Prevalence of Antifungal Resistance, Genetic Basis of Acquired Azole and Echinocandin Resistance, and Genotyping of Candida krusei recovered from an International Collection

This study was designed to evaluate the prevalence of antifungal resistance, genetic mechanisms associated with in vitro induction of azole and echinocandin resistance and genotyping of Candida krusei , which is intrinsically resistant to fluconazole and is recovered from clinical and non-clinical sources from different countries. Our results indicated that all the isolates were susceptible or had the wild phenotype (WT) to azoles, amphotericin B, and only 1.27% showed non-WT for flucytosine. Although 70.88% of the isolates were resistant to caspofungin, none of them were categorized as echinocandin-resistant as all were susceptible to micafungin and no FKS1 hotspot 1 (HS1) or HS2 mutations were detected. In vitro induction of azole and echinocandin resistance confirmed the rapid development of resistance at low concentrations of fluconazole (4 μg/ml), voriconazole (0.06 μg/ml) and micafungin (0.03 μg/ml), with no difference between clinical and non-clinical isolates in the resistance development. Overexpression of ABC1 gene and FKS1 HS1 mutations were the major mechanisms responsible for azole and echinocandin resistance, respectively. Genotyping of our 79 isolates coupled with 217 other isolates from different sources and geography confirmed that the isolates belong to two main subpopulations, with isolates from human clinical material and Asia being more predominant in cluster 1, and environmental and animals isolates and those from Europe in cluster 2. Our results are of critical concern, since realizing that the C. krusei resistance mechanisms and their genotyping are crucial for guiding specific therapy and for exploring the potential infection source.

Presumptive Identification of Candida spp. from Clinical Isolates and Anticandidal Effect of Essential Oils

Candida is normal flora of human skin, mouth, vagina, colon and causes mycosis worldwide. Candida causes oral thrush, vaginitis and other potentially life-threatening diseases. This study assesses anticandidal potential of essential oils against Candida species isolated from clinical samples obtained from Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, Maharashtra. On the basis of morphological characterization on Sabouraud dextrose agar, CHROMagar, Germ tube test and biochemical characterization, Candida species identified as Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis. Clove, thyme, cinnamon and eucalyptus essential oils with concentrations of 6.2, 12.5, 25, 50 and 100 μg/ml were used to study their anticandidal activity. C. albicans demonstrated zone of inhibition (ZOI) 29, 20, 32, 29 mm, Candida krusei 25, 25, 30, 21 mm, Candida glabrata 24, 22, 31, 32mm,Candida parapsilosis 33,31,33, 29mm towards clove, thyme, cinnamon and eucalyptus oil (each at 100 μg/ml) respectively which are highest ZOI as compared with antifungal agent Amphotericin-B, which showed 16, 13, 12, 8 mm against C. albicans, C. krusei, C.glabrata and C. parapsilosis respectively. All Candida spp. produced biofilms and enzyme lipase. This study will facilitate the development of novel broad-spectrum key molecules against a range of Candida species.

Development of a multiplex PCR short tandem repeat typing scheme for Candida krusei

Candida krusei is a human pathogenic yeast that can cause candidemia with the lowest 90-day survival rate in comparison to other Candida species. Infections occur frequently in immunocompromised patients and several C. krusei outbreaks in health care facilities have been described. Here, we developed a short tandem repeat (STR) typing scheme for C. krusei to allow for fast and cost-effective genotyping of an outbreak and compared identified relatedness of ten isolates to SNP calling from whole-genome sequencing (WGS). From a selection of 14 novel STR markers, six were used to develop two multiplex PCRs. Additionally, three previously reported markers were selected for a third multiplex PCR. In total, 119 C. krusei isolates were typed using these nine markers and 79 different genotypes were found. STR typing correlated well with WGS SNP typing, as isolates with the same STR genotype varied by 8 and 19 SNPs, while isolates that differed in all STR markers varied at least tens of thousands of SNPs. The STR typing assay was found to be specific for C. krusei , stable in 100 subcloned generations, and comparable to SNP calling by WGS. In summary, this newly developed C. krusei STR typing scheme is a fast, reliable, easy-to-interpret and cost-effective method compared to other typing methods. Moreover, the two newly developed multiplexes showed the same discriminatory power as all nine markers combined, indicating that multiplexes M3-1 and M9 are sufficient to type C. krusei .

