Diploid Sequence Types Research Articles

206.3: Utility of multilocus sequence typing (MLST) in the investigation of a cluster of Candida albicans infection in recipients of solid organs from a common donor.

Background: Invasive candidiasis in solid organ transplant recipients can be of endogenous or exogenous origin and is a cause of significant morbidity and mortality; cross contamination of grafts in abdominal multi-organ donation is a possible source of recipient infection. MLST is a highly discriminatory method based on the analysis of single nucleotide polymorphisms within the sequences of seven PCR-generated regions of housekeeping genes, which are combined to produce diploid sequence types (DST). This technique was used to analyse C. albicans isolates from three solid organ recipients. Methods: A 21-year-old woman died of hypoxic brain injury secondary to hanging and became a multi-organ donor. The right kidney recipient in transplant unit “A” lost their graft 26 days post-transplant (PT) due to Candida arteritis; C. albicans was grown from blood and perinephric clots. The simultaneous pancreas-kidney (SPK) recipient in transplant unit “B” received anti-fungal prophylaxis following isolation of C. albicans from the OTF and remained well. The liver recipient in transplant unit “C” succumbed to a ruptured aortic pseudoaneursym on PT day +23; C. albicans was isolated from the vascular tissue. Another liver recipient in the same transplant unit had recently died of the same complications from Candida infection. Isolates from all three recipients were analysed by MLST at the University of Aberdeen. Results: Isolates from two recipients linked to donor A were genetically indistinguishable and of the same unique strain type (DST 2940); isolates from the unrelated liver recipient were also identical but of a different strain type (DST 2941). These two novel DSTs differed at 5 of 7 loci and were not yet represented in the C. albicans MLST database (2939 strain types in the database at the time of analysis). Discussion: Rates of yeast contamination of abdominal OTF varies, falling between 0.4 to 4%; C. albicans is the commonest species found. Contamination from the GI tract of a colonized donor is the most likely explanation for the infection in recipients in cluster A. In the absence of an isolate from the donor, our results do not unequivocally confirm the origin of the strain of C. albicans but the strain type isolated from cluster A recipients had not been previously described, indicating that it is not a common type and highly unlikely to have arisen in the two organ recipients by chance; the isolate from the SPK transport fluid was not available for analysis. Conclusions: The C. albicans-related events in cluster A were linked through detailed investigation following early post-transplant kidney graft loss, illustrating the importance of prompt reporting. Imputability of source of infection is not always possible and availability of isolates for analysis and access to appropriate techniques can be limiting factors. Addition of unrelated, control samples adds strength to the analysis and should be included whenever possible.

Genetic diversity of Candida albicans isolates recovered from hospital environments and patients with severe acquired brain injuries

Although the epidemiology of pathogenic Candida species causing invasive human diseases is changing, Candida albicans still remains the most common cause of bloodstream infections worldwide. The propensity of this pathogen to cause infections is undoubtedly the result of its unique genetic plasticity that allow it to adapt and respond quickly to a myriad of changing conditions both in the host and in the environment. For this reason, we decided to investigate the genetic diversity of this important fungal pathogen in a particular category of patients with severe neurological deficits including the hospital environments where they are hospitalized.Genetic diversity of 21 C. albicans isolates recovered from blood, hands of healthcare workers and hospital environments was evaluated by using multilocus sequence typing (MLST) which revealed a high genetic heterogeneity with a set of 18 diploid sequence types (DSTs) recovered among 21 isolates investigated. Interestingly, 13 of these 18 MLST genotypes were completely new and added to the C. albicans MLST central database. Six eBURST clonal complexes (CC-1, CC-2, CC-6, CC-9, CC-27 and CC-42) and three singletons contained all DSTs found in this study. Among all the new DSTs identified, DST3388 was the most intriguing as this genotype was recovered from a typical C. albicans isolate clustering within the MLST-Clade 13, the most divergent evolutionary lineage within C. albicans population containing only isolates with unusual phenotypes originally known as Candida africana.In conclusion, the results of this study expand our understanding of the molecular epidemiology and global population structure of C. albicans suggesting that further studies on different categories of patients and hospital environments are needed to better understand how the population of this species adapts and evolves in heterogeneous hosts and changing environments.

