1. bookTom 76 (2022): Zeszyt 1 (January 2022)
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1732-2693
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The association of air pollutants (CO2, MTBE) on Candida albicans and Candida glabrata drug resistance

Data publikacji: 29 Jun 2022
Tom & Zeszyt: Tom 76 (2022) - Zeszyt 1 (January 2022)
Zakres stron: 243 - 253
Otrzymano: 09 Dec 2020
Przyjęty: 19 Aug 2021
Informacje o czasopiśmie
License
Format
Czasopismo
eISSN
1732-2693
Pierwsze wydanie
20 Dec 2021
Częstotliwość wydawania
1 raz w roku
Języki
Angielski
Abstract Introduction

Therapeutic methods are very important in the prevalence of opportunistic fungal infections, which are an important cause of human diseases. In this study, air pollution agents that are in direct contact with microorganisms, and the effects of carbon sources using CO2 and MTBE on growth of fungi, and particularly the evaluation of changes in the expression of interfering genes in susceptibility and drug resistance in these fungi, were investigated.

Materials and Methods

Collecting samples and isolating Candida glabrata and Candida albicans with phenotypic methods were accomplished. We then evaluated the minimum inhibitory concentration (MIC) with the M27A4 protocol of CLSI. We adjusted 20 strains of C. albicans and 10 strains of C. glabrata whose sensitivity was evaluated in the MIC test with 5% CO2 and 5mg/ml methyl tert-butyl ether (MTBE) considered as air pollutants, and followed by re-evaluating MIC testing to separate azole-resistant strains. Interfering agents were also considered.

Results

Upregulation of some genes on the two mentioned yeasts had led to drug resistance in them; they were previously sensitive to both drugs. Correspondingly, 41% of C. glabrata samples in sputum showed sensitivity to these drugs. Upregulation of ERG11 (71%) and EPA1 (90%) were observed in resistant strains. Upregulation of genes associated with aspartate proteins and downregulation of SAP3 genes were recognized in C. glabrata in sputum and a 15% downregulation of bronchoalveolar lavage (BAL) isolate and 50% upregulation of SAP1 gene in C. albicans sensitive samples were observed and compared to fluconazole and itraconazole with the oral and joint sources. Remarkably, decreased SAP2 expression in oral sources and a 60% increase in resistant strains in C. albicans were observed. The downregulation of SAP3 expression showed in the joint samples. An increase in HWP1 expression (30%) was noted in isolated and drug-sensitive samples at the sputum and BAL source. CDR1 expression was increased in MTBE-affected species; however, it decreased in the vicinity of CT.

Conclusions

Air pollutants such as CO2 and MTBE eventually caused drug resistance in Candida, which can be one of the causes of drug resistance in candidiasis infections.

Keywords

Introduction

Air pollution has become a major environmental challenge facing humanity in this century. Many agents, such as environmental elements, play an important role in human life. Geographical conditions, temperature, humidity, and pollution also have a major effect on the health or illness of humans and are considered to be a global threat [1].

