Tropicalis Infections Research Articles

Rapid identification of the predominant azole-resistant genotype in Candida tropicalis.

Candida tropicalis is a leading cause of non-albicans candidemia in tropical/sub-tropical areas and a predominant genotype of azole-resistant C. tropicalis clinical isolates belongs to clade 4. The aim of this study was to reveal markers for rapidly identifying the predominant azole-resistant Candida tropicalis genotype. We analysed XYR1, one of the six genes used in the multilocus sequence typing analysis, and SNQ2, an ATP-binding cassette (ABC) transporter in 281 C. tropicalis, including 120 and 161 from Taiwan and global areas, respectively. Intriguingly, the first 4-mer of codon sequences ATRA of CTRG_05 978 (96/119vs. 21/162, p<0.001, at phi=0. 679) and the SNQ2 A2977G resulting in amino acid I993V alternation (105/118vs.12/163, p<0.001, at phi=0.81) was significantly associated with the clade 4 genotype. The sensitivity and specificity of the clade 4 genotype detection with a combination of SNPs of CTRG_05 978 and SNQ2 were 0.812 and 0.994, respectively, at phi=0.838. Furthermore, we successfully established a TaqMan SNP genotyping assay to identify the clade 4 genotype. Our findings suggest that to improve the management of C. tropicalis infections, rapidly identifying azole-resistant C. tropicalis by detecting SNPs of CTRG_05 978 and SNQ2 is promising.

Geraniol Potentiates the Effect of Fluconazole against Planktonic and Sessile Cells of Azole-Resistant Candida tropicalis: In Vitro and In Vivo Analyses.

Candida tropicalis is regarded as an opportunistic pathogen, causing diseases ranging from superficial infections to life-threatening disseminated infections. The ability of this yeast to form biofilms and develop resistance to antifungals represents a significant therapeutic challenge. Herein, the effect of geraniol (GER), alone and combined with fluconazole (FLZ), was evaluated in the planktonic and sessile cells of azole-resistant C. tropicalis. GER showed a time-dependent fungicidal effect on the planktonic cells, impairing the cell membrane integrity. Additionally, GER inhibited the rhodamine 6G efflux, and the molecular docking analyzes supported the binding affinity of GER to the C. tropicalis Cdr1 protein. GER exhibited a synergism with FLZ against the planktonic and sessile cells, inhibiting the adhesion of the yeast cells and the viability of the 48-h biofilms formed on abiotic surfaces. C. tropicalis biofilms treated with GER, alone or combined with FLZ, displayed morphological and ultrastructural alterations, including a decrease in the stacking layers and the presence of wilted cells. Moreover, neither GER alone nor combined with FLZ caused toxicity, and both treatments prolonged the survival of the Galleria mellonella larvae infected with azole-resistant C. tropicalis. These findings indicate that the combination of GER and FLZ may be a promising strategy to control azole-resistant C. tropicalis infections.

Successful combined treatment with surgery and ALA-PDT for cutaneous infection by candida tropicalis: A case report and literature review

Candidiasis is a common fungal infection, with Candida tropicalis infections being relatively rare. These infections develop into skin fungal infections under conditions such as broad-spectrum antibiotic use, diabetes, and immunosuppression. Traditionally, antifungal drugs have been the primary treatment; however, issues like hepatotoxicity and the need for long-term use persist. Consequently, new treatment approaches are essential. Most previous reports have focused on Candida albicans, with fewer addressing Candida tropicalis. This study reports a case where a patient with a skin Candida tropicalis infection was treated with a combination of surgery and ALA photodynamic therapy(635 nm, 120 J/cm2, 177 mW/cm2). After just one session of this combined treatment, the patient achieved significant clinical remission. Therefore, the combination of surgery and ALA-PDT may offer a safe and effective treatment option.

Antifungal activity of nisin against clinical isolates of azole-resistant Candida tropicalis.

