Candida tropicalis is an emerging major human pathogen in nosocomial infections, and it is considered the second or third species of Candida most isolated from urine cultures.
AimsThe study aimed at characterizing genotypically C. tropicalis strains from patients with candiduria in a university hospital, and assessed the antifungal susceptibility profile.
MethodsThe study was conducted with hospitalized patients who developed urinary tract infection from C. tropicalis from June 2010 to June 2011 at the Grande Dourados University Hospital of the Federal University, Dourados, MS, Brazil. Susceptibility to the antifungal agents amphotericin B and fluconazole was determined by broth microdilution. The genotypic variability of isolates of C. tropicalis was analyzed by microsatellite markers and RAPD-PCR.
ResultsOnly one isolate was resistant to amphotericin B (MIC→16μg/ml); the others were susceptible to fluconazole and amphotericin B. The genotypic variability by RAPD-PCR resulted in distinct profiles for RAPD markers. A total of 10 alleles were observed for the microsatellite loci, URA3 and CT14, which were grouped differently, and four associations were observed for locus URA3 and eight for locus CT14.
ConclusionsC. tropicalis isolates from urine were susceptible to the antifungal agents tested. The genotyping techniques make possible proving the similarity and genetic diversity among isolates of C. tropicalis involved in nosocomial infections. This knowledge is important for the control and prevention of nosocomial infections caused by this yeast species.
Candida tropicalis es un patógeno humano emergente en las infecciones nosocomiales y es considerado la segunda o tercera especie de Candida más aislada en cultivos de orina.
ObjetivosEl objetivo del estudio fue caracterizar genotípicamente aislamientos de C. tropicalis procedentes de pacientes con candiduria de un hospital universitario, y evaluar su perfil de sensibilidad a los antifúngicos.
MétodosLa investigación fue realizada con pacientes hospitalizados que desarrollaron una infección urinaria por C. tropicalis desde junio de 2010 hasta junio de 2011 en el Hospital Universitario de la Universidad Federal de Grande Dourados, Dourados, MS, Brasil. La sensibilidad a los agentes antifúngicos anfotericina B y fluconazol fue determinada mediante el método de microdilución en caldo. La variabilidad genotípica de los aislamientos de C. tropicalis se analizó mediante marcadores microsatélites y RAPD-PCR.
ResultadosSólo un aislamiento fue resistente a la anfotericina B (MIC→16mg/ml); los restantes aislamientos fueron sensibles al fluconazol y la anfotericina B. La variabilidad genotípica por RAPD-PCR dio como resultado perfiles distintos para los marcadores utilizados. Se observó un total de 10 alelos de los loci microsatélites, URA3 y CT14 fueron agrupados de manera diferente y se observaron cuatro asociaciones para el locus URA3 y ocho para el locus CT14.
ConclusionesLos aislamientos de C. tropicalis obtenidos de orina fueron sensibles a los antifúngicos probados. Las técnicas de genotipificación permiten demostrar la similitud y la diversidad genética de los aislamientos de C. tropicalis implicados en las infecciones nosocomiales. Este conocimiento es importante para el control y la prevención de las infecciones hospitalarias causadas por esta especie de levadura.
Nosocomial infections caused by Candida yeasts have increased significantly worldwide in recent years and have been the growing cause of morbidity and mortality in hospitalized patients, especially those severely ill and immunocompromised.5,13,27,33,35
Nosocomial urinary tract infections caused by Candida spp. frequently occur in hospitalized patients, and account for 10–15% of all urinary tract infections.4 Episodes of candiduria in hospitalized patients increase morbidity and offer the risk of mortality.14,34 Furthermore, candiduria can be considered a risk factor for candidemia in adult patients.16
Candida tropicalis is an emerging major human pathogen in nosocomial infections5,21,22,31 and it is considered the second or third Candida species most frequently isolated in urine cultures.15,18,24 This species has some important virulence characteristics such as increased resistance to azole antifungal drugs, significant adherence to the epithelium, more so than to the silicone, biofilm formation and expression of total hemolytic activity.23,27,32
Currently several molecular typing methods have been used for molecular characterization and correlation of Candida species in hospital infections. Among them, the Randomly Amplified Polymorphic DNA (RAPD) and microsatellite markers stand out.3,10,30 Genotyping of clinical isolates by RAPD technique involves the amplification of DNA fragments by polymerase chain reaction using short primers of random sequence.11 However, microsatellite markers also amplify DNA fragments by polymerase chain reaction and are co-dominant markers which allow the heterozygous loci to be differentiated from the homozygous ones.12 The application of these molecular tools in infections within a hospital environment is to characterize genetic variations and demonstrate the degree of similarity between the isolates. Genotyping of different Candida species is important due to its prognostic and therapeutic significance, thus generating information for clinical epidemiology, allowing the correct treatment and control of infections in hospitals.