Allergic syndrome, hypersensitivity syndrome, inflammation of paranasal sinusitis, itching, respiratory infection, and many other diseases have resulted from these elements. In this way, pollution has an impact on eukaryotic and prokaryote cells, and on humans [2]. Correspondingly, Candida is a prevalent micro-organism in the reproductive and gastrointestinal mucosa that can be isolated from the oral cavity. For this reason, most of the healthy population are susceptible to the most prevalent fungal infections such as candidiasis. Candida species generate infections ranging from superficial diseases as well as systemic infections, especially in immunocompromised patients [3]. More than 150 species of Candida have been identified, but few were known to cause infections in humans, including C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, C. dubliniensis, C. kefyr, C. guilliermondii, and C. stellatoidea [4]. Among all of these, C. albicans is the most pathogenic; C. glabrata is considered to have the highest frequency of drug resistance among all non-C. albicans species [5]. After C. albicans, C. glabrata is the most prevalent agent of mucosal and disseminated candidiasis in adults [6]. C. albicans generate extremely organized biofilms consisting of various cells, such as round, budding yeast-form cells; oval pseudohyphal-cells; long, cylindrical hyphal-cells enclosed in an extracellular matrix [7]. Presently, biofilm infection happens in more than 50% of these catheters even with newly enhanced clinical procedures, and these infections can cause serious health and financial implications. As fungal biofilms are mainly resistant to currently used antifungal drugs, the treatment of diseases usually takes higher doses of antifungals with removal of colonized medical devices [8]. In recent years, extensive studies have been performed on many virulence factors in C. albicans, including hyphal formation, phenotypic switching, and extracellular hydrolytic enzyme production. Hydrolytic enzyme production, which is recognized as one of the key agents in yeast pathogenicity, is a factor contributing to the process of virulence. Among the different types of hydrolytic enzymes found in microorganisms, the most common ones related to virulence are proteinases [9]. Secreted aspartyl proteinases (SAP), phospholipase B enzyme, and lipases are the most important extracellular hydrolytic enzymes produced by C. albicans [10]. The expression and regulation of 10 SAP genes increases the number of questions regarding the role and impact of these proteinases throughout the infection procedure. Temporary activation of 10 SAP genes throughout the various infection phases provides that parts of this gene family have an important function in C. albicans’ response to the surroundings, especially its host [11]. Stresses and environmental agents, geographical condition, temperature, humidity, and pollution are associated with the presence of CO2; MTBE is present due to unusual use of synthetic agents such as fuel, paint or polish. Many other environmental factors such as CO2 and O2 concentrations in high doses, PH, and other parameters can contribute by changes in normal growth, virulence factors, hydrophobicity, biofilm formation, secretory enzymes, and alteration of fungal drug resistance and other factors that relate to microorganisms [12].

Nowadays air pollution has affected many organisms such as fungi, and has created many changes in their features. One of these pollutants is MTBE (methyl tert-butyl ether), used as a fuel additive to improve and reduce greenhouse gases and other hazardous pollutants, used instead of lead in gasoline all around the world. This substance has a very high solubility that has a high risk for contamination of drinking water [13]. The addition of MTBE improves the physical characteristic of liquid fuels (used at 15% by volume). MTBE has aqueous solubility (>5 g L−1), low sorption to soil and sediments, and volatile. Some microbial genera can use the compound and metabolize MTBE completely to CO2; it is used as a carbon source by some prokaryotic organisms [14]. To find out the relation of CO2 and MTBE exposure and drug resistance identification, the effects of CO2 and methyl tertiary butyl ether (C5H12O-MTBE) concentration on C. albicans and C. glabrata growth and their virulence factors, this project was accomplished.

Materials and Methods
Candida isolation

In this study, clinical isolates obtained from two hospitals (Imam Khomeini and Shariati Hospitals, Tehran, Iran) were used. Previously isolated samples maintained in mycology collection in the school of public health at Tehran University of Medical Sciences, and standard strains of C. albicans (ATCC10231) and C. glabrata (ATCC90030) were involved as well.

About 105 patients with cutaneous, mucous, and deep infections of candidiasis were obtained in 2016–2017 from the mentioned centers. The samples included: nails, sputum, stool, BAL, groin, skin, and mouth. We investigated 33 samples of C. albicans and 17 samples of C. glabrata which were obtained from different locations in the patient’s body. All samples were then cultured on SDA (sabouraud dextrose agar, Sigma) and incubated at 35 °C for 48 hours.

Morphological identification

All obtained yeasts were detected by morphological and molecular trends. For morphological identification, we used CHROMagar Candida medium (Paris, France) to identify C. albicans from multiple other Candida species based on the colors produced after 48 h of incubation. All yeast samples that were identified as non-albicans on the CHROMagar Candida medium have been forwarded for molecular identification by RFLP-PCR.

Besides, RFLP-PCR was utilized for verifying the identification of C. albicans and C. glabrata species used in this study.