The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p < 0.0001), while no significant differences were observed in the expression levels of the MDR1 and CDR1 genes. The minimum inhibitory concentration of nisin against C. tropicalis ranged from 2 to 8 μg/mL. Nisin treatment inhibited the growth of azole-resistant C. tropicalis, with over a four-fold reduction in OD600 nm values observed at the 8-h time point, while it promoted the transition of C. tropicalis from the spore phase to the hyphal phase, as observed on cryo-scanning electron microscopy. The results of biofilm quantification using crystal violet staining indicated a significant decrease in OD570 nm values in the nisin-treated group compared to the controls (p < 0.0001). Among the 21 azole-resistant C. tropicalis strains, the biofilm formation was inhibited in 17 strains (17/21, 81%), and more than 85% inhibition of biofilm formation was observed in the representative strains. With regard to the molecular mechanisms, the expression of the BCR1 and UPC2 genes in the azole-resistant strains was down-regulated on nisin treatment (p < 0.05). In conclusion, we demonstrated, for the first time, that nisin has antifungal activity and significant anti-biofilm activity against clinical isolates of azole-resistant C. tropicalis strains. Based on the findings, nisin could be a promising alternative antifungal agent for combating azole-resistant C. tropicalis infections.

Fungal Spectrum and Susceptibility Against Nine Antifungal Agents in 525 Deep Fungal Infected Cases.

Deep fungal infection has become an important cause of infection and death in hospitalized patients, and this has worsened with increasing antifungal drug resistance. A 3-year retrospective study was conducted to investigate the clinical characteristics, pathogen spectrum, and drug resistance of deep fungal infection in a regional hospital of Guangzhou, China. Non-duplicate fungi isolates recovered from blood and other sterile body fluids of in-patients of the clinical department were identified using biochemical tests of pure culture with the API20C AUX and CHROMagar medium. Antifungal susceptibilities were determined by Sensititre YeastOne® panel trays. In this study, 525 patients (283 female, 242 male) with deep fungal infection were included, half of them were elderly patients (≥60 years) (54.67%, n=286). A total of 605 non-repetitive fungi were finally isolated from sterile samples, of which urine specimens accounted for 66.12% (n=400). Surgery, ICU, and internal medicine were the top three departments that fungi were frequently detected. The mainly isolated fungal species were Candida albicans (43.97%, n=266), Candida glabrata (20.00%, n=121), and Candida tropicalis (17.02%, n=103), which contributed to over 80% of fungal infection. The susceptibility of the Candida spp. to echinocandins, 5-fluorocytosine, and amphotericin B remained above 95%, while C. glabrata and C. tropicalis to itraconazole were about 95%, and the dose-dependent susceptibility of C. glabrata to fluconazole was more than 90%. The echinocandins had no antifungal activity against Trichosporon asahi in vitro (MIC90>8 μg/mL), but azole drugs were good, especially voriconazole and itraconazole (MIC90 = 0.25 μg/mL). The main causative agents of fungal infection were still the genus of Candida. Echinocandins were the first choice for clinical therapy of Candida infection, followed with 5-fluorocytosine and amphotericin B. Azole antifungal agents should be used with caution in Candida glabrata and Candida tropicalis infections.

Azole non-susceptible C. tropicalis and polyclonal spread of C. albicans in Central Vietnam hospitals.

Candida spp. are responsible for infections ranging from local to systemic, and resistance to antifungal first-line therapy is increasing in non-albicans Candida species. We aimed to determine the etiology of candidiasis and the antifungal resistance of Candida spp. isolated in Hue hospitals, Central-Vietnam. Species identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry supported by fungal internal-transcribed-spacer amplification and sequencing. Antifungal susceptibility testing was performed by disk diffusion method and minimum inhibitory concentrations of azoles, caspofungin, and amphotericin B against C. tropicalis were determined by broth microdilution. Polymorphism of erg11 gene associated with fluconazole resistance was carried out by polymerase chain reaction and sequencing. Multilocus sequence typing (MLST) was used for typing selected C. albicans isolates. Overall, 196 Candida isolates were detected, mostly C. albicans (48%), followed by C. tropicalis (16%), C. parapsilosis (11%), C. glabrata (9%), C. orthopsilosis (6%) and to a lesser extent another eight species. High rates of resistance to fluconazole and voriconazole (18.8%) were observed in C. tropicalis with five isolates co-resistant to both agents. Y132F and S154F missense mutations in the ERG11 protein were associated with fluconazole-resistance in C. tropicalis (67.7%). Resistance to caspofungin was found in one isolate of C. albicans. MLST identified a polyclonal population of C. albicans with multiple diploid sequence types, and with few lineages showing potential nosocomial spread. Resistance to triazole agents should be considered in C. tropicalis infections in the studied hospitals, and surveillance measures taken to avoid Candida diffusion.