Improving SCOBY starter using co-culture of tapai and bakery yeast

Urbahillah A, Jayus J, Nurhayati N. 2021. Improving SCOBY starter using co-culture of tapai and bakery yeast. Biodiversitas 22: 4617-4624. Kombucha is a beverage fermented by a symbiotic bacteria and yeast known as SCOBY (Symbiotic Culture of Bacteria and Yeast). Bacteria and yeast contribute to the formation of organic acids, aroma, taste, and flavor of kombucha. The commercial yeasts used in Indonesian are baker’s yeast and tapai yeast. This study was conducted to develop SCOBYco-culture with tapai yeast and baker’s yeast and evaluate its activity. The ingredients for the kombucha were cascara, water, and sugar, which were fermented with three formula starter, i.e. original SCOBY 10% w/v (SN), co-culture SCOBY 10% w/v with 0.1% w/v of baker’s yeast (SNR), and co-culture SCOBY 10% w/v with tapai yeast 0.1% w/v (SNT). The starter activity were determined based on the OD (Optical Density) value. Yeast screening was carried out on the dominant starter population. Furthermore, morphologically yeast was identified based on colony type, color, and shape of cell. Then yeast was identified by their fermentation profile using API 20C Aux Kit. Isolate A showed white colony with convex elevation and the cell was round-shaped. Colony of isolate B and isolate C were creamy in color and oval cell shaped. The API results revealed that the first isolate was identified as Candida famata, second isolate was as Candida krusei, and the third isolate was as Candida magnoliae. Three types of fungi were found from SCOBY, namely Mucor sp., Trichoderma sp., and Fusarium sp. Mucor sp. has non-septate hyphae, and round black spores. Trichoderma sp. has septate hyphae, greenish-white spores, and the conidia have the shape of globose to ellipsoidal Fusarium sp. has a mold with septate hyphae, yellowish-white colonies, and the conidia have the shape of obovoid. Bacteria, yeast, and mold present in the medium form a powerful symbiosis for produce metabolite.

Cinnamaldehyde and α-terpineol inhibit the growth of planktonic cultures of Candida albicans and non albicans

Agents based in natural products have been investigated for the treatment of oral candidiasis. This study aims to evaluate the antifungal effect of phytoconstituent cinnamaldehyde and α-terpineol in planktonic cultures of Candida albicans, Candida glabrata, Candida krusei and clinical isolates of C. albicans. Reference strains of C. albicans (ATCC 90028 and ATCC 60193), C. glabrata (ATCC 2001), C. krusei (ATCC 34135) and four clinical isolates were used. Nistatin 100,000UI was used as a positive control. After preparation of the inoculum (1 × 103 CFU / mL), serial microdilution technique was performed using RPMI 1640 medium. Results: in reference strains, the MIC for α-terpineol ranged from 312,5 μg / mL (C. albicans 90028) to 40 μg / mL (C. krusei); and the cinnamaldehyde ranged from 40 μg / mL (C. albicans 90028, C. albicans 60193 and C. glabrata) to 20 μg / mL (C. krusei). Whereas for clinical strains, the MIC for α-terpineol ranged from 156 μg / mL to 78 μg / mL and cinnamaldehyde ranged from 78 μg / mL to 40 μg / mL. Therefore, the cinnamaldehyde and α-terpineol present an inhibitory effect against planktonic cultures of Candida albicans and not albicans.

The A756T Mutation of the ERG11 Gene Associated With Resistance to Itraconazole in Candida Krusei Isolated From Mycotic Mastitis of Cows