Studies aimed at assessing the genetic diversity of species of Candida non-Candida albicans (CNCA) isolated from hospitalized patients, as well as the evaluation of antifungal susceptibility profile, are relevant for improving therapeutic approaches and control hospital fungal infections. Therefore, the present study aimed at evaluating the molecular genetic diversity of C. tropicalis and studying its susceptibility to antifungal agents in patients with candiduria hospitalized in a university hospital.
Materials and methodsSamplesUrine samples from patients with urinary infection hospitalized at the University Hospital of the Grande Dourados Federal University (Dourados, State of Mato Grosso do Sul, Brazil) from June 2010 to June 2011, whose results were positive for C. tropicalis, were processed in this study.
In order to characterize a urinary tract infection caused by C. tropicalis, the presence of more than 105 colony forming units per milliliter (CFU/mL) in urine was considered.
Isolation and identification of isolatesThe yeasts were screened by cultivation in CHROMagar Candida® (Difco, BD, Franklin Lakes, NJ, USA) through routine laboratorial analysis and stored in Sabouraud Dextrose Broth (Difco, BD, Franklin Lakes, NJ, USA) with 20% glycerol in a freezer at −70°C. Isolates of C. tropicalis were identified phenotypically by way of macroscopic, microscopic and biochemical features described in the classical method, including colony morphology, micromorphological analyses, and carbohydrate assimilation and fermentation tests.37
Antifungal susceptibility testingThe antifungal susceptibility was determined by broth microdilution method, performed according to the document M27-A3 of the Clinical and Laboratory Standards Institute.7
The antifungal agents used were amphotericin B and fluconazole, and the susceptibility cutoffs were in accordance to the parameters established by Yang et al.36 with MIC values ≤1μg/ml considered susceptible and ≥2μg/ml resistant to amphotericin, and by the supplement document M27-A3–M27-S38 to fluconazole.
Genomic DNA extractionYeasts were cultured in Sabouraud Dextrose Broth (Difco, BD, Franklin Lakes, NJ, USA) and maintained overnight at 25°C. Genomic DNA was extracted as described by Chong et al.6 The amount and purity of genomic DNA were determined by optical density in a spectrophotometer (Genesys 10, Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Molecular identification by species-specific PCR primerThe identification of the species C. tropicalis was confirmed by the amplification of the internal transcribed regions 1 and 2 of the rRNA gene of Candida species.20 The PCR reaction, adapted from Alves et al.,2 was performed with genomic DNA (10–20ng), forward and reverse primers (10pmol each), PCR Master Mix (Axygen Scientific, Union City, CA, USA) (12.5μl), and water for a final volume of 25μl. The primers used for the amplification reaction of the internal transcribed spacer 1 region (ITS1)-5.8S-ITS2 of the rRNA gene were: forward (C. tropicalis, 5-AAGAATTTAACGTGGAAACTTA-3) and reverse (5-TCCTCCGCTTATTGATATGC-3) (GenBank Accession No.: EU2888196.1). All PCR reactions were performed in Mastercycler Gradient Thermal Cycler (Eppendorf, Hamburg, Germany). Amplification conditions were initial denaturation at 94°C for 3min, 35 cycles of denaturation at 94°C for 1min, annealing at 63°C for 1min, extension at 72°C for 1min, and final extension at 72°C for 5min.