Cutaneous candidiasis in Tehran-Iran: from epidemiology to multilocus sequence types, virulence factors and antifungal susceptibility of etiologic Candida species

Cutaneous candidiasis is a multipicture fungal infection caused by members of the genus Candida which is considered as a public health problem all over the world with urgency of effective treatment and control. This study was performed to analyze the clinical epidemiology and molecular aspects of cutaneous candidiasis in Tehran-Iran in relation to antifungal susceptibility and virulence factors of etiologic Candida species. Candida species were isolated from skin (27.3%) and nail scrapings (72.7%) of suspected patients and identified by ITS sequencing. Phylogeny of the isolates was evaluated using multilocus sequence typing (MLST) and antifungal susceptibility and virulence factors of the isolates were determined in relation to clinical presentation. Candida albicans was the most prevalent species (39.8%), followed by C. parapsilosis (32.9%), C. orthopsilosis (10.4%), C. tropicalis (7.9%), C. glabrata and C. guilliermondii, each (4.5%). Molecular typing of 35 C. albicans isolates by MLST revealed 28 novel sequence types with 11 singletons with 80.0% new diploid sequence types (DSTs). Majority of the isolates were susceptible to amphotericin B (91.5%), followed by posaconazole (90.3%), fluconazole (84.3%), itraconazole (74.1%), caspofungin (53.6%), and voriconazole (26.8%). Biofilm formation, yeast-to-hyphae transformation and phospholipase activity were reported species-dependent. Our results demonstrated clinical epidemiology of various Candida species from cutaneous candidiasis distributed in new molecular types with increasing importance of drug resistant of non-albicans Candida species. Our results showed that drug susceptibility and genetic variability of Candida species may be attributed to their clinical features and source of isolation.

Genotyping of Candidaalbicans isolates from oropharyngeal candidiasis in head and neck cancer patients in Iran: Molecular epidemiology and SAP2 gene expression

ObjectiveMultilocus sequence typing is a powerful method for genotyping of clinical isolates of Candidaalbicans. Cross-contamination between the patients is an important reason for nosocomial infections. Oropharyngeal candidiasis (OPC) caused by C. albicans is an important problem in patients with head and neck cancer, in Cancer Institute of Tehran. Here we studied the endemic genotypes of C. albicans isolates and the relationship between geographic distributions, potential cross-contaminations and the expression of SAP2 gene, an important gene in oral candidiasis, with MLST groups. Material and methodsA total of 32 clinical strains of C. albicans isolated from head and neck cancer patients with oropharyngeal candidiasis were subjected to MLST analysis and SAP2 gene expression was analyzed by Real-time PCR. ResultsWe identified 75 polymorphic sites in 7 loci of C. albicans isolates and 30 diploid sequence types which 27 of them were found as new. After eBURST analysis, our results determined that CC 124 was the most prevalent group among all CCs. SAP2 gene showed high expression in almost all OPC patients’ isolates, compared to the control. ConclusionWe found few genetically-related as well as identical isolates among the 32 Candida strains which indicated low cross-contaminations among the patients. There was no relationship between C. albicans MLST profiles and their geographic distribution, cancer type and SAP2 gene expression. This lack of correlation was possibly due to the small understudy population; hence, finding more relevance requires studies with a higher number of samples.

Clonality of Fluconazole-NonsusceptibleCandida tropicalisin Bloodstream Infections, Taiwan, 2011–2017

Candida tropicalis is the leading cause of non–C. albicans candidemia in tropical Asia and Latin America. We evaluated isolates from 344 patients with an initial episode of C. tropicalis candidemia. We found that 58 (16.9%) patients were infected by fluconazole-nonsusceptible (FNS) C. tropicalis with cross resistance to itraconazole, voriconazole, and posaconazole; 55.2% (32/58) of patients were azole-naive. Multilocus sequence typing analysis revealed FNS isolates were genetically closely related, but we did not see time- or place-clustering. Among the diploid sequence types (DSTs), we noted DST225, which has been reported from fruit in Taiwan and hospitals in Beijing, China, as well as DST376 and DST505–7, which also were reported from hospitals in Shanghai, China. Our findings suggest cross-boundary expansion of FNS C. tropicalis and highlight the importance of active surveillance of clinical isolates to detect dissemination of this pathogen and explore potential sources in the community.