Molecular identification
DNA extraction

For DNA extraction, 103 cells/ml of all isolates from fresh colonies were harvested, then the glass bead disruption method was done. Briefly, cultured yeasts were dissolved in 1.5 ml micro-centrifuge tube and 300 mg of 0.5 mm diameter glass beads, 300 μl of lysis buffer (100mM Tris-HCl pH 8, 10 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulphate), and 300 μl of phenol chloroform-isoamyl alcohol (25:24:1) were added. Then all samples were shaken for 5 min, centrifuged for 5 min at 5000 rpm. The supernatant was transferred to a fresh tube and extracted again with chloroform. High molecular weight DNA was precipitated by adding the same volume of isopropanol and 0.1 volume of 3 M sodium acetate (pH 5.2). After that, the solution was completely vortexed and incubated for 10 min at −20° C and centrifuged for 15 min at 12000 rpm. The precipitant was washed with ice-cold 70% ethanol, dried in the air, dissolved in 50 μl of double distilled water, and stored at −20° C until used for complementary identification of isolates.

Then for detection of Candida species, the RFLP method was applied by amplification of ITS1-5.8S-ITS2 of fungal rRNA genes fragment using ITS1, ITS4 universal primers (ITS1: 5′ TCCGTAGGTGAACCTGCGC 3′, and ITS4: 5′ TCCTGGGCTTATTGATATGC 3′) and the Msp1 restriction enzyme (Fermentas, Germany). Briefly, 2.5 μl of 10× PCR buffer with MgCl2, 0.4 mM of dNTP mix, 1 μl of Taq polymerase, 2 μl of DNA template were used for PCR. The PCR amplification was performed in Veriti 96 Thermal Cycler (Applied Biosystems, USA) based on the program: initial denaturation at 94° C for 3 min then 40 cycles at 94° C for 20 s, 55° C for 30 s and 72° C for 45 s, and subsequently final extension at 72° C for 5 min. The PCR amplicons were resolved with DNA markers in 2% agarose with ethidium bromide (0.5 μg/ml) by gel electrophoresis and for definition, Candida isolates. The PCR product was digested by Msp1 enzyme. Obtained C. albicans and C. glabrata species were selected to continue the procedure.

MIC (minimum inhibitory concentration) of two azole drugs

This test was performed based on broth microdilution Clinical and Laboratory Standard Institute (M27A4) method of CLSI with RPMI1640 (Gibco, USA). Briefly, Candida species were harvested from 24–48 hours of SDA culture and diluted with sterile phosphate buffer saline (PBS) to prepare 1- 5 × 103 CFU/ml by spectrophotometer (530 nm). MIC tests were then conducted based on CLSI protocol for fluconazole and itraconazole antifungals (Janssen Research Foundation, Beerse, Belgium). Drugs were used as reagent-grade powders dissolved in dimethyl sulfoxide (DMSO) and diluted in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) buffered to pH 7.0 with 0.165 mol · L−1 morpholine propane sulfonic acid buffer with L-glutamine without bicarbonate (MOPS, Sigma-Aldrich, St. Louis, MO, USA).

Only sensitive isolates were selected for being confronted with interference agents. We have had the same condition of CLSI protocol for all samples before and after they were confronted with the interference agents CO2 and MTBE.

Interference agents
CO2

CO2 atmosphere was provided by using a 20-L cell culture CO2 incubator (SLS, USA). The amount of CO2 injection was monitored by the calibrated automatic control panel in this incubator.

In this study, we cultured all samples (30 samples) on SDA medium and incubated them at 37° C in a 5% CO2 incubator for 1–4 weeks alternatively. Simultaneously, the samples were also cultured in the same condition without CO2 for comparing the obtained morphological results.

MTBE

In this study, all samples were cultured on SDB containing 5 mg/ml MTBE and incubated at 37 degrees for 1–4 weeks alternatively.