The amplified products were subjected to electrophoresis in 1.5% agarose gel (110V, 40min), stained with ethidium bromide (0.5mgml−1) and visualized under ultraviolet light. The size of the amplified product (149bp), specific to C. tropicalis, was determined by using the molecular weight marker 50bp.
RAPD-PCR assayThe molecular characterization of C. tropicalis isolates from urine was performed by RAPD-PCR based on the methodology of Bautista-Muñoz et al.3 The reaction contained 10ng of genomic DNA, 0.4μM of the primer, 2mM of MgCl2, 1.2U of Taq polymerase and 0.8mM dNTP. The primers used for the reaction were OPA-18 (5-AGCTGACCGT-3), OPE-18 (5-GGACTGCAGA-3) and P4 (5-AAGAGCCCGT-3). The reaction conditions for RAPD-PCR were initial denaturation at 94°C for 5min, 45 cycles of denaturation at 94°C for 1min, annealing at 36°C for 1min, extension at 72°C for 2min and final extension at 72°C for 10min.
The amplified products were subjected to electrophoresis in agarose gel at 1.2% in 0.5× TBE buffer (110V, 60min), stained with ethidium bromide (0.5mgml−1), and visualized under ultraviolet light. The determination of the sizes of the fragments was performed using the molecular weight marker 100bp.
Genetic variability analyses were performed with the Bionumerics® software, version 4.6 (Applied Maths, Sint-Martens-Latem, Belgium). The similarity was verified by the coefficient (SAB) between the patterns for each pair of isolates A and B and was calculated using the formula SAB=2E/(2E+a+b), where E is the number of common bands in the patterns of A and B, a being the number of bands in the pattern of a with no correlates in B pattern, and b is the number of bands in pattern B with no correlation in pattern A.
From the similarity matrix, units were grouped by the method UPGMA (Unweighted Pair-Group Method with arithmetical Average). SAB value of 1.00 indicates that the band pattern for lineage A is identical to B; values between 0.80 and 0.99 means very similar clinical isolates, but not identical, and may suggest a microevolution of a single strain; SAB values lower than 0.80 represent independent strains.3,29
Microsatellite assayThe reaction was adapted from the method described by Desnos-Ollivier et al.10 Two microsatellite markers were used, one upstream of the URA3 genes (URA3, GenBank Accession No. EU288195.1) and one non-annotated sequence CT14. Each reaction contained 50ng of genomic DNA, 0.1μM of each primer pair, 5mM of MgCl2, 1.25U of Taq polymerase and 0.8mM of dNTP. The forward and reverse primers used for the reactions were: URAF (5-ATTGGATAGTCCCTCTAAACTCACTACTA-3)/CTU2R (5-GTTGGAACATCAATTGATGCACATAAAT-3) and CT14a (5-GTAAATCTTGTATACCGTGGA-3)/CT14b (5-TAGCCCATTTTCTAGTTTTGC-3). The conditions for amplification were: 27 cycles of denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 7s and final extension at 72°C for 5min.
The amplification products were subjected to electrophoresis in 8% polyacrylamide gel in 1× TBE buffer (140V, 5h and 30min), stained with ethidium bromide (0.5mgml−1), and visualized under ultraviolet light. The determination of the sizes of the fragments was performed using the molecular weight marker 100bp.
The characteristics of co-dominance of microsatellite markers allow the identification of homozygotes and heterozygous genotypes, enabling the estimation of the discriminating power of each marker.12 The latter was calculated according to the Simpson index17 for each of the markers (URA3 and CT14).
ResultsThe phenotypic analysis for the identification of the Candida species from the 12 patients was able to identify 15 isolates of C. tropicalis obtained from cases of candiduria. The method of species-specific PCR of the regions ITS1 and ITS2 of the rRNA gene confirmed the species C. tropicalis in all the 15 isolates.
Antifungal susceptibility testThe results for the susceptibility test are shown in Table 1. The range of the Minimal Inhibitory Concentration (MIC) value was 0.125-16μg/ml for amphotericin B and 0.25–4μg/ml for fluconazole. Only one isolate was resistant to amphotericin B and the others were susceptible to fluconazole and amphotericin B (Table 2).
