Unusual plant-extract based media for the differentiation between species in the Candida albicans complex: A comparative study

Abstract

Candida albicans is the most common agent in human fungal infections; nevertheless, in the last decades, the closely related yeasts Candida dubliniensis and Candida africana have emerged as pathogens. The purpose of this study was to compare tobacco agar with another five agars prepared from plant extracts (Origanum vulgare, Rosmarinus officinalis, Solanum rudepannum, Solanum oblongifolium and Brugmansia arborea) on the differentiation of C. albicans complex. The hyphae and chlamyconidia formation and the color and margin of the colonies of 200 clinical isolates of C. albicans, C. dubliniensis and C. africana were evaluated. After seven days of incubation at 28 °C, Tobacco agar, S. rudepannum and B. arborea agars allowed the differentiation of 100 % C. dubliniensis. Additionally, 24% of C. africana isolates produced brownish colonies in the medium prepared from Rosmarinus officinalis (rosemary) extract. These results indicate that S. rudepannun, B. arborea and rosemary agar could be used as screening for the phenotypic differentiation between the species of C. albicans complex. Rosemary agar could be used to aid in the differentiation of C. albicans from C. africana. These culture media based on plants, could be used as simple and inexpensive screening methods in the phenotypic differentiation of C. dubliniensis and C. africana.

Key words
Brugmansia arborea; Candida africana; Candida albicans; Candida dubliniensis; Solanum oblongifolium; Solanum rudepannum

INTRODUCTION

The taxonomy of the genus Candida has changed revealing that the most significant species of the genus (Candida albicans, Candida parapsilopsis and Candida glabrata) are closely related to other cryptic species and are therefore recognized as “species complexes” (Nnadi et al. 2012). These new sister-yeasts were reported as “atypical species”, since they shared phenotypic characteristics with the type species. The routine discrimination between the closely related species has been problematic, and the most accurate method for their identification are the PCR-based tests (Arastehfar et al. 2018, Theill et al. 2016).

Currently, C. albicans complex includes Candida albicans sensu stricto; C. africana and C. stellatoidea, biovars of C. albicans (Tietz et al. 2001, Langeron & Guerra 1939, Jacobsen et al. 2008); and Candida dubliniensis (Sullivan et al. 1995), recognized as a genetically and phenotypically differentiable specie of C. albicans (Hu et al. 2015, Romeo et al. 2013). While C. albicans is the main fungal pathogen, the true occurrence of C. africana, C. stellatoidea and C. dubliniensis might be underestimated (Jorgensen & Pfaller 2015).

C. dubliniensis has demonstrated enhanced resistance to fluconazole, out of all available drugs and isolates that were sensitive to fluconazole would go on to generate resistant derivatives (Reginato el al. 2016, Sullivan et al. 2004). The primary mechanism of fluconazole resistance, which involves the upregulation of the key facilitator efflux pumps MDR1 and CDR1, is comparable to that of C. albicans (Pristov & Ghannoum 2019). Meanwhile, C africana isolates are still susceptible to widely used antifungal agents despite developing an increasing resistance to them. (Romeo & Criseo 2010).

A small number of selective complex media have been designed to distinguish members of the C. albicans complex based on colony morphology and physiological assimilation tests. On some of them, such as rosemary agar and oregano agar, C. dubliniensis isolates formed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 h of incubation at 25 °C, but on which C. albicans isolates grew as smooth colonies without chlamydospores (Loreto et al. 2008).

On media such as Staib agar (Guizotia abyssinica), Sunflower (Helianthus annuus) and Tobacco agar (Nicotiana tabacum), it is also expressed, the property of producing brown colonies is associated with the action of the laccase enzyme on melanin substrates (Al Mosaid et al. 2001, Khan et al. 2005, 2004).

All these media have proven to be inexpensive, simple, and could potentially allow an early detection of C. dubliniensis colonies; however, several authors have reported atypical isolates. Furthermore, none of these media has been used to differentiate isolates of C. africana and the use of new substrates (based on other solanaceous plants) has not been studied either.

The aim of this work is to compare the differentiation of C. albicans, C. dubliniensis and C. africana on Tobacco extract agar and another five plant extracts: Origanum vulgare, Rosmarinus officinalis, Solanum rudepannum, Solanum oblongifolium and Brugmansia arborea. Isolates of Candida stellatoidea are scarce and they were not included in this study.

MATERIALS AND METHODS

Strains

A total of 200 clinical isolates were used in this study, including C. albicans (n: 146), C. dubliniensis (n: 25) and C. africana (n: 25). Four atypical Candida albicans (with phenotypical characteristics compatible with C. africana, but with HWP1 sequences consistent with C. albicans) were included too.

