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1.  Multilocus Sequence Typing for Candida albicans Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis 
We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting.
A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S).
The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns.
MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.
PMCID: PMC3115997  PMID: 21474986
Candia albicans; Multilocus sequence typing; Pulsed-field gel electrophoresis; Southern hybridization; Genotyping
2.  Microevolution of Candida albicans Strains during Catheter-Related Candidemia 
Journal of Clinical Microbiology  2004;42(9):4025-4031.
We examined microevolution in a series of Candida albicans strains isolated from patients with catheter-related candidemia. Sixty-one isolates (29 from blood, 18 from catheters, 10 from urine, and 4 from other sites) were obtained from 15 patients who were admitted to the same hospital over a 3-year period. Isolates were analyzed by using Southern hybridization with the C1 fragment of Ca3 as a probe (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG) by using SfiI (REAG-S) and BssHII (REAG-B). When catheter isolates were compared with blood isolates from the same patient, catheter isolates from 5 of 14 patients (36%) exhibited minor band differences (microevolution) relative to blood isolates in either C1 fingerprinting (n = 4), REAG-S (n = 3), or REAG-B (n = 5) profiles, although they had identical EK patterns. However, the other sequential isolates from each patient, which had identical EK patterns, showed the same REAG and C1 fingerprinting patterns. Both fingerprinting methods revealed that two distinct genotypes were shared by isolates from seven patients in a neonatal intensive care unit, suggesting two nosocomial clusters. Except for two catheter isolates from the index patients of each cluster, no consecutive isolates collected from each of the two clusters showed any microevolution during the 2- or 7-month cluster periods. The findings suggest that in catheter-related candidemia, some C. albicans strains undergo microevolution during catheter colonization.
PMCID: PMC516333  PMID: 15364985
3.  Investigation of the sequence of colonization and candidemia in nonneutropenic patients. 
Journal of Clinical Microbiology  1994;32(4):975-980.
Among neutropenic patients with hematologic malignancies, candidemia has been shown to arise typically from autoinfection after colonization. In patients without neutropenia, we examined the similarities of strains colonizing or infecting various body sites and those subsequently causing Candida bloodstream infections. Strain similarity was examined by karyotyping and restriction endonuclease analysis of genomic DNA (REAG) by using two restriction enzymes (SfiI and BssHII). The banding patterns of 42 isolates from 19 patients were independently evaluated in a blinded fashion by three observers. The interobserver reliability measured with a generalized kappa statistic was 0.59 for karyotyping, 0.84 for REAG with SfiI, and 0.88 for REAG with BssHII (P < 0.001 for each). REAG classified the initial colonizing or infecting isolate and subsequent blood isolates as identical in 16 patients (84%). The mean duration of colonization or infection prior to a positive blood culture was 5 and 23 days in patients infected with related and unrelated isolates, respectively (P = 0.14; 95% confidence interval = -14.5 to 50.5). Karyotyping results matched the REAG results for isolates from 14 of the 19 patients (74%). In patients infected with identical isolates, the initial isolate was most frequently recovered from the urine (n = 5) or vascular catheter tips (n = 4). In the five subjects with organisms showing disparate results between the methods, karyotyping revealed different banding patterns, whereas REAG suggested that the isolates were identical. Candida colonization or infection with an identical strain frequently precedes bloodstream infection in nonneutropenic patients. Future studies should evaluate whether patients at high risk for candidemia and who have vascular catheter or urine samples that are positive for a Candida on culture should be treated empirically.
PMCID: PMC267165  PMID: 8027353
4.  Genetic Diversity among Korean Candida albicans Bloodstream Isolates: Assessment by Multilocus Sequence Typing and Restriction Endonuclease Analysis of Genomic DNA by Use of BssHII▿† 
Journal of Clinical Microbiology  2011;49(7):2572-2577.
Multilocus sequence typing (MLST) has been successfully applied to the epidemiology of Candida albicans isolates not only within the hospital setting but also in multiple locations nationwide. We performed MLST to investigate the genetic relatedness among bloodstream infection (BSI) isolates of C. albicans recovered from 10 Korean hospitals over a 12-month period. The 156 isolates yielded 112 unique diploid sequence types (DSTs). While 95 DSTs were each derived from a single isolate, 17 DSTs were shared by 61 isolates (39.1%). Interestingly, 111 (71.1%) isolates clustered within previously known clades, and 29 (18.6%) clustered within a new clade that includes strains of Asian origin previously typed as singletons. This MLST study was complemented by restriction endonuclease analysis of genomic DNA using BssHII (REAG-B) in order to evaluate whether strains with identical DSTs and originating from the same hospital corresponded to nosocomial clusters. Importantly, only those isolates with a strong epidemiological relationship showed ≥95% identical REAG-B types. Our results indicate that REAG-B typing can be complementary to MLST but should be limited to the investigation of isolates of identical DSTs and when interhuman transmission is suspected.