A Predominance of Clade 17 Candida albicans Isolated From Hemocultures in a Tertiary Care Hospital in Thailand.

Candida albicans is one of the most common human fungal pathogens. Candidemia has significant mortality globally. No epidemiological study of C. albicans based on multilocus sequence typing (MLST) has been conducted in Thailand. Therefore, MLST was used to study the molecular epidemiology of C. albicans blood strains in a large Thai teaching hospital. In vitro virulence phenotypes and antifungal susceptibility testing by broth microdilution were also conducted. Forty-six C. albicans blood strains from 37 patients were collected from the Department of Microbiology, Siriraj Hospital, in 2016 and 2017. Most patients (71.8%) were more than 60 years old, and the case fatality rate was 54.8%. The male-to-female ratio was 5:3. Thirty-four diploid sequence types (DSTs), including six new DSTs, were identified, with DST2514 (8.7%) and DST2876 (8.7%) as the most common DSTs. Strains were clustered into nine clades. Unlike other studies of C. albicans blood strains in Asia, clade 17 was the most common (13 strains, 28.3%). Sequential allelic changes were evident in sequential strains from one patient. All strains produced phospholipase and hemolysin, while none produced proteinase. The ability to form biofilm was found in 82.6% of the strains. Clade 17 strains showed significantly stronger hemolytic activity than non–clade 17 strains (69.2% versus 27.3%; p = 0.022). However, no significant association existed between clades and patient mortalities. All were susceptible or wild type to anidulafungin (MIC range = 0.015–0.12 and GM = 0.030), micafungin (MIC range = ≤ 0.008–0.015 and GM = 0.008), caspofungin (MIC range = 0.008–0.12 and GM = 0.036), and amphotericin B (MIC range = 0.25–0.5 and GM = 0.381). Only one strain was resistant to voriconazole (MIC range = ≤ 0.008 to ≥ 8 and GM = 0.010) and fluconazole (MIC range = 0.12–16 and GM = 0.398). In conclusion, a high prevalence of clade 17 C. albicans blood strains was found in Thailand, in contrast to other Asian countries. This unique finding might be explained by the strong hemolytic activity that is required for bloodstream infection of C. albicans.

Dissecting the Heterogeneous Population Genetic Structure of Candida albicans: Limitations and Constraints of the Multilocus Sequence Typing Scheme

Candida albicans is a fungal opportunistic pathogen of significant public health importance mainly due to the recent emergence of strains with increased aggressiveness and antifungal resistance. Here, we aimed to describe the epidemiological profiles and approximate the population structure of C. albicans by analyzing the C. albicans multilocus sequence typing (MLST) database (Calb-MLST-DB), which contains the largest publically available dataset for this species. Based on 4,318 database isolates, we confirmed the ubiquitous nature of C. albicans including a group of diploid sequence types (DSTs) obtained from Healthy individuals exclusively (taken as an indicator of lack of association with illnesses in its host), until isolates established from Non-Healthy individuals (potentially associated with pathogenic processes) and other DSTs reported in both types (Healthy and Non-Healthy). The highest number of reported DSTs was related to blood, oral and vaginal swabs (32.4, 20.5, and 13.8%, respectively). High genetic diversity was observed in the seven housekeeping genes included in the MLST scheme, with a diverse population structure (154 clonal complexes, CCs; and a high number of singletons, n = 1,074). Phylogenetic reconstruction on the concatenated alignment of these housekeeping genes for all the reported DSTs (n = 3,483) was partially concordant with the CC assignment, however, an absence of bootstrap threshold supported nodes or p-distance, and the lack of association with the other epidemiological variables, evidenced the limitations of the MLST scheme. Marked genetic admixture signals were identified by STRUCTURE, with the majority being attributable to recombination events according to the RDP program results, although another type of exchange event cannot be ruled out. Our results reaffirm the genetic diversity inherent in the genes used for the MLST scheme, which are associated with the chromosomal remodeling already proposed for C. albicans. This was also corroborated with an internal validation at a micro geographical scale. Despite these results are biased due to the unavailability of considering the broad global spectrum of C. albicans isolates around the world. This suggests that the strategy used to population type this pathogen should be reevaluated to improve epidemiological monitoring of its health impact.