Determination of MIC after treatment by CO2 and MTBE

Alternatively, isolates were confronted with CO2 and MTBE for four weeks. MIC tests were repeated to detect possible resistances to fluconazole and itraconazole. The same condition described previously was followed for MIC and all tests were performed in duplicate.

RNA extraction

The RNA molecules were extracted by the RNX-plus kit (Sinaclon Co., Iran) from all Candida species which gained resistance after being confronted with CO2 and MTBE. Briefly, 103 cells/ml from fresh colonies were prepared and treated with the isolation re-agent. The quantity and quality of the extracted RNA were then confirmed by absorption measurement at 260 and 280 nm using the spectrophotometer (Beckman Coulter, CA, USA) and electrophoresis on 1% agarose gel. The extracted RNA was stored at −80° C and used for cDNA synthesis with easy cDNA reverse transcription kit (Sinaclon Co., Iran) according to the instructions.

Real-time PCR after effect of agents

The obtained cDNAs were then utilized in real-time PCR assay with specific primers and by using AMPLICQON (Real Q plus 2x master mixes Green High Rox) in the ABI one step (Biosystems, Rotkreus, Switzerland) instrument. The mixture contained 10 µl of master mix (Green High Rox), 1 µl of each specific primer (ERG11, CDR1, HWP1, EPA1, SAP1-3), and 2 µl of each cDNA sample. The mixture was adjusted to the final volume of 20 µl applying DEPC water. The program of real-time PCR was as follows: initial denaturation at 95° C for 2 minutes and followed by 40 cycles including in 95° C for 20 seconds, 59–60° C for 20 seconds, and 72° C for 30 seconds.

Primers

The specific primers (ERG11, CDR1, HWP1, EPA1, and SAP1-3) were designed by applying all-ID design software (Table 1) [15, 16, 17, 18, 19, 20].

Sequences of primers used in Real-time PCR reaction in C. albicans and C. glabrata

Gene Primer Sequence (5′->3′)
Sap1 Forward TGGGTTCCTGATGCTTCTGTT
Reverse TCGGCAAAGACTTGCTTTGTG
Sap2 Forward GGGGACATATGATCCAAGTGGT
Reverse CCACCGGCTTCATTGGTTTT
Sap3 Forward ATGTTACTGGTCCCCAAGGTG
Reverse CCTTGACCAGCTTGACATGAA
HWP1 Forward AATCATCAGCTCCTGCCACTG
Reverse GTCGTAGAGACGACAGCACTA
CDR1 Forward GGTGCTAATATCCAATGTTGG
Reverse GTAATGGTTCTCTTTCAGCTG
EPA1 Forward GGTCACTTACCCGCAAGCTA
Reverse CCAGATGGCGTAGGCTTGAT
ERG11 Forward GAGATTGCACCACCCATTGC
Reverse TGGAGATAGCACCGAAACCG
β-actin Forward ACGGTATTGTTTCCAACTGGGACG
Reverse TGGAGCTTCGGTCAACAAAACTGG

The β-actin gene was used for the optimization of the real-time PCR as a housekeeping gene. The reactions were performed in duplicate and the analysis was done by REST2009 software.

Results

The effect of MTBE and CO2 on drug sensitivity and some virulence factors in C. albicans and C. glabrata were assessed. C. albicans and C. glabrata isolates were then recognized based on the result of morphological and molecular tests. The CHROM agar candida medium was applied for morphological diagnosis and C. albicans with green color and C. glabrata with pink color were selected. Performed molecular complementary identification based on RFLP-PCR revealed all 105 isolated as C. albicans and C. glabrata. The total of 105 Candida species which were isolated from different sources of candidiasis involved patients in the mentioned hospitals, which are shown in Table 2.