PMCID: PMC3147862  PMID: 21562112
5.  Genetic Relationships between Respiratory Pathogens Isolated from Dental Plaque and Bronchoalveolar Lavage Fluid from Patients in the Intensive Care Unit Undergoing Mechanical Ventilation 
Ventilator-associated pneumonia (VAP) is a leading cause of morbidity and mortality in patients hospitalized in intensive care units. Recent studies suggest that dental plaque biofilms serve as a reservoir for respiratory pathogens. The goal of this study was to determine the genetic relationship between strains of respiratory pathogens first isolated from the oral cavity and later isolated from bronchoalveolar lavage fluid from the same patient undergoing mechanical ventilation with suspected VAP.
Plaque and tracheal secretion samples were obtained on the day of hospital admission and every other day thereafter until discharge from the intensive care unit from 100 patients who underwent mechanical ventilation. Bronchoalveolar lavage was performed for 30 patients with suspected VAP. Pulse-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relatedness of strains obtained from oral, tracheal, and bronchoalveolar lavage samples.
Isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species, and enteric species recovered from plaque from most patients were indistinguishable from isolates recovered from bronchoalveolar lavage fluid (i.e., had >95% similarity of pulse-field gel electrophoresis patterns). Nearly one-half of the Pseudomonas strains showed identical genetic profiles between patients, which suggested a common environmental source of infection.
Respiratory pathogens isolated from the lung are often genetically indistinguishable from strains of the same species isolated from the oral cavity in patients who receive mechanical ventilation who are admitted to the hospital from the community. Thus, dental plaque serves as an important reservoir for respiratory pathogens in patients who undergo mechanical ventilation.
Trial registration identifier: NCT00123123
PMCID: PMC3582026  PMID: 18991508
6.  Investigation of Candida albicans transmission in a surgical intensive care unit cluster by using genomic DNA typing methods. 
Journal of Clinical Microbiology  1995;33(3):576-580.
An apparent outbreak of serious Candida albicans infections (n = 6) occurred in a surgical intensive care unit over a 4-week period. Four patients developed C. albicans bloodstream infections. An additional patient developed catheter-related C. albicans infection; the sixth patient developed an infection of cerebrospinal fluid. C. albicans was isolated from the hands of five health care workers (17%) and the throat of one health care worker (3%) during the outbreak investigation. Karyotyping and restriction endonuclease analysis of genomic DNA with BssHII of 23 C. albicans isolates from patients and the 6 health care worker isolates revealed 9 and 12 different patterns, respectively. Three of six patients appeared to be infected with the same C. albicans strain (two bloodstream infections and one cerebrospinal fluid infection). The hands of a health care worker were colonized with strain that appeared identical to an isolate from a patient prior to infection of the patient. However, restriction endonuclease analysis with SfiI found differences among the isolates determined to be identical by the other two methods. Karyotyping alone does not appear to be sufficient to differentiate between outbreak and control isolates. Restriction endonuclease analysis typing may be a more sensitive method than karyotyping alone in the investigation of a cluster of C. albicans infections. Furthermore, the use of more than one restriction enzyme may be necessary for optimal strain discrimination in restriction endonuclease analysis of genomic DNA.
PMCID: PMC227993  PMID: 7751360
7.  Multilocus Sequence Typing for Analyses of Clonality of Candida albicans Strains in Taiwan 
Journal of Clinical Microbiology  2006;44(6):2172-2178.
