Candida auris is an emerging, multidrug-resistant yeast, causing outbreaks in healthcare facilities. Echinocandins are the antifungal drugs of choice to treat candidiasis, as they cause few side effects and resistance is rarely found. Previously, immunocompromised patients from Kuwait with C. auris colonisation or infection were treated with echinocandins, and within days to months, resistance was reported in urine isolates. To determine whether the development of echinocandin resistance was due to independent introductions of resistant strains or resulted from intra-patient resistance development, whole genome sequencing (WGS) single-nucleotide polymorphism (SNP) analysis was performed on susceptible (n = 26) and echinocandin-resistant (n = 6) isolates from seven patients. WGS SNP analysis identified three distinct clusters differing 17-127 SNPs from two patients, and the remaining isolates from five patients, respectively. Sequential isolates within patients had a maximum of 11 SNP differences over a time period of 1-10 months. The majority of isolates with reduced susceptibility displayed unique FKS1 substitutions including a novel FKS1M690V substitution, and nearly all were genetically related, ranging from only three to six SNP differences compared to susceptible isolates from the same patient. Resistant isolates from three patients shared the common FKS1S639F substitution; however, WGS analysis did not suggest a common source. These findings strongly indicate that echinocandin resistance is induced during antifungal treatment. Future studies should determine whether such echinocandin-resistant strains are capable of long-term colonisation, cause subsequent breakthrough candidiasis, have a propensity to cross-infect other patients, or remain viable for longer time periods in the hospital environment.
Matched MeSH terms: Drug Resistance, Fungal/genetics
Biofilms play an essential role in chronic and healthcare-associated infections and are more resistant to antimicrobials compared to their planktonic counterparts due to their (1) physiological state, (2) cell density, (3) quorum sensing abilities, (4) presence of extracellular matrix, (5) upregulation of drug efflux pumps, (6) point mutation and overexpression of resistance genes, and (7) presence of persister cells. The genes involved and their implications in antimicrobial resistance are well defined for bacterial biofilms but are understudied in fungal biofilms. Potential therapeutics for biofilm mitigation that have been reported include (1) antimicrobial photodynamic therapy, (2) antimicrobial lock therapy, (3) antimicrobial peptides, (4) electrical methods, and (5) antimicrobial coatings. These approaches exhibit promising characteristics for addressing the impending crisis of antimicrobial resistance (AMR). Recently, advances in the micro- and nanotechnology field have propelled the development of novel biomaterials and approaches to combat biofilms either independently, in combination or as antimicrobial delivery systems. In this review, we will summarize the general principles of clinically important microbial biofilm formation with a focus on fungal biofilms. We will delve into the details of some novel micro- and nanotechnology approaches that have been developed to combat biofilms and the possibility of utilizing them in a clinical setting.
Matched MeSH terms: Drug Resistance, Fungal/drug effects*
Candida africana is a pathogenic species within the Candida albicans species complex. Due to the limited knowledge concerning its prevalence and antifungal susceptibility profiles, a comprehensive study is overdue. Accordingly, we performed a search of the electronic databases for literature published in the English language between 1 January 2001 and 21 March 2020. Citations were screened, relevant articles were identified, and data were extracted to determine overall intra-C. albicans complex prevalence, geographical distribution, and antifungal susceptibility profiles for C. africana. From a total of 366 articles, 41 were eligible for inclusion in this study. Our results showed that C. africana has a worldwide distribution. The pooled intra-C. albicans complex prevalence of C. africana was 1.67% (95% CI 0.98-2.49). Prevalence data were available for 11 countries from 4 continents. Iran (3.02%, 95%CI 1.51-4.92) and Honduras (3.03%, 95% CI 0.83-10.39) had the highest values and Malaysia (0%) had the lowest prevalence. Vaginal specimens were the most common source of C. africana (92.81%; 155 out of 167 isolates with available data). However, this species has also been isolated from cases of balanitis, from patients with oral lesions, and from respiratory, urine, and cutaneous samples. Data concerning the susceptibility of C. africana to 16 antifungal drugs were available in the literature. Generally, the minimum inhibitory concentrations of antifungal drugs against this species were low. In conclusion, C. africana demonstrates geographical variation in prevalence and high susceptibility to antifungal drugs. However, due to the relative scarcity of existing data concerning this species, further studies will be required to establish more firm conclusions.