Dispersion the sources of Candida isolates

Nature of specimen C. albicans C. glabrata Other spices Number of Candida spp %
BAL 8 3 8 19 18.02
SPUTUM 13 10 31 54 51.42
NAIL 6 2 4 12 11.42
MOUTH 2 - 2 4 3.8
GROIN 3 - 4 7 6.68
SKIN 1 1 4 6 5.81
STOOL - 1 2 3 2.85
Total 33 17 60 105

The minimum inhibitory concentrations (MICs) against fluconazole and itraconazole were evaluated based on the M27-A4 method. Based on the obtained results, 20 samples of C. albicans and 10 samples of C. glabrata which were completely sensitive to both mentioned antifungals, were selected for continuing our research (Table 3, Figure 1). C. albicans selected sensitive species were isolated from BAL, sputum, mouth, skin, joint, and nail samples. C. glabrata selected sensitive species were isolated from BAL, stool, skin, and sputum samples. All these isolates were sensitive to both itraconazole and fluconazole. All 30 samples were cultured on SDA and SDB mediums simultaneously and were confronted with 5% CO2 and 5mg/ml MTBE respectively. The cultured media were then incubated at 37° C for up to 4 weeks and were tested for MIC after two and four weeks of incubation. The initial changes were observed after 2–4 weeks of confronting with mentioned agents, and the resistant isolates against fluconazole and itraconazole were then recognized. Regarding C. albicans, the species that grew in ≥8 µg/ml of fluconazole was mentioned as resistant. However, this concentration has been mentioned as ≥ 64 µg/ml for C. glabrata. Furthermore, the MIC criteria for being mentioned as resistant against itraconazole was ≥1 µg/ml in both C. albicans and C. glabrata (Table 4).

Fig. 1

Sensitivity pattern of C. albicans and C. glabrata

Isolates of C. albicans and C. glabrata which were sensitive for both drugs with PCR-RFLP

TMML no Source sample MIC (µg/ml) Itr MIC (µg/ml) Flu Candida spp
TMML1 BAL 0.016 0.125 albicans
TMML2 sputum 0.25 2 albicans
TMML3 sputum 0.125 1 albicans
TMML4 sputum 0.5 1 albicans
TMML5 mouth 0.016 1 albicans
TMML6 nail 0.5 1 albicans
TMML7 nail 1 2 albicans
TMML8 BAL 0.016 1 albicans
TMML9 nail 0.062 0.5 albicans
TMML10 groin 0.016 0.5 albicans
TMML11 BAL 0.016 0.5 albicans
TMML12 BAL 0.062 0.5 albicans
TMML13 sputum 0.125 0.25 albicans
TMML14 sputum 0.125 0.25 albicans
TMML15 sputum 0.016 0.5 albicans
TMML16 sputum 0.016 0.25 albicans
TMML17 skin 0.5 2 albicans
TMML18 sputum 0.125 0.5 albicans
TMML19 groin 0.016 1 albicans
TMML20 sputum 0.031 0.5 albicans
TMML21 stool 1 8 glabrata
TMML22 sputum 0.5 8 glabrata
TMML23 sputum 1 16 glabrata
TMML24 sputum 1 8 glabrata
TMML25 sputum 0.25 8 glabrata
TMML26 sputum 1 16 glabrata
TMML27 sputum 0.125 16 glabrata
TMML28 sputum 1 8 glabrata
TMML29 skin 1 8 glabrata
TMML30 BAL 0.062 1 glabrata