Multilocus sequence typing (MLST) was used to characterize the genetic profiles of 51 Candida albicans isolates collected from 12 hospitals in Taiwan. Among the 51 isolates, 16 were epidemiologically unrelated, 28 were isolates from 11 critically ill, human immunodeficiency virus (HIV)-negative patients, and 7 were long-term serial isolates from 3 HIV-positive patients. Internal regions of seven housekeeping genes were sequenced. A total of 83 polymorphic nucleotide sites were identified. Ten to 20 different genotypes were observed at the different loci, resulting, when combined, in 45 unique genotype combinations or diploid sequence types (DSTs). Thirty (36.1%) of the 83 individual changes were synonymous and 53 (63.9%) were nonsynonymous. Due to the diploid nature of C. albicans, MLST was more discriminatory than the pulsed-field gel electrophoresis-BssHII-restricted fragment method in discriminating epidemiologically related strains. MLST is able to trace the microevolution over time of C. albicans isolates in the same patient. All but one of the DSTs of our Taiwanese strain collections were novel to the internet C. albicans DST database ( The DSTs of C. albicans in Taiwan were analyzed together with those of the reference strains and of the strains from the United Kingdom and United States by unweighted-pair group method using average linkages and minimum spanning tree. Our result showed that the DNA type of each isolate was patient specific and associated with ABC type and decade of isolation but not associated with mating type, anatomical source of isolation, hospital origin, or fluconazole resistance patterns.
PMCID: PMC1489451  PMID: 16757617
8.  Coaggregation of Candida dubliniensis with Fusobacterium nucleatum 
Journal of Clinical Microbiology  1999;37(5):1464-1468.
The binding of microorganisms to each other and oral surfaces contributes to the progression of microbial infections in the oral cavity. Candida dubliniensis, a newly characterized species, has been identified in human immunodeficiency virus-seropositive patients and other immunocompromised individuals. C. dubliniensis phenotypically resembles Candida albicans in many respects yet can be identified and differentiated as a unique Candida species by phenotypic and genetic profiles. The purpose of this study was to determine oral coaggregation (CoAg) partners of C. dubliniensis and to compare these findings with CoAg of C. albicans under the same environmental conditions. Fifteen isolates of C. dubliniensis and 40 isolates of C. albicans were tested for their ability to coaggregate with strains of Fusobacterium nucleatum, Peptostreptococcus micros, Peptostreptococcus magnus, Peptostreptococcus anaerobius, Porphyromonas gingivalis, and Prevotella intermedia. When C. dubliniensis and C. albicans strains were grown at 37°C on Sabouraud dextrose agar, only C. dubliniensis strains coaggregated with F. nucleatum ATCC 49256 and no C. albicans strains showed CoAg. However, when the C. dubliniensis and C. albicans strains were grown at 25 or 45°C, both C. dubliniensis and C. albicans strains demonstrated CoAg with F. nucleatum. Heating the C. albicans strains (grown at 37°C) at 85°C for 30 min or treating them with dithiothreitol allowed the C. albicans strains grown at 37°C to coaggregate with F. nucleatum. CoAg at all growth temperatures was inhibited by mannose and α-methyl mannoside but not by EDTA or arginine. The CoAg reaction between F. nucleatum and the Candida species involved a heat-labile component on F. nucleatum and a mannan-containing heat-stable receptor on the Candida species. The CoAg reactions between F. nucleatum and the Candida species may be important in the colonization of the yeast in the oral cavity, and the CoAg of C. dubliniensis by F. nucleatum when grown at 37°C provides a rapid, specific, and inexpensive means to differentiate C. dubliniensis from C. albicans isolates in the clinical laboratory.
PMCID: PMC84803  PMID: 10203506
9.  Physical and genetic map of Streptococcus thermophilus A054. 
Journal of Bacteriology  1994;176(24):7413-7422.
The three restriction endonucleases SfiI, BssHII, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054. A total of 5, 8, and 24 fragments were produced with SfiI, BssHII, and SmaI, respectively. An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes. Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfiI, BssHII, and SmaI fragments. All restriction fragments were arranged in a unique circular chromosome. Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map. Among them, six rrn loci were precisely located. The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites. Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome. One was associated with the tandem rrnD and rrnE loci, and the other was mapped in the region of the lactose operon.
PMCID: PMC197195  PMID: 8002562
10.  Construction of an SfiI macrorestriction map of the Candida albicans genome. 
Journal of Bacteriology  1993;175(20):6637-6651.
The opportunistic fungal pathogen, Candida albicans, is diploid as usually isolated and has no apparent sexual cycle. Genetic analysis has therefore been very difficult. Molecular genetics has yielded important information in the past few years, but it too is hampered by the lack of a good genetic map. Using the well-characterized strain 1006 and strain WO-1, which undergoes the white-opaque phenotypic transition, we have developed a genomic restriction map of C. albicans with the enzyme SfiI. There are approximately 34 SfiI restriction sites in the C. albicans genome. Restriction fragments were separated by pulsed-field electrophoresis and were assigned to chromosomes by hybridization of complete and partial digests with known chromosome-specific probes as well as by digestion of isolated chromosomes. Telomeric fragments were identified by hybridization with a telomere-specific probe (C. Sadhu, M.J. McEachern, E.P. Rustchenko-Bulgac, J. Schmid, D.R. Soll, and J.B. Hicks, J. Bacteriol. 173:842-850, 1991). WO-1 differs from 1006 in that it has undergone three reciprocal chromosomal translocations. Analysis of the translocation products indicates that each translocation has occurred at or near an SfiI site; thus, the SfiI fragments from the two strains are similar or identical. The tendency for translocation to occur at or near SfiI sites may be related to the repeated sequence RPS 1, which contains four such sites and could provide homology for ectopic pairing and crossing over. The genome size of both strains is about 16 to 17 megabases, in good agreement with previous determinations.