All C. albicans and C. dubliniensis isolates came from vaginal and oropharyngeal samples from Colombia and they were obtained from the culture collection of the Microbiology Laboratory of Universidad Popular del Cesar (Valledupar, Colombia); and C. africana isolates came from vaginal samples from different geographical origins, including America, Europe and Asia. Atypical C. albicans were previously described by Rodríguez-Leguizamón (Bogotá, Colombia) (Rodriguez-leguizamon et al. 2015). All the strains were previously identified by phenotypic, proteomic and molecular methods (Lachance et al. 2011, Romeo & Criseo 2008, Roberts et al. 2016).

C. albicans ATCC 68548, ATCC 28367, ATCC 90028 and C. dubliniensis CBS 7987 were used as control strains. Besides, 100 clinical isolates including Cryptococcus neoformans (10), Nakaseomyces glabratus (C. glabrata) (10), C. parapsilosis (10), C. tropicalis (10), C. auris (30), C. pseudohaemulonii (10), Clavispora lusitaniae (C. lusitaniae) (5), C. metapsilosis (5), Kluyveromyces marxianus (C. kefyr) (5), and Meyerozyma guilliermondii (C. guilliermondii) (5) were used.

Plants

All plants were acquired from Mogambo Sendero Ambiental (http://mogambosenderoambiental.com) and voucher specimens were deposited at Herbario Nacional de Colombia. Plant material was dried at 50 °C in a convection oven and stored at room temperature (25 °C).

N. tabacum, R. officinalis (rosemary) and O. vulgare (oregano) agar have been used before by other authors (Khan et al. 2004, De Loreto et al. 2008). S. rudepannum (turkey berry), S. oblongifolium (cucubo) and B. arborea (white angel’s trumpet) were used as they are plants of the Solanacea family, as is N. tabacum.

Culture media

Tobacco agar was prepared according to Khan et al. 2004, and rosemary agar and oregano agar were prepared according to De Loreto et al. 2008. Three more agars were prepared from the extracts of plants in the Solanaceae family: turkey berry, cucubo and white angel’s trumpet, which were obtained by 2 h of boiling using a Clevenger apparaturs (200 g of dried stems and leaves per liter of destilled water) that reduced the loss of water by condensation of water vapor. Later, filtration through several layers of gauze was done and 200 mL of each extract were added to 800 mL of a basal medium (dextrose 1%; creatinine 0.078%; agar 2%, and 800 mL of distilled water) and sucrose 5 g/L was added. The solutions were autoclaved separately at 121 °C for 15 min and then allowed to cool to 50 – 55 °C. These conditions are similar to those used by De Loreto et al. 2008.

Finally, all the media were dispensed in 25 mL into Petri dishes (90 mm-diameter) and the agar plates were inoculated with 48-hour-old culture (Sabouraud agar) C. albicans, C. dubliniensis and C. africana isolates; followed by incubation at 28 °C and 37 °C for 7 days. 28° C (25 °C – 30 °C) is the temperature used in previous studies; but 37ºC is the most used in clinical laboratories, and in fact, many laboratories only have one incubator, which is usually used at 37° C.

Plates were examined daily looking for variations in macromorphology (color and margin of the colony) and micromorphology (single conidia, blastoconidia, chlamydoconidia and hyphae). Analysis was carried out three times on different days. The microscopic characteristics were observed under 100 and 400 x magnifications.

The authors declare that in this work no experiments were carried out in humans or animals.

RESULTS

C. albicans and C. dubliniensis isolates grew well on all the culture media and at both incubation temperatures; however, C. africana isolates grew poorly in all media when the incubation temperature was 28 °C.

In both temperatures and after 7 days of incubation, C. albicans isolates (typical and atypical) showed white-to-cream color colonies in all media (Figure 1a-b, A); C. dubliniensis isolates showed white-to-cream color colonies in all media (Figure 1a-1b, B), except Tobacco agar, where it produced yellowish brown colonies; and C. africana presented white-to-cream colonies in media prepared from oregano and white angel’s trumpet (Figure 1a, C). Six strains of C. africana, showed brown colonies in media prepared from rosemary (Figure 1b, C).

Figure 1
C. albicans (A), C. dubliniensis (B) and C. africana (C) isolates in media prepared from a) Solanum oblongifolium (cucubo) and b) Rosmarinus officinalis (rosemary) after 7 days of incubation.

In all culture, C. dubliniensis isolates showed conidia, blastoconidia and pseudomycelia; while C. albicans and C. africana showed solitary conidia and few blastoconidia. Chlamydoconidia were absent in C. albicans and C. africana isolates and C. dubliniensis produced chlamydoconidia on media prepared from tobacco, turkey berry and white angel’s trumpet. In terms of colony appearance, production of conidia and micromorphology, no differences were observed between isolates of the same species. (Figure 2 and Table I).