Matched MeSH terms: Drug Resistance, Fungal/drug effects; Drug Resistance, Fungal/genetics
Candida albicans is an opportunistic fungal pathogen, which causes a plethora of superficial, as well as invasive, infections in humans. The ability of this fungus in switching from commensalism to active infection is attributed to its many virulence traits. Biofilm formation is a key process, which allows the fungus to adhere to and proliferate on medically implanted devices as well as host tissue and cause serious life-threatening infections. Biofilms are complex communities of filamentous and yeast cells surrounded by an extracellular matrix that confers an enhanced degree of resistance to antifungal drugs. Moreover, the extensive plasticity of the C. albicans genome has given this versatile fungus the added advantage of microevolution and adaptation to thrive within the unique environmental niches within the host. To combat these challenges in dealing with C. albicans infections, it is imperative that we target specifically the molecular pathways involved in biofilm formation as well as drug resistance. With the advent of the -omics era and whole genome sequencing platforms, novel pathways and genes involved in the pathogenesis of the fungus have been unraveled. Researchers have used a myriad of strategies including transcriptome analysis for C. albicans cells grown in different environments, whole genome sequencing of different strains, functional genomics approaches to identify critical regulatory genes, as well as comparative genomics analysis between C. albicans and its closely related, much less virulent relative, C. dubliniensis, in the quest to increase our understanding of the mechanisms underlying the success of C. albicans as a major fungal pathogen. This review attempts to summarize the most recent advancements in the field of biofilm and antifungal resistance research and offers suggestions for future directions in therapeutics development.
The in vitro susceptibility of clinical Candida isolates towards fluconazole and voriconazole was determined using the E-test method. A total of 41 clinical isolates recovered from patients since 2004 until 2009 from two local hospitals in Kuala Lumpur, Malaysia were used. These comprised Candida tropicalis, Candida albicans, Candida krusei, Candida parapsilosis, Candida rugosa, Candida dubliniensis and Candida glabrata. Strains from American Type Culture Collection were used as quality control. Lawn cultures of the isolates on RPMI-1640 agar medium supplemented with 2% glucose were incubated with the E-test strips at 35ºC for 48 h. Our results show that 71% were susceptible to fluconazole and 90% were susceptible to voriconazole. All strains of C. krusei were resistant to fluconazole and 50% were susceptible in a dose-dependent manner to voriconazole. There were 66% and 33% of C. glabrata that were resistant to fluconazole and voriconazole. Our study revealed that majority of the clinical Candida isolates was susceptible to fluconazole and voriconazole with a small percentage being resistant to both the drugs.
Evolution of fungicide resistance is a major threat to food production in agricultural ecosystems. Fungal pathogens rapidly evolved resistance to all classes of fungicides applied to the field. Resistance to the commonly used azole fungicides is thought to be driven mainly by mutations in a gene (CYP51) encoding a protein of the ergosterol biosynthesis pathway. However, some fungi gained azole resistance independently of CYP51 mutations and the mechanisms leading to CYP51-independent resistance are poorly understood. We used whole-genome sequencing and genome-wide association studies (GWAS) to perform an unbiased screen of azole resistance loci in Rhynchosporium commune, the causal agent of the barley scald disease. We assayed cyproconazole resistance in 120 isolates collected from nine populations worldwide. We found that mutations in highly conserved genes encoding the vacuolar cation channel YVC1, a transcription activator, and a saccharopine dehydrogenase made significant contributions to fungicide resistance. These three genes were not previously known to confer resistance in plant pathogens. However, YVC1 is involved in a conserved stress response pathway known to respond to azoles in human pathogenic fungi. We also performed GWAS to identify genetic polymorphism linked to fungal growth rates. We found that loci conferring increased fungicide resistance were negatively impacting growth rates, suggesting that fungicide resistance evolution imposed costs. Analyses of population structure showed that resistance mutations were likely introduced into local populations through gene flow. Multilocus resistance evolution to fungicides shows how pathogen populations can evolve a complex genetic architecture for an important phenotypic trait within a short time span.