PMCID: PMC206775  PMID: 8407841
11.  Changes in Karyotype and Azole Susceptibility of Sequential Bloodstream Isolates from Patients with Candida glabrata Candidemia▿  
Journal of Clinical Microbiology  2007;45(8):2385-2391.
We examined the changes in genotypes and azole susceptibilities among sequential bloodstream isolates of Candida glabrata during the course of fungemia and the relationship of these changes to antifungal therapy. Forty-one isolates were obtained from 15 patients (9 patients who received antifungal therapy and 6 patients who did not) over periods of up to 36 days. The isolates were analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and tested for antifungal susceptibility to fluconazole, itraconazole, and voriconazole. PFGE typing consisted of electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA by use of NotI (REAG-N). The 41 isolates yielded 23 different karyotypes and 11 different REAG-N patterns but only 3 MLST types. The sequential strains from each patient had identical or similar REAG-N patterns. However, they had two or three different karyotypes in 6 (40%) of 15 patients. The isolates from these six patients exhibited the same or similar azole susceptibilities, and five patients did not receive antifungal therapy. Development of acquired azole resistance in sequential isolates was detected for only one patient. For this patient, an isolate of the same genotype obtained after azole therapy showed three- or fourfold increases in the MICs of all three azole antifungals and exhibited increased expression of the CgCDR1 efflux pump. This study shows that karyotypic changes can develop rapidly among sequential bloodstream strains of C. glabrata from the same patient without antifungal therapy. In addition, we confirmed that C. glabrata could acquire azole resistance during the course of fungemia in association with azole therapy.
PMCID: PMC1951250  PMID: 17581937
12.  Characterization of genetically distinct subgroup of Candida albicans strains isolated from oral cavities of patients infected with human immunodeficiency virus. 
Journal of Clinical Microbiology  1995;33(3):696-700.
During the course of a study of oral Candida albicans strains from 60 human immunodeficiency virus-infected patients over a 2.5-year period, 18 of the 295 C. albicans isolates had genomes that failed to hybridize with a C. albicans-specific DNA probe (27A). These strains were germ tube positive and chlamydospore positive and were identified as C. albicans by the ID 32C test (API Systems, Montlieu, France). These strains were analyzed for the presence of two other C. albicans-specific DNA segments by PCR. The first was a C. albicans 1,348-bp species-specific sequence, and the second was a 1,059-bp C. albicans repetitive element. The probe 27A-hybridizing strains yielded PCR products which differed from those of the nonhybridizing strains. Five of these genetically atypical C. albicans strains and 98 of the C. albicans strains were then analyzed for purported virulence factors. The genetically atypical C. albicans strains, in comparison with typical C. albicans strains, produced greater amounts of extracellular proteinase (P = 0.038, Student's t test), adhered to a greater degree to buccal epithelial cells (P = 0.018, Student's t test), and were less susceptible to the antifungal drug 5-flucytosine (P = 0.0003, Mann-Whitney test). Analysis of these strains with other common antifungal drugs showed no statistically significant variation in susceptibility. The results of this study indicated that these genetically atypical C. albicans strains possess increased virulence in comparison with typical C. albicans strains.
PMCID: PMC228016  PMID: 7751379
13.  Strain variation among and antifungal susceptibilities of isolates of Candida krusei. 
Journal of Clinical Microbiology  1996;34(7):1856-1858.