Figure 2
Chlamydoconidias produced by C. dubliniensis in media prepared from Solanum rudepannum (friegaplatos) (Lactophenol cottom blue, 400x).

Table I
Macroscopic and microscopic characteristics of Candida albicans complex in different plant extract media, after incubation at 28 °C.

Plant extract	Characteristics	C. albicans (n: 146)	C. africana(n: 25)*	C. dubliniensis(n: 25)
Brugmansia arborea (borrachero)	Colony color	White to cream	White to cream	White to cream
	Colony margin	Entire	Entire	Scalloped
	Chlamydoconidia	Absent	Absent	Present
Nicotiana tabacum (tobacco)	Colony color	White to cream	White to cream	Brown
	Colony margin	Entire	Entire	Scalloped
	Chlamydoconidia	Absent	Absent	Present
Rosmarinus officinalis (rosemary)	Colony color	White to cream	White to cream (76 % ) and Brown (24 %)	White to cream
	Colony margin	Entire	Entire	Scalloped
	Chlamydoconidia	Absent	Absent	Absent
Origanum vulgare (oregano)	Colony color	White to cream	White to cream	White to cream
	Colony margin	Entire	Entire	Scalloped
	Chlamydoconidia	Absent	Absent	Absent
Solanum rudepannum. (turkey berry)	Colony color	White to cream	White to cream	White to cream
	Colony margin	Entire	Entire	Scalloped
	Chlamydoconidia	Absent	Absent	Present
Solanum oblongifolium (“cucubo”)	Colony color	White to cream	White to cream	White to cream
	Colony margin	Entire	Entire	Scalloped
	Chlamydoconidia	Absent	Absent	Absent

• The differential characteristics are marked in bold type.
  *C. africana shows poor growth (less than 15 CFU per plate) at 28 °C.

Cryptococcus neoformans, C. glabrata, C. parapsilosis, C. tropicalis, C. auris, C. pseudohaemulonii, C. lusitaniae, C. metapsilosis, C. kefyr, and C. guilliermondii isolates grew poorly at 28 °C. At 37 °C the growth was abundant.

Candida glabrata, C. auris, C. tropicalis, C. lusitaniae, C. parapsilosis and C. metapsilosis produced white-to-cream color colonies in all media. In Tobacco agar, C. neoformans and C. guilliermondii produced brownish colonies, and C. pseudohaemulonii produced khaki color colonies.

Brown color colonies were also produced by C. neoformans in oregano, rosemary (figure 3a), borrachero and cucubo agar, while khaki color colonies were also produced in oregano and cucubo agar by C. guilliermondii and C. kefyr; in turkey berry agar by C. guilliermondii, C. kefyr and C. pseudohaemulonii, and in rosemary agar by C. kefyr (figure 3f), C. guilliermondii and C. pseudohaemulonii (figure 3d). Isolates did not produce chlamydospore on any media. C. parapsilosis (figure 3b), C. africana (figure 3c) and C. glabrata (figure 3e), isolates are showed in Rosemary agar at 37 °C.

Figure 3
Cryptococcus neoformans (a), C. parapsilosis (b), C. africana (c), C. pseudohaemulonii (d), C. glabrata(e), C. kefyr (f), isolates in Rosemary agar at 37 °C.

DISCUSSION

Tobacco agar allowed the differentiation of C. dubliniensis isolates. Similar results were reported by other authors (Bosco-Borgeat et al. 2011, Loreto et al. 2010, Liverio et al. 2017). S. rudepannun, B. arborea agar also allowed the differentiation of this specie. These two plant-extract based agar have not been reported before as culture media. On the other hand, although the taxonomic status of C. africana has been controversial, this yeast is recognized as a varietal distinction of C. albicans (Hu et al. 2015).

Candida africana has been linked to various human pathologies and has been isolated mainly from vaginal samples (Borman et al. 2013). Clinical isolates were reported in China (Shan et al. 2014), Argentina (Theill et al. 2016), India (Sharma et al. 2014), Iran (Khedri et al. 2018, Nikmanesh et al. 2020, Majdabadi et al. 2018, Naeimi et al. 2018, Yazdanparast et al. 2015), Africa (Nnadi et al. 2012, Tietz et al. 2001, Ngouana et al. 2015), USA (Romeo et al. 2013), Chile (Odds et al. 2007, Odds & Jacobsen 2008)and Europe (Borman et al. 2013, Romeo & Criseo 2009, Alonso-Vargas et al. 2008, Mendling et al. 2004).