Candida krusei (C. krusei) has been recently recognized as an important pathogen involved in mycotic mastitis of cows. The phenotypic and molecular characteristics of 15 C. krusei clinical isolates collected from cows with clinical mastitis in three herds of Yinchuan, Ningxia, were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry. In addition to sequencing analysis, the ERG11 gene that encodes 14α-demethylases, the expression of the ERG11 gene, and efflux transporters ABC1 and ABC2 in itraconazole-susceptible (S), itraconazole-susceptible dose dependent (SDD), and itraconazole-resistant (R) C. krusei isolates was also quantified by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. Sequencing analysis revealed three synonymous codon substitutions of the ERG11 gene including T939C, A756T, and T642C in these C. krusei clinical isolates. Among them, T642C and T939C mutations were detected in itraconazole-resistant and -susceptible C. krusei isolates, but the A756T substitution was found only in itraconazole-resistant isolates. Importantly, the expression of the ERG11 gene in itraconazole-resistant isolates was significantly higher compared with itraconazole-SDD and itraconazole-susceptible isolates (p = 0.052 and p = 0.012, respectively), as determined by the qRT-PCR assay. Interestingly, the expression of the ABC2 gene was also significantly higher in itraconazole-resistant isolates relative to the itraconazole-SDD and itraconazole-susceptible strains. Notably, the expression of ERG11 was positively associated with resistance to itraconazole (p = 0.4177 in SDD compared with S, p = 0.0107 in SDD with R, and p = 0.0035 in S with R, respectively). These data demonstrated that mutations of the ERG11 gene were involved in drug resistance in C. krusei. The A756T synonymous codon substitution of the ERG11 gene was correlated with an increased expression of drug-resistant genes including ERG11 and ABC2 in itraconazole-resistant C. krusei isolates examined in this study.

Atividade antifúngica de isolados clínicos de Candida não-albicans aos óleos essenciais de Syzygium aromaticum (cravo-da-índia) e Eucalyptus globulus (eucalipto-comum)

Introdução: O gênero Candida infecta o ser humano com alta incidência, sendo a espécie Candida albicans a mais isolada em infecções invasivas e superficiais. Porém, tem sido relatado um aumento considerável de espécies de Candida não-albicans em infecções fúngicas. Os óleos essenciais, por serem voláteis, podem agir como sinais de comunicação química e arma de defesa. Objetivo: Avaliar a eficácia, in vitro, dos óleos essenciais de Syzygium aromaticum e Eucalyptus globulus na inibição do crescimento de espécies de Candida não-albicans. Métodos: Para avaliação da atividade antifúngica de S. aromaticum e de E. globulus e do efeito dos seus óleos essenciais sobre a micromorfologia das espécies Candida krusei, Candida parapsilosis e Candida glabrata, foram empregadas, nesta ordem, as técnicas de difusão em ágar e microcultivo para leveduras. Resultados: Na técnica de difusão, o óleo essencial de S. aromaticum apresentou formação de halo de inibição para Candida krusei, Candida parapsilosis e Candida glabrata. O óleo de E. globulus, por sua vez, não apresentou crescimento de halos de inibição em nenhuma das concentrações testadas frente as três espécies de Candida não-albicans. Todavia, com o microcultivo, ambos os óleos essenciais se provaram, in vitro, eficazes antimicrobianos tendo apresentado estruturas indicativas de atividade antifúngica na maior concentração dos óleos e diferentes graus de destruição celular nas demais concentrações. Conclusão: Nas condições deste estudo, concluiu-se que os produtos avaliados exerceram atividade antifúngica contra cepas de Candida não-albicans, destacando-se o óleo essencial de S. aromaticum que apresentou atividade antimicrobiana em ambas as metodologias.

Avaliação do extrato floral de Fridericia platyphylla (Cham.) L. G. Lohmann sobre Candida krusei, C. tropicalis, C. guilliermondii e C. albicans

O objetivo do estudo, foi verificar a ação antifúngica sobre Candida a partir de diferentes concentrações de extrato hidroetanólico 70% floral de Fridericia platyphylla. As flores foram coletadas no Cerrado sentido restrito, e o extrato produzido por maceração. Foram realizadas análises organolépticas para cor, aroma e homogeneidade da solução, pH e densidade relativa. O ensaio antifúngico foi realizado em diferentes concentrações de extrato floral utilizando o método de difusão em disco sobre Candida, C. albicans, C. guilliermondii, C. krusei e C. tropicalis. O extrato apresentou coloração vinho acastanhado, aromático, límpido e homogêneo, pH de 5,25 e densidade relativa igual a 0,9699 g mL-1 20 °C. A atividade antifúngica mostrou potencial fungistático entre as cepas de Candida testadas, com resultados de 16,9-8,7 mm para C. tropicalis, entre 19,7-4,6 mm para C. guilliermondii, entre 26,1-9,1 mm para C. albicans e de 11,8-8,7 mm para C. krusei entre as concentrações de extrato floral 500-15,62 mg mL-1. O extrato hidroetanólico floral de Fridericia platyphylla demonstrou bons resultados de inibição fúngica in vitro. Novos trabalhos deverão ser realizados testando o sinergismo entre os fármacos antifúngicos e o extrato floral.