Candida krusei is an emerging pathogen that is well known for its propensity to develop resistance to fluconazole and other azoles. Despite the potential clinical significance of C. krusei, little is known of its epidemiology and genetic diversity as defined by the newer DNA-based typing methods. We investigated the genotypic diversity and antifungal susceptibility of 67 clinical isolates from 44 patients and 5 health care workers from six different medical centers. Strain delineation was performed by restriction endonuclease analysis of genomic DNA (REAG) with the restriction enzyme HinfI followed by conventional electrophoresis. The susceptibility of the isolates to the antifungal agents amphotericin B, flucytosine, fluconazole, and itraconazole was determined by methods recommended by the National Committee for Clinical Laboratory Standards. The MICs at which 90% of the isolates were inhibited ranged from 1.0 microgram/ml for itraconazole to 64 micrograms/ml for fluconazole. In general, isolates from a given patient, or epidemiologically related isolates from a single institution, were identical by molecular typing methods. Epidemiologically unrelated isolates were distinctly different by the REAG typing method employed. These data document the genetic diversity and antifungal susceptibility of clinical isolates of C. krusei.
PMCID: PMC229137  PMID: 8784612
14.  Oral Candida albicans isolates from HIV-positive individuals have similar in vitro biofilm-forming ability and pathogenicity as invasive Candida isolates 
BMC Microbiology  2011;11:247.
Candida can cause mucocutaneous and/or systemic infections in hospitalized and immunosuppressed patients. Most individuals are colonized by Candida spp. as part of the oral flora and the intestinal tract. We compared oral and systemic isolates for the capacity to form biofilm in an in vitro biofilm model and pathogenicity in the Galleria mellonella infection model. The oral Candida strains were isolated from the HIV patients and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. norvegensis, and C. dubliniensis. The systemic strains were isolated from patients with invasive candidiasis and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, and C. kefyr. For each of the acquired strains, biofilm formation was evaluated on standardized samples of silicone pads and acrylic resin. We assessed the pathogenicity of the strains by infecting G. mellonella animals with Candida strains and observing survival.
The biofilm formation and pathogenicity in Galleria was similar between oral and systemic isolates. The quantity of biofilm formed and the virulence in G. mellonella were different for each of the species studied. On silicone pads, C. albicans and C. dubliniensis produced more biofilm (1.12 to 6.61 mg) than the other species (0.25 to 3.66 mg). However, all Candida species produced a similar biofilm on acrylic resin, material used in dental prostheses. C. albicans, C. dubliniensis, C. tropicalis, and C. parapsilosis were the most virulent species in G. mellonella with 100% of mortality, followed by C. lusitaniae (87%), C. novergensis (37%), C. krusei (25%), C. glabrata (20%), and C. kefyr (12%).
We found that on silicone pads as well as in the Galleria model, biofilm formation and virulence depends on the Candida species. Importantly, for C. albicans the pathogenicity of oral Candida isolates was similar to systemic Candida isolates, suggesting that Candida isolates have similar biofilm-forming ability and virulence regardless of the infection site from which it was isolated.
PMCID: PMC3217868  PMID: 22053894
15.  Oral Colonization, Phenotypic, and Genotypic Profiles of Candida Species in Irradiated, Dentate, Xerostomic Nasopharyngeal Carcinoma Survivors 
Journal of Clinical Microbiology  2000;38(6):2219-2226.
The aim of this study was to investigate oral yeast colonization and oral yeast strain diversity in irradiated (head and neck), dentate, xerostomic individuals. Subjects were recruited from a nasopharyngeal carcinoma clinic and were segregated into group A (age, <60 years [n = 25; average age ± standard deviation {SD}, 48 ± 6 years; average postirradiation time ± SD, 5 ± 5 years]) and group B (age, ≥60 years [n = 8; average age ± SD, 67 ± 4 years; average postirradiation time ± SD, 2 ± 2 years]) and were compared with age- and sex-matched healthy individuals in group C (age, <60 years [n = 20; average age ± SD, 44 ± 12 years] and group D (age, ≥60 years [n = 10; average age, 70 ± 3 years]). Selective culture of oral rinse samples was carried out to isolate, quantify, and speciate yeast recovery. All test subjects underwent a 3-month comprehensive oral and preventive care regimen plus topical antifungal therapy, if indicated. A total of 12 subjects from group A and 5 subjects from group B were recalled for reassessment of yeast colonization. Sequential (pre- and posttherapy) Candida isolate pairs from patients were phenotypically (all isolate pairs; biotyping and resistotyping profiles) and genotypically (Candida albicans isolate pairs only; electrophoretic karyotyping by pulsed-field gel electrophoresis, restriction fragment length polymorphism [RFLP], and randomly amplified polymorphic DNA [RAPD] assays) evaluated. All isolates were Candida species. Irradiated individuals were found to have a significantly increased yeast carriage compared with the controls. The isolation rate of Candida posttherapy remained unchanged. A total of 9 of the 12 subjects in group A and 3 of the 5 subjects in group B harbored the same C. albicans or Candida tropicalis phenotype at recall. Varying degrees of congruence in the molecular profiles were observed when these sequential isolate pairs of C. albicans were analyzed by RFLP and RAPD assays. Variations in the genotype were complementary to those in the phenotypic characteristics for some isolates. In conclusion, irradiation-induced xerostomia seems to favor intraoral colonization of Candida species, particularly C. albicans, which appeared to undergo temporal modifications in clonal profiles both phenotypically and genotypically following hygienic and preventive oral care which included topical antifungal therapy, if indicated. We postulate that the observed ability of Candida species to undergo genetic and phenotypic adaptation could strategically enhance its survival in the human oral cavity, particularly when salivary defenses are impaired.