MIC result after CO2 exposure and MTBE in C. albicans and C. glabrata

No Spp After 2 weeks with %5 CO2 After 4 weeks with %5 CO2 After 2 weeks with 5mg/dl MTBE After 4 weeks with 5mg/dl MTBE
Flu Itra Flu Itra Flu Itra Flu Itra
TMML1 C. glabrata 4 1 4 1 2 2 32 8
TMML2 C. glabrata 2 0.5 2 0.5 1 0.5 16 2
TMML3 C. glabrata 2 0.5 4 0.5 0.5 1 16 2
TMML4 C. glabrata 2 0.5 4 1 0.5 0.25 16 4
TMML5 C. glabrata 2 0.5 2 0.5 2 0.5 16 4
TMML6 C. glabrata 4 0.5 64 16 0.5 1 32 2
TMML7 C. glabrata 2 0.5 2 0.5 0.5 0.062 0.5 0.125
TMML8 C. glabrata 2 0.5 4 0.5 1 0.5 32 4
TMML9 C. glabrata 4 1 64 16 1 1 16 8
TMML10 C. glabrata 2 0.125 64 16 0.5 0.25 32 2
TMML11 C. albicans 0.125 0.062 0.5 0.125 1 0.4 16 4
TMML12 C. albicans 64 16 64 16 2 2 64 16
TMML13 C. albicans 64 16 64 16 0.125 0.125 0.125 0.25
TMML14 C. albicans 64 16 64 16 0.5 0.25 1 0.25
TMML15 C. albicans 64 16 64 16 1 0.25 16 8
TMML16 C. albicans 64 16 64 16 4 2 64 16
TMML17 C. albicans 64 16 64 16 2 2 64 16
TMML18 C. albicans 0.025 0.025 64 16 1 0.25 16 16
TMML19 C. albicans 64 16 64 16 1 0.25 2 0.5
TMML20 C. albicans 64 16 64 16 0.5 0.5 1 0.5
TMML21 C. albicans 64 16 64 16 0.5 0.5 1 0.5
TMML22 C. albicans 64 16 64 16 0.25 0.25 1 0.5
TMML23 C. albicans 64 16 64 16 2 0.125 64 16
TMML24 C. albicans 64 16 64 16 0.5 1 0.5 0.5
TMML25 C. albicans 1 0.5 2 0.5 0.25 0.125 2 0.5
TMML26 C. albicans 0.062 0.031 64 16 0.25 0.125 0.25 0.125
TMML27 C. albicans 1 0.25 2 1 0.25 0.5 1 0.5
TMML28 C. albicans 64 16 64 16 0.25 0.062 0.25 0.25
TMML29 C. albicans 64 16 64 16 0.5 1 16 4
TMML30 C. albicans 64 16 64 16 0.25 0.062 0.25 0.5

The resistant species of both C. albicans and C. glabrata were then tested for possible changes in gene regulation by RT-PCR. The results indicated the upregulation of biofilm-associated genes in 57.1% of the sensitive C. glabrata species which isolated from sputum and became resistant against both antifungals.

In this study, most species increased the expression of genes associated with biofilm formation. 58.8% of all C. glabrata samples were sputum samples. Among the sputum samples, 42.8% were sensitive to both drugs, while 57.1% of the sensitive sputum samples became resistant to itraconazole after exposure to only interfering agents. Furthermore, an increase in expression of the EPA1 gene has been observed in about 70% of such sensitive samples. Regarding ERG11 gene, upregulation and downregulation were observed respectively in 71% and 20% of resistant C. glabrata isolates. Increasing the gene expression in aspartyl pro-tease gene and decrease of gene expression in the SAP3 gene were observed in two groups, each consisting of 30% of C. glabrata isolates (Table 5, Figure 2).

Expression of ERG11, EPA1, SAP3 genes in comparison with β-actin in C. glabrata

Gene Type Reaction Efficiency Expression Std. Error 95% C.I. P(H1) Result
ERG11 TRG 1.0 0.876 0.045 - 29.445 0.000 - 123.640 0.894
EPA1 TRG 1.0 64.669 1.893 - 866.949 0.438 - 8,060.454 0.000 UP
SAP3 TRG 1.0 0.745 0.009 - 17.387 0.000 - 434.218 0.784
B-act REF 1.0 1.000

Fig. 2

Total result of C. glabrata. genes expression (ERG11, EPA1, SAP3)

In C. albicans, in 30% of the resistant species against both drugs, an upregulation in the HWP1 gene was observed. Different changes in the regulation of other genes, which may be due to exposure to different interfering factors, were observed and revealed (Table 6, Figure 3).