Matched MeSH terms: Drug Resistance, Fungal/genetics*
Recurrent vulvovaginal candidiasis affects women worldwide and the resistance to azole drugs may be an important factor. The extent of strain-to-strain variation within a species and its relationship to the ability of the organism to colonize the vulvovaginal mucosa is not well established. The aims of this study were to compare: (i) the genotypes of Candida strains in sequential infections in patients with recurrent vaginitis, (ii) the genotypes of strains in patients with only one episode of infection in a period of 1 year and (iii) determine the in vitro antifungal susceptibilities of strains that cause recurrent vaginitis. Fifty-one cultured specimens from six distinct Candida species were genotyped via random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method using the ERIC1 and ERIC2 primers (ERIC, enterobacterial repetitive intergenic consensus). Statistical analyses allowed three different scenarios to be discerned for recurrent cases: (i) strain maintenance without genetic variation, (ii) strain maintenance with minor genetic variation and (iii) outright strain replacement. The genetic relatedness between strains from patients with recurrent vaginitis and patients with single episode of vaginitis were demonstrated by the dendogramme and the mean pairwise similarity coefficient S(AB) for the intergroup comparison was 0.223. However, intragroup genetic relatedness was slightly higher than intergroup comparison, with mean S(AB) of 0.261 and 0.331 for Groups I and II, respectively. A high proportion of Group I isolates (87.5%) causing recurrent infections were resistant to ketoconazole, whereas 41.7% of these isolates were cross-resistant to both clotrimazole and ketoconazole as shown by the in vitro antifungal susceptibility test, especially for C. glabrata isolates. Pregnancy status of patients displayed a highly significant association with C. albicans species whereas non-albicans species had a markedly higher prevalence in non-pregnant patients (p<0.001). These results may have a profound impact on the management of vaginal candidiasis, especially in recurrent cases.
Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases.
Matched MeSH terms: Drug Resistance, Fungal/drug effects
Flexibility in carbon metabolism is pivotal for the survival and propagation of many human fungal pathogens within host niches. Indeed, flexible carbon assimilation enhances pathogenicity and affects the immunogenicity of Candida albicans. Over the last decade, Candida glabrata has emerged as one of the most common and problematic causes of invasive candidiasis. Despite this, the links between carbon metabolism, fitness, and pathogenicity in C. glabrata are largely unexplored. Therefore, this study has investigated the impact of alternative carbon metabolism on the fitness and pathogenic attributes of C. glabrata. We confirm our previous observation that growth on carbon sources other than glucose, namely acetate, lactate, ethanol, or oleate, attenuates both the planktonic and biofilm growth of C. glabrata, but that biofilms are not significantly affected by growth on glycerol. We extend this by showing that C. glabrata cells grown on these alternative carbon sources undergo cell wall remodeling, which reduces the thickness of their β-glucan and chitin inner layer while increasing their outer mannan layer. Furthermore, alternative carbon sources modulated the oxidative stress resistance of C. glabrata as well as the resistance of C. glabrata to an antifungal drug. In short, key fitness and pathogenic attributes of C. glabrata are shown to be dependent on carbon source. This reaffirms the perspective that the nature of the carbon sources available within specific host niches is crucial for C. glabrata pathogenicity during infection.
A 2-year-old, exotic shorthair cat presented with baldness and mild scaling on trunk that was confirmed as Microsporum canis (M. canis) infection by the following methods. Wood's lamp and trichogram were used to demonstrate fungal elements suggestive of dermatophytosis consistent with M. canis. Dermatophyte test medium (DTM) and polymerase chain reaction (PCR) were used for identification. E-test and broth microdilution test were then utilized to estimate antifungal minimal inhibitory concentrations (MICs) towards ITZ and TRF respectively. The strain was isolated from the patient and revealed TRF MIC >32 µg/ml and ITZ MIC 0.023 µg/ml. Patient was cured of dermatophytosis with systemic ITZ.
The incidence of candidaemia among immunocompromised patients in Malaysia is increasing at an alarming rate. Isolation of clinical strains that are resistant to fluconazole has also risen markedly. We report here the repeated isolation of Candida tropicalis from the blood of a neonatal patient with Hirschsprung's disease. In vitro fluconazole susceptibility tests of the eight isolates obtained at different time points showed that seven of the isolates were resistant and one isolate was scored as susceptible dose-dependent. Random amplification of polymorphic DNA fingerprinting of the isolates using three primers and subsequent phylogenetic analysis revealed that these isolates were highly similar strains having minor genetic divergence, with a mean pairwise similarity coefficient of 0.893+/-0.041. The source of the infectious agent was thought to be the central venous catheter, as culture of its tip produced fluconazole-resistant C. tropicalis. This study demonstrates the utility of applying molecular epidemiology techniques to complement traditional mycological culture and drug susceptibility tests for accurate and appropriate management of recurrent candidaemia and highlights the need for newer antifungals that can combat the emergence of fluconazole-resistant C. tropicalis strains.