PMCID: PMC86768  PMID: 10834980
16.  Particular Candida albicans Strains in the Digestive Tract of Dyspeptic Patients, Identified by Multilocus Sequence Typing 
PLoS ONE  2012;7(4):e35311.
Candida albicans is a human commensal that is also responsible for chronic gastritis and peptic ulcerous disease. Little is known about the genetic profiles of the C. albicans strains in the digestive tract of dyspeptic patients. The aim of this study was to evaluate the prevalence, diversity, and genetic profiles among C. albicans isolates recovered from natural colonization of the digestive tract in the dyspeptic patients.
Methods and Findings
Oral swab samples (n = 111) and gastric mucosa samples (n = 102) were obtained from a group of patients who presented dyspeptic symptoms or ulcer complaints. Oral swab samples (n = 162) were also obtained from healthy volunteers. C. albicans isolates were characterized and analyzed by multilocus sequence typing. The prevalence of Candida spp. in the oral samples was not significantly different between the dyspeptic group and the healthy group (36.0%, 40/111 vs. 29.6%, 48/162; P > 0.05). However, there were significant differences between the groups in the distribution of species isolated and the genotypes of the C. albicans isolates. C. albicans was isolated from 97.8% of the Candida-positive subjects in the dyspeptic group, but from only 56.3% in the healthy group (P < 0.001). DST1593 was the dominant C. albicans genotype from the digestive tract of the dyspeptic group (60%, 27/45), but not the healthy group (14.8%, 4/27) (P < 0.001).
Our data suggest a possible link between particular C. albicans strain genotypes and the host microenvironment. Positivity for particular C. albicans genotypes could signify susceptibility to dyspepsia.
PMCID: PMC3335024  PMID: 22536371
17.  Comparison of the Hydrophobic Properties of Candida albicans and Candida dubliniensis 
Infection and Immunity  2001;69(2):779-786.
Although Candida dubliniensis is a close genetic relative of Candida albicans, it colonizes and infects fewer sites. Nearly all instances of candidiasis caused by C. dubliniensis are restricted to the oral cavity. As cell surface hydrophobicity (CSH) influences virulence of C. albicans, CSH properties of C. dubliniensis were investigated and compared to C. albicans. Growth temperature is one factor which affects the CSH status of stationary-phase C. albicans. However, C. dubliniensis, similar to other pathogenic non-albicans species of Candida, was hydrophobic regardless of growth temperature. For all Candida species tested in this study (C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis), CSH status correlated with coaggregation with the anaerobic oral bacterium Fusobacterium nucleatum. Previous studies have shown that CSH status of C. albicans involves multiple surface proteins and surface protein N-glycans. The hydrophobic surface glycoprotein CAgp38 appears to be expressed by C. albicans constitutively regardless of growth temperature and medium. C. dubliniensis expresses a 38-kDa protein that cross-reacts with the anti-CAgp38 monoclonal antibody; however, expression of the protein was growth medium and growth temperature dependent. The anti-CAgp38 monoclonal antibody has been shown to inhibit adhesion of C. albicans to extracellular matrix proteins and to vascular endothelial cells. Since protein glycosylation influences the CSH status of C. albicans, we compared the cell wall mannoprotein content and composition between C. albicans and C. dubliniensis. Similar bulk compositional levels of hexose, phosphate, and protein in their N-glycans were determined. However, a component of the C. albicans N-glycan, acid-labile phosphooligomannoside, is expressed much less or negligibly by C. dubliniensis, and when present, the oligomannosides are predominantly less than five mannose residues in length. In addition, the acid-labile phosphooligomannoside profiles varied among the three strains of C. dubliniensis we tested, indicating the N-glycan of C. dubliniensis differs from C. albicans. For C. albicans, the acid-labile phosphooligomannoside influences virulence and surface fibrillar conformation, which affects exposure of hydrophobic surface proteins. Given the combined role in C. albicans of expression of specific surface hydrophobic proteins in pathogenesis and of surface protein glycosylation on exposure of the proteins, the lack of these virulence-associated CSH entities in C. dubliniensis could contribute to its limited ability to cause disseminated infections.