Analyses of the Global Multilocus Genotypes of the Human Pathogenic Yeast Candida tropicalis.

Candida tropicalis is a globally distributed human pathogenic yeast, especially prevalent in tropical and sub-tropical regions. Over the last several decades, a large number of studies have been published on the genetic diversity and molecular epidemiology of C. tropicalis from different parts of the world. However, the global pattern of genetic variation remains largely unknown. Here we analyzed the published multilocus sequence data at six loci for 876 isolates from 16 countries representing five continents. Our results showed that 280 of the 2677 (10.5%) analyzed nucleotides were polymorphic, resulting in a mean of 82 (a range of 38–150) genotypes per locus and a total of 633 combined diploid sequence types (DSTs). Among these, 93 combined DSTs were shared by 336 strains, including 10 by strains from different continents. Analysis of Molecular Variance (AMOVA) showed that 89% of the observed genetic variations were found within regional and national populations while < 10% was due to among-country separations. Pairwise geographic population analyses showed overall low but statistically significant genetic differentiation between most geographic populations, with the Singaporean and Indian populations being the most distinct from other populations. However, the Mantel test showed no significant correlation between genetic distance and geographic distance among the geographic populations. Consistent with high genetic variation within and limited variations among geographic populations, results from STRUCTURE analyses showed that the 876 isolates could be grouped into 15 genetic clusters, with each cluster having a broad geographic distribution. Together, our results suggest frequent gene flows among certain regional, national, and continental populations of C. tropicalis, resulting in abundant regional and national genetic diversities of this important human fungal pathogen.

Multilocus sequence typing for Candida albicans strains from the intestinal tract of patients with cirrhosis.

Multilocus sequence typing (MLST) was applied to investigate the genetic diversity of Candida albicans in the intestinal tract of cirrhosis patients. We used CHROM agar Candida medium to obtain 105 Candida sp. isolates from fecal samples (276 subjects), including 63 isolates from the cirrhosis group (141 subjects) and 42 isolates from the healthy control group (135 subjects). Among the 105 Candida strains isolated, 60 strains were identified as Candida albicans. Patients with cirrhosis had significantly higher rates of colonization by Candida sp. (44.68% vs. 31.11%, p < 0.05) and C. albicans (27.66% vs. 15.56%, p < 0.05) relative to healthy controls. In the cirrhosis group, the rate of colonization further increased with disease progression and antibiotic treatment (p < 0.01). Sixty C. albicans isolates were analyzed by MLST. Fifty diploid sequence types (DST) were observed, and 26 new DSTs and 3 novel alleles were found. The majority of isolates were distributed among three clades, clade 8 (31.67%), clade 14 (15.00%) and clade 18 (21.67%). Among 39 strains from the cirrhosis group, 16 strains (41.02%) belonged to clade 8, while only 3 strains (14.29%) from healthy group belonged to clade 8 (p < 0.05). In addition, concatenated sequences of the 7 housekeeping gene fragments were analyzed for all the different DSTs in clade 8 to evaluate the loss of heterozygosity (LOH), which indicates C. albicans microvariation in the gut of cirrhosis patients. This study suggests that cirrhosis disease progression and antibiotic treatment is associated with increased colonization by Candida sp. and C. albicans. We are the first to provide MLST-based genotype profiles for C. albicans Guizhou China, and to identify clade 8 as the potential main clade of C. albicans colonization in the gut of cirrhosis patients.

Multilocus sequence typing reveals clonality in Saprolegnia parasitica outbreaks.

The molecular epidemiology of fish pathogen Saprolegnia parasitica is still largely unknown. We developed a multilocus sequence typing scheme based on seven housekeeping genes to characterize 77 S.parasitica strains isolated from different fish host species at different times and from different geographic areas in Switzerland between 2015 and 2017. Ten different diploid sequence types (DSTs) were identified. The majority (52%) of outbreaks in Switzerland seemed to be caused by one genotype, namely DST3, which was recovered from farm-raised and wild-caught fish in all the geographic areas and river basins included in the study. DST3 was also recovered from the rivers Bienne (eastern France) and Doubs, where the episodes of massive mortality due to saprolegniosis started in 2009. Another genotype (DST7) showed, to a lesser extent, a distribution across different river basins, while eight DSTs were unique to a defined geographic area or river basin. The occurrence of sporadic DSTs indicates a certain degree of diversity within S.parasitica in the environment. The wide distribution of DST3 suggests that a clonal population may have spread in eastern France and Switzerland across geographic barriers, with strong implications for the management of both captive and wild fish populations.