Four fungal isolates: Simplicillium chinense (iso 9, accession no. KX425621), Penicillium simplicissimum (iso 10, KP713758), Trichoderma asperellum (iso 11, KP792512), and Coriolopsis sp. (1c3, KM403574) were subjected to a series of induced-tolerance training under high metal concentrations to determine if greater tolerance could be achieved from constant exposure to such conditions. Adaptive tolerance assay (Tolerance Index, TI) and Field-Emission Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX) characterized their metal tolerance. "Untrained" S. chinense, P. simplicissimum and T. asperellum showed tolerance towards 4000-4500ppm Al(III) (TI: 0.64-0.71), 1000ppm Cr(III) (0.52-0.83) and Pb(II) (0.32-0.88). With tolerance training, tolerance towards 2000-6000ppm Al(III), 500-3000ppm Pb(II) and 2000-3000ppm Cr(III) were achieved (TI: 0.01-0.82) compared to untrained cultures (0.00-0.59). In contrast, tolerance training for Coriolopsis sp. and P. simplicissimum was less successful, with TI values similar or lower than untrained cultures. SEM-EDX analysis proposed biosorption and bioaccumulation as mechanisms for metal removal. The latter was demonstrated with the removal of Cr(III) and Pb(II) by S. chinense (12.37 and 11.52mgg-1, respectively) and T. asperellum (10.44 and 7.50mgg-1). Induced-tolerance training may render benefit in the long run, but this delicate approach is suggestively species and metal dependent.
The in vitro antifungal susceptibilities of 159 clinical isolates of Candida species from patients with invasive candidiasis in Kuala Lumpur Hospital, Malaysia, were determined against amphotericin B, fluconazole, voriconazole, itraconazole and caspofungin. The most common species were Candida albicans (71 isolates), Candida parapsilosis (42 isolates), Candida tropicalis (27 isolates) and Candida glabrata (12 isolates). The susceptibility tests were carried out using an E-test. The MIC breakpoints were based on Clinical Laboratory Standards Institute criteria. Amphotericin B and voriconazole showed the best activities against all the isolates tested, with MIC(90) values of ≤1 µg ml(-1) for all major species. Only one Candida lusitaniae isolate was resistant to amphotericin B, and all the isolates were susceptible to voriconazole. In total, six isolates were resistant to fluconazole, comprising two isolates of C. albicans, two of C. parapsilosis, one C. tropicalis and one C. glabrata, and all of these isolates showed cross-resistance to itraconazole. The MIC(90) of itraconazole was highest for C. glabrata and C. parapsilosis. Caspofungin was active against most of the isolates except for five isolates of C. parapsilosis. The MIC(90) of caspofungin against C. parapsilosis was 3 µg ml(-1). In conclusion, amphotericin B remains the most active antifungal agent against most Candida species except for C. lusitaniae. Voriconazole is the best alternative for fluconazole- or itraconizole-resistant isolates. Although five of the C. parapsilosis isolates showed in vitro resistance to caspofungin, more clinical correlation studies need to be carried out to confirm the significance of these findings. Currently, despite the increase in usage of antifungals in our hospitals, especially in the management of febrile neutropenia patients, the antifungal-resistance problem among clinically important Candida isolates in Kuala Lumpur Hospital is not yet worrying. However, continued antifungal-susceptibility surveillance needs to be conducted to monitor the antifungal-susceptibility trends of Candida species and other opportunistic fungal pathogens.
The in vitro susceptibilities of Malaysian clinical isolates of Cryptococcus neoformans var. grubii and C . gattii to five antifungal drugs (amphotericin B, flucytosine, fluconazole, itraconazole and ketoconazole) were determined using the Etest method. None of the Malaysian isolates was resistant to amphotericin B and ketoconazole. Isolates resistant to flucytosine, fluconazole and itraconazole were observed in this study. Minimum inhibition concentrations (MICs) of > or = 32 microg ml(-1) against flucytosine, > or = 64 microg ml(-1) against fluconazole and > or = 1 microg ml(-1) against itraconazole were noted in four (8.3%), two (4.2%) and one (2.1%) isolates respectively. There was no significant difference in the MICs for both Cryptococcus species (P > 0.05), indicating that C. gattii was as susceptible as var. grubii to all the antifungal drugs tested. No significant difference in the MICs for both Cryptococcus species collected from 1980 to 1990 and 2002 to 2004 were observed (P > 0.05).