At present, it is difficult to distinguish C. africana from C. albicans using routine laboratory methods and this circumstance has prevented the epidemiology to be well defined. Furthermore, recent genomic analyses have suggested that C. stellatoidea like C. africana, descended from the same hybrid ancestor as other C. albicans strains and has undergone a parallel massive loss of heterozygosity (Mixao et al. 2021). The agar media prepared from rosemary allowed to differenciate some strains of C. africana from C. dubliniensis and C. albicans. C. africana was the only specie that produced brown colonies on this medium, but only six strains of twenty-five did so.

Rosemary extract agar and oregano extract agar had previously been reported by De Loreto for the differentiation of C. dubliniensis and C. albicans. These authors described that in both media, C. dubliniensis isolates formed rough colonies with hyphal fringes and abundant chlamydospores after 24 to 48 h of incubation at 25 °C. In our case, C. dubliniensis isolates formed smooth colonies with no hyphal fringes or chlamydospores.

Considering that the chemical composition of plant extracts is influenced by several factors, information about the chemical composition of the extracts would provide valuable information to understand its influence on the culture media; however, this information is not available and could be a limitation in this study like the small sample size of C. africana.

De Loreto provide no information regarding the differentiation of C. africana of C. albicans. Nevertheless, in our study, 24 % C. africana isolates produced brown colonies on rosemary agar. In this work, incubation lasted for up to a week, which could explain the differences observed in the color of C. africana colonies compared to that report.

Media containing plant or seed extracts such as niger (Guizottia abyssinica), sunflower, sesame seed and tomato juice, have also been used as differential culture media for the Candida albicans complex; however, all of them have been used for the discrimination of C. dubliniensis and C. albicans (Alves et al. 2006, Al Mosaid et al. 2003, Staib & Morschhauser 1999). Another media as DRBC (Dichloran-rose bengal-chloramphenicol) agar and Sabouraud-triphenyltetrazolium agar also allow the differentiation between C. dubliniensis and C. albicans (Theill et al. 2016).

Although Tobacco agar is widely known as a medium for the differentiation of C. albicans and C. dubliniensis (Bosco-Borgeat et al. 2011, Liverio et al. 2017, Monteiro et al. 2011) there are no reports on the growth description of C. africana in this medium. Furthermore, to our knowledge, the solanaceous plants turkey berry, cucubo and white angel’s trumpet had not been previously described for the production of agar media.

None of the isolates of C. africana grew abundantly on the media at 28 °C. In addition, at 37 °C, chlamydospores formation by C. dubliniensis was reduced. De Loreto et al. 2008, found that the temperature was a determinant factor to produce chlamydospores in C. dubliniensis isolates on rosemary and oregano extract agar.

CONCLUSIONS

In conclusion, appropriate differentiation of cryptic species is clinically relevant. C. dubliniensis and C. albicans var. africana may be underreported in clinical samples because most identification methods fail in their recognition. The antifungal susceptibility profiles, phenotypic, clinical, and ecologic similarity data for non-Candida albicans to help better understand the pathogenic mechanisms and best treatment options. Here, we showed that previously reported rosemary extract agar is an easy, simple to prepare and inexpensive method that could also be used as a further test in the presumptive differentiation between C. africana and C. albicans. However, further studies, including a larger number of C. africana isolates, are crucial to confirm the present results.

ACKNOWLEDGMENTS

Authors acknowledge Claudia Parra and Andrés Ceballos (Laboratory of proteomics from Universidad Javeriana, Colombia), Guillermo García Effron (Laboratory for Mycology and Molecular Diagnosis from Universidad Nacional del Litoral, Argentina), Patrice Le Pape (Laboratoire de Parasitologie-Mycologie et immunologie parasitaire Institut de Biologie- CHU de Nantes, France), Shangrong Fan (Dept of Obstetrics and Gynecology, Peking University Shenzhen Hospital, China), Sebastien Bertout (Laboratoire de Parasitologie et Mycologie Medicale UFR Pharmacie, Universite de Montpellier), Andrew Borman (UK National Mycology Reference Laboratory from Public Health England South West Laboratory, England) and Jacques Meis (Centre of Expertise in Mycology Radboudumc/CWZ, The Netherlands) for providing Candida africana isolates. This study was financed by the call 891 de MINCIENCIAS (Colombia) and the financing agreement of research projects number 169 of the Universidad Popular del Cesar (Valledupar, Colombia).

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Publication Dates

• Publication in this collection
  09 Aug 2024
• Date of issue
  2024

History

• Received
  27 May 2023
• Accepted
  14 Mar 2024