Data from isolates of C. tropicalis, RAPD profiles and microsatellite genotypes.
| Patients | Isolated | Date of isolation | MIC | RAPD | Microsatellites | ||||
|---|---|---|---|---|---|---|---|---|---|
| AMB | FLU | OPA-18 | OPE-18 | P4 | URA3 | CT14 | |||
| P1 | 1 | 21/06/2010 | 0.5 | 0.5 | B | B | D | 178:199 | 151:157 |
| P2 | 2 | 20/07/2010 | 1 | 1 | D | B | B | 176:176 | 148:157 |
| P3 | 3 | 22/09/2010 | >16 | 0.5 | B | B | A | 172:172 | 151:154 |
| P4 | 4 | 19/12/2010 | 1 | 1 | B | A | B | 172:172 | 148:148 |
| P5 | 5 | 24/01/2011 | 0.5 | 1 | B | A | B | 172:172 | 148:148 |
| P6 | 6 | 03/02/2011 | 1 | 0.25 | A | A | B | 172:172 | 145:151 |
| P6 | 7 | 07/02/2011 | 0.5 | 4 | A | B | A | 172:172 | 145:151 |
| P7 | 8 | 04/03/2011 | 0.5 | 2 | C | A | B | 172:172 | 145:145 |
| P8 | 9 | 19/03/2011 | 1 | 0.5 | D | B | B | 172:172 | 145:151 |
| P8 | 10 | 23/03/2011 | 0.5 | 2 | D | B | B | 172:172 | 148:154 |
| P8 | 11 | 30/03/2011 | 1 | 1 | C | A | B | 172:172 | 145:151 |
| P9 | 12 | 05/04/2011 | 0.5 | 1 | C | B | B | 172:172 | 154:154 |
| P10 | 13 | 25/04/2011 | 1 | 1 | D | B | B | 172:172 | 154:154 |
| P11 | 14 | 18/05/2011 | 1 | 0.5 | F | B | C | 172:191 | 148:148 |
| P12 | 15 | 19/05/2011 | 1 | 1 | E | B | B | 172:172 | 145:151 |
The isolates showed six (A–F), two (A and B) and four (A–D) different molecular profiles for the markers OPA-18, OPE-18 and P4 respectively (Table 2 and Fig. 1).
Dendrogram of cluster isolates from candiduria by UPGMA determined by RAPD-PCR using primers (A) OPA-18, (B) OPE-18, and (C) P4. The similarity among genotypes was determined with the Dice coefficient and calculated by the BioNumerics software version 4.6. The roman algorithms (I–IV) represent the cluster, and the letters (A–F) represent profiles.
The amplification using the primer OPA-18 allowed the formation of four clusters named I, II, III and IV, which grouped 13.3%, 26.7%, 20% and 26.7% of the samples respectively. Clusters I and II were more related to each other with a similarity coefficient of approximately 90%. Cluster IV correlated to the others in approximately 70%. Samples 14 and 15 had a correlation to the other with a similarity coefficient of 65 and 87.6%, respectively. The values found were: SAB 0.73±0.11, mean 73.86 and standard deviation of 11.08 (Fig. 1a).
The results of amplified products by the primer OPE-18 led to the formation of two clusters (I and II), which grouped 33.3% and 66.6% of the samples, respectively. The similarity coefficient between the two clusters was 93.3%. The values found were: SAB 0.96±0.33, mean 96.82 and standard deviation of 3.33 (Fig. 1b).
Analyzing the clusters constructed by means of amplified products of the primer P4, the formation of clusters I and II, which grouped 13.3% and 73.33% of the samples, respectively, was observed. The similarity coefficient between the two clusters was 80%. Samples 14 and 1 correlated to the others with a coefficient of similarity of 78% and 63.1%, respectively. The values found were: SAB 0.74±0.08, mean 74.63 and standard deviation 8.66 (Fig. 1c).
Microsatellite analysisAmplified products from microsatellites were observed for all C. tropicalis isolates. The differences in size of the amplified fragments between the two markers are related to the different number of repeats of microsatellites. Isolates that showed two fragments were heterozygous and those that showed one fragment were homozygous.