MDR1 overexpression combined with ERG11 mutations induce high-level fluconazole resistance in Candida tropicalis clinical isolates

BackgroundMarked increases in fluconazole resistance in Candida tropicalis have been recently reported. In this study, the molecular mechanisms behind fluconazole resistance were investigated.MethodsTwenty-two C. tropicalis clinical isolates, including 12 fluconazole-resistant isolates and 10 fluconazole-susceptible isolates, were collected from a tertiary care teaching hospital in Beijing between 2013 and 2017. Antifungal susceptibility testing, multilocus sequence typing, ERG11 amplification and sequencing, quantitative real-time reverse transcription-polymerase chain reaction (ERG11, UPC2, MDR1, and CDR1), and clinical data collection were performed for all C. tropicalis isolates.ResultsMultilocus sequence typing revealed that the 10 fluconazole-susceptible isolates and 12 fluconazole-resistant isolates were divided into nine and seven diploid sequence types, respectively. Of the 12 patients with fluconazole-resistant isolates, six had been previously exposed to azole and four had a fatal outcome. Y132F and S154F amino acid substitutions in Erg11p were found in all fluconazole-resistant isolates except one. MDR1 gene overexpression was identified in fluconazole-resistant isolates. In particular, seven high-level fluconazole resistant isolates (minimum inhibitory concentration ≥ 128 mg/L) and three pan-azole resistant isolates were identified. CDR1, ERG11, and UPC2 gene expression levels in fluconazole-resistant isolates were not significantly different from the control isolates (P = 0.262, P = 0.598, P = 0.114, respectively).ConclusionsThis study provides evidence that the combination of MDR1 gene overexpression and ERG11 missense mutations is responsible for high-level fluconazole resistance and pan-azole resistance in C. tropicalis clinical isolates. To the best of our knowledge, this is the first study investigating the relationship between MDR1 gene overexpression and increased fluconazole resistance.

Multilocus Sequence Typing Reveals a New Cluster of Closely Related Candida tropicalis Genotypes in Italian Patients With Neurological Disorders.

Candida tropicalis is a pathogenic yeast that has emerged as an important cause of candidemia especially in elderly patients with hematological malignancies. Infections caused by this species are mainly reported from Latin America and Asian-Pacific countries although recent epidemiological data revealed that C. tropicalis accounts for 6–16.4% of the Candida bloodstream infections (BSIs) in Italy by representing a relevant issue especially for patients receiving long-term hospital care. The aim of this study was to describe the genetic diversity of C. tropicalis isolates contaminating the hands of healthcare workers (HCWs) and hospital environments and/or associated with BSIs occurring in patients with different neurological disorders and without hematological disease. A total of 28 C. tropicalis isolates were genotyped using multilocus sequence typing analysis of six housekeeping (ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1) genes and data revealed the presence of only eight diploid sequence types (DSTs) of which 6 (75%) were completely new. Four eBURST clonal complexes (CC2, CC10, CC11, and CC33) contained all DSTs found in this study and the CC33 resulted in an exclusive, well-defined, clonal cluster from Italy. In conclusion, C. tropicalis could represent an important cause of BSIs in long-term hospitalized patients with no underlying hematological disease. The findings of this study also suggest a potential horizontal transmission of a specific C. tropicalis clone through hands of HCWs and expand our understanding of the molecular epidemiology of this pathogen whose population structure is still far from being fully elucidated as its complexity increases as different categories of patients and geographic areas are examined.

Clonal spread and azole-resistant mechanisms of non-susceptible Candida albicans isolates from vulvovaginal candidiasis patients in three Shanghai maternity hospitals.