Morphological characteristics of Candida albicans, Candida krusei, Candida guilliermondii, and Candida glabrata biofilms, and response to farnesol

Background and Aim: Different Candida species isolated in humans and animals have different types of parasite activity. The most pathogenic species is Candida albicans followed by Candida tropicalis. However, the effects of the morphology of Candida krusei, Candida guilliermondii, and Candida glabrata biofilms on the pathogenicity of these species have not been fully characterized. To the best of our knowledge, there is no literature on the effect of farnesol on rare Candida species. This study aimed to check the effect of different farnesol concentrations on the species C. krusei, C. guilliermondii, and C. glabrata compared with the strain C. albicans ATCC 10231, which has been widely studied, and is a strong producer of biofilms. Materials and Methods: We studied the morphological and densitometric parameters of biofilms produced by Candida species under the influence of the drug farnesol (Sigma-Aldrich, St. Louis, MO). We used a heart brain broth with the addition of 2% bovine blood serum in 96-well plates. To each well, we added 100 μL of C. albicans, C. krusei, C. guilliermondii, or C. glabrata culture, and 0.2-400 μM farnesol. The microliter plates were cultured with the lid closed at 37°C for 48 h. Then, the liquid was removed, and the wells were washed 3 times with 200 μL phosphate buffer solution (pH 7.3). Biofilm fixation was performed using 150 μL of 96% ethanol for 15 min. Then, the microliter plates were dried for 20 min at 37°C, a 0.5% solution of crystalline violet was added, and the plates were placed in an incubator at 37°C. After 5 min, the contents of the wells were removed, washed 3 times with 200 μL of phosphate buffer solution (pH 7.2), and dried. The dye was extracted by washing with 200 μL of 96% ethanol for 30 min. The results were obtained using a photometric analyzer of enzyme immunoassay reactions at an optical density (OD) wavelength of 450 nm. Results: All of Candida spp. strains tested were susceptible to farnesol at concentrations ranging from 0.8 to 400 μM for C. albicans, C. krusei, and C. guilliermondii, and 12.5 to 400 μM for C. glabrata. Conclusion: This study provides new insights into the use of farnesol against biofilms produced by Candida species, but further studies in vivo are necessary to evaluate the effectiveness of the reduction of OD. To the best of our knowledge, the antimicrobial activity of farnesol against C. krusei, C. guilliermondii, and C. glabrata has not been reported previously, although studies have confirmed the inhibitory effect of farnesol on the growth of different microorganisms.

Extração, caracterização e avaliação microbiológica de óleos essenciais de rejeitos de frutas cítricas comerciais

Com o aumento da produção de resíduos oriundos de processamento de alimentos diversas medidas estão sendo implementadas gerando benefícios que ultrapassam as questões ambientais. Este trabalho propôs o reaproveitamento de cascas de frutas cítricas excedentes de estabelecimentos comerciais, por meio da extração de óleos essenciais (OEs). Avaliou-se sua eficácia antimicrobiana, os parâmetros físico-químicos, sua constituição química e os padrões de agrupamento dos OEs com base em análises estatísticas. Os OEs foram extraídos das cascas pelo método de hidrodestilação. Determinou-se o índice de refração, densidade relativa e solubilidade em etanol 90%. A identificação dos constituintes foi realizada por meio da GC-MS e para os agrupamentos das amostras utilizou-se PCA e HCA. Para a atividade antimicrobiana utilizou-se a técnica de difusão de disco, utilizando duas leveduras (Candida parapsilosis e Candida krusei) e duas bactérias (Staphylococcus aureus e Escherichia coli). Constatou-se que os OEs dos cultivares de laranja apresentam maior rendimento de extração e as análises físico-químicas foram similares entre os cultivares de laranja e limão. Identificou-se 61 componentes nos OEs, sendo o d-limoneno majoritário. A PCA e a HCA revelaram agrupamentos que corroboram com as cultivares laranjas, limões e mexerica. Todos os microrganismos foram sensíveis aos OEs dos limões e da mexerica e a E. coli foi sensível a todos os OEs. Por sua vez, a bactéria S. aureus se mostrou resistente aos OEs dos cultivares de laranja. Constatou-se que o reaproveitamento do excedente das cascas de frutas cítricas é viável, sendo os OEs eficazes contra determinados microrganismos.