Development and Application of a Short Tandem Repeat Multiplex Typing Assay for Candida tropicalis.

Candida tropicalis is a clinically important yeast that causes candidemia in humans with a high mortality rate. The yeast primarily infects immunocompromised patients, and causes outbreaks in health care facilities. Antifungal resistant isolates have been reported. We developed a short tandem repeat (STR) typing scheme for C. tropicalis to enable fast, cost-effective, and high-resolution genotyping. For the development of the typing scheme, 6 novel STR markers were selected, combined into 2 multiplex PCRs. In total, 117 C. tropicalis isolates were typed, resulting in the identification of 104 different genotypes. Subsequently, the outcome of STR typing of 10 isolates was compared to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). Isolates with more than 111 SNPs were differentiated by the typing assay. Two isolates, which were identical according to SNP analysis, were separated by STR typing in 1 marker. To test specificity, the STR typing was applied to 15 related yeast species, and we found no amplification of these targets. For reproducibility testing, 2 isolates were independently typed five times, which showed identical results in each experiment. In summary, we developed a reliable and multiplex STR genotyping for C. tropicalis, which was found to correlate well to SNP calling by WGS. WGS analysis from and extensive collection of isolates is required to establish the precise resolution of this STR assay. IMPORTANCE Candida tropicalis frequently causes candidemia in immunocompromised patients. C. tropicalis infections have a high mortality rate, and the yeast is able to cause outbreaks in health care facilities. Further, antifungal resistant isolates are on the rise. Genotyping is necessary to investigate potential outbreaks. Here, we developed and applied a STR genotyping scheme in order to rapidly genotype isolates with a high-resolution. WGS SNP outcomes were highly comparable with STR typing results. Altogether, we developed a rapid, high-resolution, and specific STR genotyping scheme for C. tropicalis.

Design of a multi-epitope vaccine against the pathogenic fungi Candida tropicalis using an in silico approach

BackgroundCandida tropicalis causes tropical invasive fungal infections, with a high mortality. This fungus has been found to be resistant to antifungal classes such as azoles, echinocandins, and polyenes in several studies. As a result, it is vital to identify novel approaches to prevent and treat C. tropicalis infections. In this study, an in silico technique was utilized to deduce and evaluate a powerful multivalent epitope-based vaccine against C. tropicalis, which targets the secreted aspartic protease 2 (SAP2) protein. This protein is implicated in virulence and host invasion. ResultsBy focusing on the Sap2 protein, 11 highly antigenic, non-allergic, non-toxic, and conserved epitopes were identified. These were subsequently paired with RS09 and flagellin adjuvants, as well as a pan HLA DR-binding epitope (PADRE) sequence to create a vaccine candidate that elicited both cell-mediated and humoral immune responses. It was projected that the vaccine design would be soluble, stable, antigenic, and non-allergic. Ramachandran plot analysis was applied to validate the vaccine construct’s 3-dimensional model. The vaccine construct was tested (at 100 ns) using molecular docking and molecular dynamics simulations, which demonstrated that it can stably connect with MHC-I and Toll-like receptor molecules. Based on in silico studies, we have shown that the vaccine construct can be expressed in E. coli. We surmise that the vaccine design is unrelated to any human proteins, indicating that it is safe to use. ConclusionsThe vaccine design looks to be an effective option for preventing C. tropicalis infections, based on the outcomes of the studies. A fungal vaccine can be proposed as prophylactic medicine and could provide initial protection as sometimes diagnosis of infection could be challenging. However, more in vitro and in vivo research is needed to prove the efficacy and safety of the proposed vaccine design.