In our multicenter study, 43 fluconazole non-susceptible and 45 fluconazole-susceptible isolates were collected from vulvovaginal candidiasis (VVC) patients from three Shanghai maternity hospitals to analyze their molecular epidemiological features and fluconazole resistant mechanisms. Cross-resistance to fluconazole, itraconazole and voriconazole was observed in 53.5% of the nonsusceptible isolates. Though we acquired 12 clonal complexes (CCs) of diploid sequence types (DSTs) in clinical isolates by a multilocus sequence typing method, fluconazole nonsusceptible isolates all belonged to CC69 with a predominant genotype of DST 79. Increased expressions of efflux pump genes (CDR1, CDR2, and MDR1) were observed only in minor fluconazole non-susceptible isolates by real-time quantitative polymerase chain reaction (PCR). However, ERG11 genes of fluconazole SDD and resistant isolates had significantly higher expression levels than fluconazole-susceptible isolates. Moreover, 13 distinct amino acid substitutions in Erg11p were found in clinical isolates. Three of the substitutions were novel amino acid substitutions (T123I, P98S, and Y286D), which were not in the susceptible isolates. Only two heterozygous amino acid substitutions (A18P/A and R365G/R) in Erg3p were found in two isolates with cross-resistance to fluconazole, itraconazole, and voriconazole. Taken together, we observed the clonal spread of CC69 in fluconazole non-susceptible isolates of Candida albicans from VVC patients with the dominant genotype DST79. ERG11 gene mutations and overexpression predominantly contributed to fluconazole resistance instead of the more common increased expressions of efflux pump genes (CDR1, CDR2, and MDR1).

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

Candida tropicalis 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 C. tropicalis strains isolated from patients at Al-Amiri Hospital in Kuwait. C. tropicalis 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 C. tropicalis 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. C. tropicalis 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, C. tropicalis strains in each cluster were isolated >3 months apart. Our data show diverse origins of C. tropicalis 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 C. tropicalis isolates from Middle East and may be useful for studying genetic relationships among global C. tropicalis strains.

Fruits as the vehicle of drug resistant pathogenic yeasts

We investigated the diversity and drug susceptibility of pathogenic yeasts on fruit surfaces. Fruits were purchased from supermarkets and washed with buffer. The pellets were re-suspended in medium after centrifugation. The cell suspensions were plated onto CHROMagar Candida medium. Yeasts were identified by ribosomal DNA sequencing and their drug susceptibilities were determined by broth microdilution assay. Of 184 isolates, comprised of 55 species, from 22 different types of fruits, 29 species, including Candida famata, Candida fermentati, Candida guilliermondii, Candida intermedia, Candida krusei, Candida orthopsilosis, Candida parapsilosis, Candida pelliculosa, Candida tropicalis, and others have been reported to cause diseases in humans. In addition to C. krusei, intrinsically resistant to fluconazole, all Rhodotorula and Rhodosporidium species were resistant to fluconazole. One each of C. tropicalis isolate was belonged to diploid sequence type (DST)149 and DST225, genotypes also detected in isolates from humans. Furthermore, the DST225 isolate was less susceptible to azole drugs. The susceptibilities to azole drugs for clinical and agricultural usage were associated to each other. It is important to be aware of the existence of pathogenic yeasts, especially drug-resistant ones, on the fruit surfaces, a potential route for pathogenic yeasts to be transmitted to humans.

Evaluation of a Multilocus Sequence Typing (MLST) scheme for Leishmania (Viannia) braziliensis and Leishmania (Viannia) panamensis in Colombia

BackgroundLeishmaniases are parasitic vector-borne diseases affecting more than 12 million people in 98 countries. In Colombia, leishmaniasis is widespread and the most common clinical manifestation is cutaneous, mainly caused by L. panamensis and L. braziliensis. Currently, the genetic diversity of these species in Colombia is unknown. To address this, we applied molecular techniques for their characterization, using multilocus sequence typing (MLST) to explore the genetic variability and phylodynamics of the disease.MethodsSeven previously described genetic markers were selected highlighting the implementation of a mitochondrial marker. Markers were applied to 163 samples from isolates obtained between 1980 and 2001.ResultsThe identification of the samples showed an excellent correlation with typing tests previously applied (MLEE, monoclonal antibodies). Isolates of L. braziliensis showed greater genetic diversity than L. panamensis, and a greater number of diploid sequence types (DSTs). In addition, the geographical distribution of DSTs for each species were obtained through georeferencing maps.ConclusionsTo our knowldge, this study represents the first description of the genetic variability of L. panamensis in Colombia and South America, and is the first to propose a scheme of MLST for epidemiological surveillance of leishmaniasis in the country.

Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait.

Candida dubliniensis and Candida albicans are two closely related species. Although C. dubliniensis 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 C. dubliniensis 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 C. dubliniensis 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 C. dubliniensis 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 CdFCA1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical C. dubliniensis isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant C. dubliniensis isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.

Whole Genome-Based Amplified Fragment Length Polymorphism Analysis Reveals Genetic Diversity in Candida africana.

This study aimed at investigating the genetic diversity of a panel of Candida africana strains recovered from vaginal samples in different countries. All fungal strains were heterozygous at the mating-type-like locus and belonged to the genotype A of Candida albicans. Moreover, all examined C. africana strains lack N-acetylglucosamine assimilation and sequence analysis of the HXK1 gene showed a distinctive polymorphism that impair the utilization of this amino sugar in this yeast. Multi-locus sequencing of seven housekeeping genes revealed a substantial genetic homogeneity among the strains, except for the CaMPIb, SYA1 and VPS13 loci which contributed significantly to the classification of our set of C. africana strains into six existing diploid sequence types. Amplified fragment length polymorphism fingerprint analysis yielded greater genotypic heterogeneity among the C. africana strains. Overall the data reported here show that in C. africana genetic diversity occurs and the existence of this intriguing group of C. albicans strains with specific phenotypes associated could be useful for future comparative studies in order to better understand the genetics and evolution of this important human pathogen.

Multilocus sequence analyses reveal extensive diversity and multiple origins of fluconazole resistance in Candida tropicalis from tropical China

Candida tropicalis is among the most prevalent human pathogenic yeast species, second only to C. albicans in certain geographic regions such as East Asia and Brazil. However, compared to C. albicans, relatively little is known about the patterns of genetic variation in C. tropicalis. This study analyzed the genetic diversity and relationships among isolates of C. tropicalis from the southern Chinese island of Hainan. A total of 116 isolates were obtained from seven geographic regions located across the Island. For each isolate, a total of 2677 bp from six gene loci were sequenced and 79 (2.96%) polymorphic nucleotide sites were found in our sample. Comparisons with strains reported from other parts of the world identified significant novel diversities in Hainan, including an average of six novel sequences (with a range 1 to 14) per locus and 80 novel diploid sequence types. Most of the genetic variation was found within individual strains and there was abundant evidence for gene flow among the seven geographic locations within Hainan. Interestingly, our analyses identified no significant correlation between the diploid sequence types at the six loci and fluconazole susceptibility, consistent with multiple origins of fluconazole resistance in the Hainan population of C. tropicalis.

Multilocus sequence typing of Candida tropicalis shows clonal cluster enrichment in azole-resistant isolates from patients in Shanghai, China

To explore the putative correlation between the multilocus sequence types (MLST) and antifungal susceptibility of clinical Candida tropicalis isolates in Mainland China. Eighty-two clinical C. tropicalis isolates were collected from sixty-nine patients at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, from July 2012 to February 2015, and antifungal susceptibility tests were performed. Genetic profiles of those 82 isolates (30 azole-resistant and 52 azole-susceptible) were characterised by multilocus sequence typing. Phylogenetic analysis of the data was conducted with the clustering method, using UPGMA (unweighted pair group method with arithmetic averages) and the minimal spanning tree algorithm. MLST clonal clusters were analysed using the eBURST V3 package. Of the six gene fragments identified in multilocus sequence typing, SAPT4 presented the highest typing efficiency, whereas SAPT2 was the least efficient. Of the 44 diploid sequence types (DSTs) differentiated, 32 DSTs and 12 genotypes were identified as new to the C. tropicalis DST database. Twenty (45.45%) of the 44 DSTs were assigned to seven major groups based on eBURST analysis. Of these, Group 6, which contained DST 376, DST 505, DST 506 and DST 507, accounted for 76.7% of the 30 azole-resistant isolates. However, the genetic relationships among the azole-susceptible isolates were relatively decentralised. This MLST analysis of the putative correlation between the MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Mainland China shows that DSTs 376, 505, 506 and 507 are closely related azole-resistant C. tropicalis clones.