The aims of our research were to investigate the gene expression of the multidrug efflux transporter, CDR1 and the major drug facilitator superfamily transporter, MDR1 gene in azole drug-resistant Candida albicans and Candida glabrata clinical isolates recovered from vaginitis patients; and to identify hotspot mutations that may be present in the C. albicans CaCDR1 gene that could be associated with drug-resistance. The relative expression of the CDR1 and MDR1 transcripts in ketoconazole and clotrimazole-resistant isolates and drug-susceptible ATCC strains were determined by semi-quantitative reverse transcription-polymerase chain reaction. Expression of CaCDR1 transcript was upregulated to varying extents in all three azole-resistant C. albicans isolates studied (1.6-, 3.7- and 3.9-fold) and all three C. glabrata isolates tested (at 1.9-, 2.3- and 2.7-fold). The overexpression level of CaCDR1 in the isolates correlated with the degree of resistance as reflected by the minimum inhibitory concentration (MIC) of the drugs. The messenger RNA for another efflux pump, MDR1, was also overexpressed in one of the azole-resistant C. albicans isolates that overexpressed CDR1. This finding suggests that drug-resistance may involve synergy between energy-dependent drug efflux pumps CDR1p and MDR1p in some but not all isolates. Interestingly, DNA sequence analysis of the promoter region of the CaCDR1 gene revealed several point mutations in the resistant clinical isolates compared to the susceptible isolates at 39, 49 and 151 nucleotides upstream from the ATG start codon. This finding provides new information on point mutations in the promoter region which may be responsible for the overexpression of CDR1 in drug-resistant isolates.
Matched MeSH terms: Drug Resistance, Fungal/genetics
In the Asia-Pacific region, Candida albicans is the predominant Candida species causing invasive candidiasis/candidemia in Australia, Japan, Korea, Hong Kong, Malaysia, Singapore and Thailand whereas C. tropicalis is the most frequently encountered Candida species in Pakistan and India. Invasive isolates of C. albicans, C. parapsilosis complex and C. tropicalis remain highly susceptible to fluconazole (>90% susceptible). Fluconazole resistance (6.8-15%), isolates with the non-wild-type phenotype for itraconazole susceptibility (3.9-10%) and voriconazole (5-17.8%), and echinocandin resistance (2.1-2.2% in anidulafungin and 2.2% in micafungin) among invasive C. glabrata complex isolates are increasing in prevalence. Moreover, not all isolates of C. tropicalis have been shown to be susceptible to fluconazole (nonsusceptible rate, 5.7-11.6% in China) or voriconazole (nonsusceptible rate, 5.7-9.6% in China).
Matched MeSH terms: Drug Resistance, Fungal/drug effects
Peritonitis is the leading complication of peritoneal dialysis, which is primarily caused by bacteria rather than fungi. Peritonitis is responsible for approximately 18% of the infection-related mortality in peritoneal dialysis patients. In this paper, we report the isolation of a rare fungus, Quambalaria cyanescens, from the peritoneal fluid of a man after he switched from continuous ambulatory peritoneal dialysis to nocturnal intermittent peritoneal dialysis. Based on the morphological examination and multigene phylogeny, the clinical isolate was confirmed as Q. cyanescens. This pathogen exhibited low sensitivity to all tested echinocandins and 5-flucytosine. Interestingly, morphological characterization revealed that Q. cyanescens UM 1095 produced different pigments at low temperatures (25°C and 30°C) on various culture media. It is important to monitor the emergence of this rare fungus as a potential human pathogen in the tropics. This study provides insight into Q. cyanescens UM 1095 phenotype profiles using a Biolog phenotypic microarray (PM). Of the 760 nutrient sources tested, Q. cyanescens UM 1095 utilized 42 compounds, and the fungus can adapt to a broad range of osmotic and acidic environments. To our knowledge, this is the first report of the isolation of Q. cyanescens from peritoneal fluid, revealing this rare fungus as a potential human pathogen that may be misidentified using conventional methods. The detailed morphological, molecular and phenotypic characterization of Q. cyanescens UM 1095 provides the basis for future studies on its biology, lifestyle, and potential pathogenicity.
Glucose is an important fuel source to support many living organisms. Its importance in the physiological fitness and pathogenicity of Candida glabrata, an emerging human fungal pathogen has not been extensively studied. The present study aimed to investigate the effects of glucose on the growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of C. glabrata. In addition, its effect on the expression of a putative high affinity glucose sensor gene, SNF3 was also investigated. Glucose concentrations were found to exert effects on the physiological responses of C. glabrata. The growth rate of the species correlated positively to the amount of glucose. In addition, low glucose environments were found to induce C. glabrata to form biofilm and resist amphotericin B. Conversely, high glucose environments promoted oxidative stress resistance of C. glabrata. The expression of CgSNF3 was found to be significantly up-regulated in low glucose environments. The expression of SNF3 gene in clinical isolates was found to be higher compared to ATCC laboratory strains in low glucose concentrations, which may explain the better survivability of clinical isolates in the low glucose environment. These observations demonstrated the impact of glucose in directing the physiology and virulence fitness of C. glabrata through the possible modulation by SNF3 as a glucose sensor, which in turn aids the species to adapt, survive and thrive in hostile host environment.
Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.