Species Distribution and Antifungal Susceptibility of Candida spp. responsible for Pulmonary Candidiasis

Fungal infection can occur in almost all parts of the human body, including the respiratory system. One group of fungi causing opportunistic infections in the lungs also known as pulmonary candidiasis is Candida spp. This study aimed to determine species diversity and antifungal sensitivity of Candida sp. causing pulmonary candidiasis from sputum specimen isolates in the Zainoel Abidin Hospital (ZAH). The sputum specimens were from inpatients and outpatients during a period of January 2019 to January 2021. Identification of Candida spp. and the antifungal sensitivity were carried out using culture and Gram Staining as well as the VITEK® 2 Compact. The results of this study indicated that there were six species of Candida sp. obtained from the study period. Of total 73 isolates, the highest percentage of species was Candida albicans (56.16%). The percentage of pulmonary candidiasis based on age was dominated by the early-late age category 46-65 years (50.68%) and based on sex was dominated by men (71.23%). Antifungal susceptibility assays revealed that the Candida species remained highly susceptible to the antifungals (amphotericin B, caspofungin, flucytosine, fluconazole, micafungin, and voriconazole), i.e., greater than 91 percent. Overall, the results of this study indicated that pulmonary candidiasis were predominantly related to C. albicans and C. tropicalis infections in ZAH and the sensitivity of antifungal drugs remained empirically and definitively effective. This research might be important as a part of infection prevention and control strategies, as well as the administration of empirical antifungals to combat Candida-mediated lung infections.

Candida Tropicalis Spondylodiscits in an Immunocompetent Host: A Case Report and Literature Review

Abstract Background Fungal spondylodiscitis is not common but should be suspected in some cases. Candida tropicalis infections are being more frequently diagnosed due to some factors related to the microorganism. Case Description A C. tropicalis spondylodiscitis is described in a 72-year-old man who was treated with a combination of echinocandin (micafungin) and surgery. Conclusion The presence of some risk factors should promptly raise the suspicion of fungal spondylodiscitis. Treatment should be instituted as early as possible for the best outcome for the patient.

Lanosterol 14α-demethylase (CYP51)/histone deacetylase (HDAC) dual inhibitors for treatment of Candida tropicalis and Cryptococcus neoformans infections.

Invasive fungal infections remain a challenge due to lack of effective antifungal agents and serious drug resistance. Discovery of antifungal agents with novel antifungal mechanism is important and urgent. Previously, we designed the first CYP51/HDAC dual inhibitors with potent activity against resistant Candida albicans infections. To better understand the antifungal spectrum and synergistic mechanism, herein new CYP51/HDAC dual inhibitors were designed which showed potent invitro and invivo antifungal activity against C.neoformans and C.tropicalis infections. Antifungal mechanism studies revealed that the CYP51/HDAC dual inhibitors acted by inhibiting various virulence factors of C.tropicalis and C.neoformans and down-regulating resistance-associated genes. This study highlights the potential of CYP51/HDAC dual inhibitors as a promising strategy for the discovery of novel broad-spectrum antifungal agents.

Novel antifungal activity of oligostyrylbenzenes compounds on Candida tropicalis biofilms.

This study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections.

Modulation of ERG Genes Expression in Clinical Isolates of Candida tropicalis Susceptible and Resistant to Fluconazole and Itraconazole.

Candida tropicalis is a non-albicans Candida specie that causes candidosis in several countries, including Brazil. However, little is known about the mechanisms of drug resistance in C. tropicalis infections. In this study, we used clinical isolates of C. tropicalis susceptible as well as resistant to either Fluconazole or Itraconazole to assess the relationship between drug resistance and the expression of ERG and efflux pump genes. Our results showed that the main mechanism of resistance against both Fluconazole and Itraconazole in this specie is through the up-regulation of ERG rather than that of the efflux pump genes. We demonstrated that, although pre-treatment with azole drugs increases the expression of both ERG6 and ERG11 genes, the resistant or susceptible dose-dependent (SDD) samples are able to maintain high expression levels of these genes for longer periods of time than the susceptible samples.