A total of 10 alleles were observed for the 15 isolates, five for each of the URA3 locus and CT14. Four allelic combinations were observed for the locus URA3 and 8 for the locus CT14 (Table 2).
Discriminatory power based on the Simpson index for the URA3 marker was 0.372, and 0.86 for CT14, while combining the two markers the discriminatory power provided was 0.88.
DiscussionThe occurrence of candiduria may be due to factors such as anatomical abnormalities in the urinary tract, comorbidities, urinary drainage devices, abdominal surgery, admission to the Intensive Care Unit (ICU), use of broad spectrum antibiotics, diabetes mellitus, increasing age and belonging to female gender.1,25
C. tropicalis has been featured among nosocomial infections as a global emerging pathogen among species of CNCA.13 It is a commensal microorganism in the human gastrointestinal tract with the potential to cause invasive infections due to virulence factors, which have greater potential to spread and cause mortality in ICU patients than C. albicans or other CNCA species.15,19,23
Studies have demonstrated the need to perform antifungal susceptibility testing for C. tropicalis, because some isolates may present a resistance to antifungal agents.15,26,32 However, the results of our study indicate that the isolates of C. tropicalis from urine were susceptible to the antifungal agents tested, except for one isolate which was resistant to amphotericin B. This profile may be related to microbiota established in this hospital or to the clinical and pathological profile of patients.
The analysis of RAPD-PCR allowed us to evaluate the genotypic profile of each isolate, making it possible to differentiate them genotypically. This method has been used for the study of clinical isolates of Candida spp., with the purpose of identification, correlation and phylogenetic analysis.28,30 The primer OPA-18 was more discriminatory than OPE-18 and P4 for genotypic analysis by RAPD-PCR; however, all indicated similarity among isolates.
With regard to microsatellites, the marker CT14 showed greater genetic difference between the isolates, as eight allelic combinations were observed. The combination of the two markers revealed nine different genotypes among the isolates, with the predominance of genotypes 172:172 and 145:151 for URA3 and CT14, respectively. However, the discriminatory power (0.88) obtained from the combination of markers is not favorable to explain the typing results with confidence.17
The occurrence of two isolates in one patient with different genotypic profiles by RAPD-PCR and microsatellite techniques was observed. Genotypic variation of these isolates may be associated to the reproduction of microorganisms during the in vivo or in vitro growth. A considerable alteration in the MIC of the isolates from patient 6 (isolates 6 and 7) to fluconazole points out the importance of antifungal susceptibility testing and warns about the tendency of decreased sensitivity of CNCA species.
Nosocomial infections caused by Candida may be due to the presence of yeasts in the patient's own microflora (endogenous) or the transmission to the patient from the microbiota in health professionals, on inanimate surfaces, catheters and probes (exogenous).9 The combination of the two techniques for genotyping revealed that isolates 4 and 5, from different patients and with different periods of hospitalization, had genotypic similarities, confirming the possibility of a spread, exogenously, of the infectious agent of a single strain from patient to patient. Thus, knowledge of the source of infection can help to prevent the spreading of resistant microorganisms and, therefore, help with the appropriate prophylactic treatment.
The results show the possibility of assessing the genetic similarity among isolates of Candida species involved in nosocomial infections, and comparing the genotypes among isolates from different sites, as already reported by other authors3,10 which aids in the investigation of outbreaks. The RAPD-PCR and microsatellite techniques allowed us to visualize the genetic diversity and molecularly define the isolates, as it was observed by other authors who used these techniques of molecular genotyping to assess the genetic diversity of species of Candida.3,10–12 This information regarding genetic diversity is important for the control and prevention of nosocomial infections of endogenous or exogenous origin caused by yeasts, especially those isolates of C. tropicalis, which have emerged as an isolated species of CNCA in hospital infections.
Conflicts of interestThe authors have no conflicts of interest to declare.
Our thanks to Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) for their financial aid for the execution of this research, to Grande Dourados Federal University for its support, and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship.