PMCID: PMC97952  PMID: 11159968
18.  Replacement of Candida albicans with C. dubliniensis in Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis Treated with Fluconazole 
Journal of Clinical Microbiology  2002;40(9):3135-3139.
Candida dubliniensis is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with Candida albicans. Although most C. dubliniensis isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of C. dubliniensis. In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar Candida medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for C. albicans) and Cd25 (for C. dubliniensis). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible C. albicans isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months). C. albicans strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, ≥64 μg/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 μg/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 μg/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 μg/ml and those for the other two were 32 and 64 μg/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.
PMCID: PMC130753  PMID: 12202543
19.  Molecular Mechanisms of Fluconazole Resistance in Candida dubliniensis Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis 
Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.
PMCID: PMC127221  PMID: 12019078
20.  Analysis of Candida albicans adhesion to salivary mucin. 
Infection and Immunity  1993;61(5):1940-1949.
Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.
PMCID: PMC280787  PMID: 8478083
21.  Candida species distribution, genotyping and virulence factors of Candida albicans isolated from the oral cavity of kidney transplant recipients of two geographic regions of Brazil 
BMC Oral Health  2014;14:20.
Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. This investigation aimed to study the prevalence of Candida spp. and to analyze the ABC genotypes of 76 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil.
We typed 48 strains with ABC genotyping and Microsatelitte using primer M13 and tested three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils phagocytosis.
C. albicans was the most prevalent species (86.4%), followed by C. tropicalis (4.5%). C. albicans genotype A was the most prevalent (58 isolates; 76.4%), followed by genotype C (15 isolates; 19.7%) and genotype B (3 isolates; 3.9%). When Microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. The majority of Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed also better ability to combat PMNs phagocytosis.
We found a high rate of C. albicans genotype C strains isolated from the oral cavity of this group of patients. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute to a better understanding of the pathogenesis of oral candidiasis.
PMCID: PMC3995545  PMID: 24628850
Candida spp; Oral candidiasis; Kidney transplant recipients; Genotyping; Virulence factors
22.  Investigating Biofilm Production, Coagulase and Hemolytic Activity in Candida Species Isolated From Denture Stomatitis Patients 
Oral candidiasis, in the form of Candida-associated denture stomatitis, represents a common disease in a large percentage of denture wearers, and Candida albicans remains the most commonly isolated species. In this study, we aimed to evaluate biofilm production, coagulase and hemolytic activity of Candida species isolated from denture stomatitis patients.
Materials and Methods:
This study included 70 patients (31 female, 39 male). Forty-eight of the patients were found to have a positive culture. A total of 48 Candida isolates representing five species, C. albicans (n=17), C. glabrata (n=10), C. krusei (n=9), C. kefyr (n=7) and C. parapsilosis (n=5), were tested. Their coagulase activities were evaluated by a classical tube coagulase test with rabbit plasma. A blood plate assay on 3% enriched sheep blood Sabouraud-dextrose agar (SDA) was used to determine their in vitro hemolytic activities. Biofilm production was determined by a visual tube method.
Twenty-one Candida isolates exhibited coagulase activity, and the coagulase activities of the C. albicans (64.7%) isolates were higher than other species. C. albicans, C. glabrata, C. kefyr and C. krusei species demonstrated beta hemolysis. C. parapsilosis strains failed to demonstrate any hemolytic activities. Fifteen (88.0%) of the C. albicans strains were biofilm positive. Six (35.2%) of these strains were strongly positive, 8 (47.0%) C. albicans strains were moderately positive and 1 (5.8%) C. albicans strain was weakly positive. Sixteen (51.6%) of the non-albicans Candida strains were biofilm positive while 15 (48.3%) did not produce biofilms.
The results of this present study indicate coagulase, hemolytic activity and biofilm production by Candida spp. isolated from patients with denture stomatitis. Investigations of these virulence factors might be helpful in gaining information about the possible virulence of oral Candida species related to denture stomatitis.
PMCID: PMC4261369  PMID: 25610156
Candida species; Coagulase activity; Denture stomatitis
23.  Oral and esophageal Candida albicans infection in hyposalivatory rats. 
Infection and Immunity  1990;58(7):2228-2236.