Breakthrough Bloodstream Infections Caused by Echinocandin-Resistant Candida tropicalis: An Emerging Threat to Immunocompromised Patients with Hematological Malignancies.

Background. Candida tropicalis is a virulent fungal pathogen for which echinocandins are the primary therapy. Emergence of resistance to echinocandins of C. tropicalis carries potentially ominous therapeutic implications. Methods. We describe herein two patients with breakthrough C. tropicalis fungemia during echinocandin therapy, characterize their molecular mechanism of resistance, and systematically review 13 previously reported cases of echinocandin-resistant C. tropicalis bloodstream infections (BSIs) and other diseases. Results. Among these 15 patients with echinocandin-resistant C. tropicalis infections, the median age was 61 years (ages 28–84 years) and 13 (86%) were immunocompromised. Thirteen (86%) of all patients had a history of pervious or concurrent exposure to echinocandins. Isolates of C. tropicalis from 11 cases, including the two index cases, underwent DNA sequencing of the FKS1 gene for mutations known to confer echinocandin resistance. The amino acid substitution Ser654Pro was shown in four cases, while other FKS1 mutations encoded Ser80S/Pro, Phe641Leu, Phe641Ser, Ser80S/Pro substitutions. These mutational events were not associated with collateral increases in minimum inhibitory concentrations to antifungal triazoles and amphotericin B. Overall mortality in patients with echinocandin-resistant C. tropicalis infections was 40%. Among those six patients who died, two received monotherapy with voriconazole, one was treated with fluconazole, one remained on caspofungin, and two were switched to liposomal amphotericin B. Nine patients (60%) survived after being treated with an antifungal agent other than an echinocandin. Conclusions. Emergence of resistance to echinocandins by C. tropicalis, occurs during antifungal therapy, is associated with high mortality, is mediated by a diverse range of FKS1 mutations, retains in vitro susceptibility to triazoles and amphotericin B, and constitutes an emerging threat to patients with hematological malignancies.

Antifungal susceptibility, genotyping, resistance mechanism, and clinical profile of Candida tropicalis blood isolates.

Candida tropicalis is one of the major candidaemia agents, associated with the highest mortality rates among Candida species, and developing resistance to azoles. Little is known about the molecular mechanisms of azole resistance, genotypic diversity, and the clinical background of C. tropicalis infections. Consequently, this study was designed to address those questions. Sixty-four C. tropicalis bloodstream isolates from 62 patients from three cities in Iran (2014–2019) were analyzed. Strain identification, antifungal susceptibility testing, and genotypic diversity analysis were performed by MALDI-TOF MS, CLSI-M27 A3/S4 protocol, and amplified fragment length polymorphism (AFLP) fingerprinting, respectively. Genes related to drug resistance (ERG11, MRR1, TAC1, UPC2, and FKS1 hotspot9s) were sequenced. The overall mortality rate was 59.6% (37/62). Strains were resistant to micafungin [minimum inhibitory concentration (MIC) ≥1 μg/ml, 2/64], itraconazole (MIC > 0.5 μg/ml, 2/64), fluconazole (FLZ; MIC ≥ 8 μg/ml, 4/64), and voriconazole (MIC ≥ 1 μg/ml, 7/64). Pan-azole and FLZ + VRZ resistance were observed in one and two isolates, respectively, while none of the patients were exposed to azoles. MRR1 (T255P, 647S), TAC1 (N164I, R47Q), and UPC2 (T241A, Q340H, T381S) mutations were exclusively identified in FLZ-resistant isolates. AFLP fingerprinting revealed five major and seven minor genotypes; genotype G4 was predominant in all centers. The increasing number of FLZ-R C. tropicalis blood isolates and acquiring FLZ-R in FLZ-naive patients limit the efficiency of FLZ, especially in developing countries. The high mortality rate warrants reaching a consensus regarding the nosocomial mode of C. tropicalis transmission.

