing (MLST), lacks sufficient resolution to distinguish among many bacterial strains within a species. Here, we show that strain typing based on the presence or absence of distributed genes is able to resolve all completely sequenced genomes of six bacterial species. This was accomplished by the development of a clustering method, neighbour grouping, which is completely consistent with the lower-resolution MLST method, but provides far greater resolving power. Because the presence/absence of distributed genes can be determined by low-cost microarray analyses, it offers a practical, high-resolution alternative to MLST that could provide valuable diagnostic and prognostic information for pathogenic bacterial species.
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Project description:The emerging pathogen Candida auris has been associated with nosocomial outbreaks on six continents. Genetic analysis indicates simultaneous and independent emergence of separate clades of the species in different geographical locations. Both invasive infection and colonization have been observed, warranting attention due to variable antifungal resistance profiles and hospital transmission. MALDI-TOF based identification methods have become routine in hospitals and research institutes. However, identification of the newly emerging lineages of C. auris yet remains a diagnostic challenge. In this study an innovative liquid chromatography (LC)-high resolution OrbitrapTM mass spectrometry method was used for identification of C. auris from axenic microbial cultures. A set of 102 strains from all five clades and different body locations were investigated. The results revealed correct identification of all C. auris strains within the sample cohort, with an identification accuracy of 99.6% from plate culture, in a time-efficient manner. Furthermore, application of the applied mass spectrometry technology provided the species identification down to clade level, thus potentially providing the possibility for epidemiological surveillance to track pathogen spread. Identification beyond species level is required specially to differentiate between nosocomial transmission and repeated introduction to a hospital.
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Project description:The emergence of a multidrug-resistant Candida species, C. auris and C. haemulonii, has been reported worldwide. In Thailand, information on them is limited. We collected clinical isolates from Thai patients with invasive candidiasis. Both species were compared with a laboratory C. albicans strain. In vitro antifungal susceptibility and thermotolerance, and pathogenesis in the zebrafish model of infection were investigated. Both species demonstrated high minimal inhibitory concentrations to fluconazole and amphotericin B. Only C. auris tolerated high temperatures, like C. albicans. In a zebrafish swim-bladder-inoculation model, the C. auris-infected group had the highest mortality rate and infectivity, suggesting the highest virulence. The case fatality rates of C. auris, C. haemulonii, and C. albicans were 100%, 83.33%, and 51.52%, respectively. Further immunological studies revealed that both emerging Candida species stimulated genes involved in the proinflammatory cytokine group. Interestingly, the genes relating to leukocyte recruitment were downregulated only for C. auris infections. Almost all immune response genes to C. auris had a peak response at an early infection time, which contrasted with C. haemulonii. In conclusion, both emerging species were virulent in a zebrafish model of infection and could activate the inflammatory pathway. This study serves as a stepping stone for further pathogenesis studies of these important emerging species.
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Project description:Candida auris-a fungus (yeast) that can cause hospital outbreaks was first recognized in 2009. The authors report data on 38 cases of C. auris bloodstream infections in multidisciplinary hospitals situated in two distantly located regions of Russia, considering predisposing factors, antifungal susceptibility of isolates, treatment, and outcomes. Interhospital transfers of patients and labor migration contributed to the spread of C. auris. The South Asian lineage of the studied strains was indicated by K143R substitution in ERG11 gene and phylogenetic analysis of internal transcribed spacer and D1-D2 domain. All isolates from C. auris candidemia cases were susceptible to echinocandins. High-level resistance to fluconazole and resistance to amphotericin B were present in the majority of strains. The overall all-cause mortality rate in C. auris bloodstream infections was 55.3% and the 30-day all-cause mortality rate 39.5%. The attributable mortality was 0%. Eradication of C. auris from blood was associated with the favourable outcomes in patients. It was achieved irrespective of whether antifungal preparations within or outside the susceptibility range were administered. Further international surveillance and studies providing consensus guidelines for the management of C. auris infections are needed.
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Project description:Molecular typing studies have shown that the predominant form of reproduction of Candida albicans is clonal and that, in a majority of situations, persistent or recurrent infections are due to a unique strain. Characterization of distinct subpopulations and correlation with clinical features may thus be important to understanding the pathogenesis of candidiasis. In a clonal model, a unique polymorphic marker may identify populations with different biological properties. We therefore compared 48 bloodstream isolates and 48 nonbloodstream matched strains of C. albicans at the elongation factor 3-encoding gene (CEF3) polymorphic microsatellite locus of C. albicans. Sizing of the alleles was performed by automated capillary electrophoresis. A new, 137-bp allele was characterized, and seven nondescribed combinations were observed, resulting in 15 and 11 distinct CEF3 profiles in bloodstream and control strains, respectively. Genotypes 126-135, 130-136, and 131-131 accounted for 60.4% of both bloodstream and control strains. Four bloodstream isolates but no control strains displayed the 135-135 combination. None of the other genotypes was present at an increased frequency in bloodstream isolates. Bloodstream and nonbloodstream strains of C. albicans thus have a heterogeneous structure at the CEF3 locus, with three major and multiple minor allelic combinations.
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