The opportunistic fungus Candida albicans is a major cause of oral and esophageal infections in immuno-compromised patients, individuals on drug therapy, and the chronically ill. Because it has been observed that persons suffering from hyposalivation have an increased prevalence of oral candididiasis, we developed an animal model of infection based on hyposalivation. The objectives of our studies were to understand the mechanisms by which C. albicans causes oral disease and to begin to elucidate the role played by saliva in controlling C. albicans in the oral cavity. Our results showed that (i) oral Candida infection was established by a small challenge inoculum, (ii) mucosal lesions developed in the oral cavities and esophagi of infected rats, and (iii) transmission of oral Candida infection from an inoculated rat to uninoculated cagemates occurred rapidly. In addition, we compared the abilities of a clinical isolate and a spontaneously derived morphological mutant from that isolate to infect hyposalivatory rats and to induce disease. Infection was induced by the morphological mutant in hyposalivatory rats; however, the morphological mutant took significantly longer to transmit oral infection to uninoculated cagemates than did the parental strain.
PMCID: PMC258801  PMID: 2194965
24.  Candida species and other yeasts in the oral cavities of type 2 diabetic patients in Cali, Colombia 
Colombia Médica : CM  null;44(1):26-30.
To determine the prevalence of Candida species and to study factors associated to oral cavity colonization in patients with type 2 diabetes mellitus.
A total of 107 diabetics were classified into controlled and uncontrolled according to glycosylated hemoglobin values. Each patient was assessed for stimulated salivary flow rates, pH, and an oral rinse to search for yeast. The study also determined the state of oral health via Klein and Palmer CPO indexes for permanent dentition, dental plaque by O'Leary, and a periodontal chart.
We found yeasts in 74.8% of the patients. A total of 36 of the 52 subjects with controlled diabetes presented yeasts and 44 in the uncontrolled; no significant differences (p = 0.2) were noted among the presence of yeasts and the control of blood glucose. The largest number of isolates corresponded to C. albicans, followed by C. parapsilosis. Uncontrolled individuals presented a significantly higher percentage of yeast different from C. albicans (p = 0.049).
We found a high percentage of Candida colonization and uncontrolled individuals had greater diversity of species. The wide range of CFU/mL found both in patients with oral candidiasis, as well as in those without it did not permit distinguishing between colonization and disease. We only found association between isolation of yeasts and the low rate of salivary flow.
PMCID: PMC4002006  PMID: 24892318
Type 2 diabetes mellitus; Candida sp; candidiasis; Prototheca; Colombia
25.  Most frequent scenario for recurrent Candida vaginitis is strain maintenance with "substrain shuffling": demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2. 
Journal of Clinical Microbiology  1996;34(4):767-777.
The following three basic scenarios have emerged for the genetic relatedness of strains in recurrent vaginal candidiasis: strain maintenance without genetic variation, strain maintenance with minor genetic variation, and strain replacement. To test the frequency of each of the three scenarios, the genetic relatedness of Candida albicans isolates from each of 18 patients with recurrent infections was assessed by sequential DNA fingerprinting with the following three probes: the Ca3 probe; the C1 probe, a subfragment of the Ca3 probe which hybridizes to hypervariable genomic fragments; and the unrelated CARE2 probe. In each of the 18 patients with recurrent infections, the same strain was responsible for sequential infections, suggesting that the predominant scenario is strain maintenance. However, in 56% of these patients, the strain exhibited minor genetic variations in sequential infections. These changes were not found to be progressive. Rather, the changes suggest that substrains of an established infecting strain are shuffled in sequential infections. Results are also presented that in 45% of patients with recurrent infections, oral and vulvovaginal isolates were identical, in 35% they were highly related but not identical, and in 20% they were unrelated. These results differ markedly from those for commensal isolates simultaneously cultured from the oral cavity and vulvovaginal region of healthy individuals. Finally, it is demonstrated that in all eight cases in which C. albicans was isolated from both the male sexual partner of the patient with a recurrent infection and the patient, an isolate from the male partner was identical or highly related to the vulvovaginal strain. These results demonstrate that in patients with recurrent vulvovaginitis, a single strain usually dominates both in the different body locations of the patient and in the male partner and that it is maintained through sequential infections. However, in patients with recurrent infections, different substrains of the established clone dominate in an apparently random fashion, a process that we refer to as "substrain shuffling".
PMCID: PMC228891  PMID: 8815082

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