Antifungal and biofilm inhibitory effect of Cymbopogon citratus (lemongrass) essential oil on biofilm forming by Candida tropicalis isolates; an in vitro study.

Cymbopogon citratus (lemongrass) essential oil has been widely used as a traditional medicine and is well known for antimicrobial properties. Therefore, it might be a potent anti-infective and biofilm inhibitive against Candida tropicalis infections. Until now, no ideal coating or cleaning method based on an essential oil has been described to prevent biofilm formation of Candida strains on silicone rubber maxillofacial prostheses, voice prostheses and medical devices susceptible to C. tropicalis infections. To investigate the antifungal and biofilm inhibitory effects of Cymbopogon citratus oil. Clinical isolates of C. tropicalis biofilms on different biomaterials were used to study the inhibitory effect. The efficacy of Cymbopogon citratus, Cuminum cyminum, Citrus limon and Cinnamomum verum essential oils were compared on biofilm formation of three C. tropicalis isolates on 24 well polystyrene plates. C. citratus oil coated silicone rubber surfaces were prepared using hypromellose ointment as a vehicle. The antifungal tests to determine minimum inhibitory and minimum fungicidal concentrations were assessed by a microbroth dilution method and biofilm formation was determined by a crystal violet binding assay. C. tropicalis strains formed more biofilm on hydrophobic materials than on hydrophilic glass. C. citratus oil showed a high antifungal effect against all C. tropicalis strains. For comparison, C. limon oil and C. cyminum oil showed minor to no killing effect against the C. tropicalis strains. C. citratus oil had the lowest minimal inhibitory concentration of all essential oils tested and inhibited biofilm formation of all C. tropicalis strains. C. citratus oil coating on silicone rubber resulted in a 45-76% reduction in biofilm formation of all C. tropicalis strains. Cymbopogon citratus oil has good potential to be used as an antifungal and antibiofilm agent on silicone rubber prostheses and medical devices on which C. tropicalis biofilms pose a serious risk for skin infections and may cause a shorter lifespan of the prosthesis.

Candida tropicalis Isolates from Mexican Republic Exhibit High Susceptibility to Bleomycin and Variable Susceptibility to Hydrogen Peroxide.

Candida sp. are found as part of the commensal flora in humans but can cause invasive candidiasis in patients with severe underlying disease, especially cancer patients. These patients are frequently subjected to nonsurgical anticancer treatments such as ionizing radiation and anticancer drugs, which kill proliferating human cells by damaging DNA but also affect the microbiota of the patient. C. tropicalis, an emerging fungal pathogen, is associated with high mortality rates of cancer patients especially in tropical regions. In this study, we have investigated the in vitro susceptibility of 38 C. tropicalis clinical isolates from several Mexican hospitals to chronic treatments with several DNA damaging agents, including oxidizing compounds and anticancer drugs. C. tropicalis isolates displayed a high variability in their susceptibility to hydrogen peroxide (H2O2) while showing a high susceptibility to bleomycin (BLM), an anticancer drug that causes double-strand breaks in DNA. This contrasted with the moderate-to-high resistance exhibited by several C. albicans laboratory strains. At least for the C. tropicalis reference strain MYA3404, this susceptibility was hardly modified by the presence of serum. Our results open the possibility of using susceptibility to BLM to differentiate between C. tropicalis and C. albicans; however, analysis of a larger number of isolates is required. The use of BLM for prevention of C. tropicalis infections in neutropenic patients with cancer should be also evaluated. Finally, the variable susceptibility to H2O2 might be due to allelic variation of the histone acetyl-transferase complex which modulates the induction kinetics of H2O2-induced genes in C. tropicalis.

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.

Candida tropicalis Biofilms: Biomass, Metabolic Activity and Secreted Aspartyl Proteinase Production.

According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24-120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation.

Identification of Azole Resistance Markers in Clinical Isolates of Candida tropicalis Using cDNA‐AFLP Method

Global reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism(') s drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs. Two susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates. Five genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)-belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes. Our findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development.