Expression of CDR1, HWP1, SAP1-3 genes in comparison with β-actin in C. albicans

Gene Type Reaction Efficiency Expression Std. Error 95% C.I. P(H1) Result
CDR1 TRG 1.0 1.623 0.160 - 20.966 0.035 - 68.781 0.342
HWP1 TRG 1.0 1.300 0.069 - 21.856 0.009 - 533.742 0.702
SAp3 TRG 1.0 2.298 0.471 - 20.190 0.077 - 48.176 0.042 UP
Ssp2 TRG 1.0 4.547 0.388 - 86.223 0.007 - 849.223 0.024 UP
Sap1 TRG 1.0 2.243 0.025 - 763.031 0.000 - 12,429.932 0.491
B-act REF 1.0 1.000

Fig. 3

Total result of C. albicans genes expression (CDR1, HWP1, SAP1-3)

The result of biofilm formation for C. albicans and C. glabrata according to the effects of CO2 and MTBE is shown in Figures 4 and 5.

Fig. 4

Result of biofilm formation for C. albicans according to the effects of CO2 and MTBE

Fig. 5

Result of biofilm formation for C. glabrata according to the effects of CO2 and MTBE

Discussion

Candida species are considered the most important fungal yeast pathogen in immunosuppressed patients and other patients who use broad-spectrum antibiotics more widely. Candidiasis infections have increased in the recent decade and the causative agents may be affected by environmental changes such as CO2 concentration, SN, MTBE, and other elements of air pollution [21]. Environmental changes including osmotic shock, carbon source, and oxygen concentration can affect the fungal cell wall. The cell walls in C. albicans consist of two layers: inner and outer. The outer layer is composed of the mannan fibrillary layer, and the inner layer contains β-glucan and chitin [22]. The vulnerability of the mannan layer emerged in response to increasing the temperature from 37° C in the early stage of biofilm formation. Mild heat stress can cause an increase in the thickness of the mycelial inner cell wall at 39° C [23]. Candida lysin secreted by C. albicans hyphae has an essential role in supporting the complicated structures of mature biofilms [24].

Air pollution such as MTBE and high concentrations of CO2 (as an ubiquitous molecule) are among important environmental agents that threaten all living organisms, including yeasts. MTBE has been established as a human carcinogen by affecting oxidative stress and mitochondrial membrane and lysosomal membrane damages [13]. It has a negative effect on humans, such as a toxicity effect on human blood lymphocytes. This substance was used in liquid form with a purity of 99.9%. Evidently, the CO2 concentration in humans is higher than air. The amount of CO2 in the air is 0.03% and CO2 concentration in human bodies is 4.5–30%. Therefore, the CO2 content in humans is hundreds of times higher than in air. Recent research has reported that a low level of CO2 can cause changes in some virulence factors in the gene level [25]. CO2 is used for antimicrobial activity, especially the maintenance of foodstuffs, as it can impact microorganisms by inactivation of the cells.

However, in some countries, using MTBE is banned due to frequent soil and groundwater pollution by accidental spills from distribution systems and storage facilities. Butyl ether methyl is a gas additive that is added to increase the octane number and produced from methanol and iso-butylene. It is mainly used as a fuel oxygenating agent. The negative advantage of using this material is leak to surface and underwater minerals, but it is one of the major sources of contamination.

CO2 could affect fungal physiology, morphology, and pathogenesis, respectively; it could also affect allergenic properties in pathogenic fungi. CO2 concentration may also be considered an important pollution agent, affecting many features of yeast, which causes changes in its physiological and morphological characteristics. CO2 is stimulus-inducing for filamentation in C. albicans and formation of pseudohyphae in C. glabrata yeasts is stimulated by CO2 [26].

Kim and colleagues have indicated some changes occurred in Cryptococcus neoformans (synthase of polysaccharide capsule was increased) after exposure to 5% CO2 atmosphere. Based on previous studies, 5% CO2 atmosphere affected metabolism and pathogenesis of C. neoformans and C. albicans. In addition, some reports have shown that filamentation and pseudohyphal formation in C. glabrata is influenced under the effect of 5% CO2 atmosphere. Synthase of C. neoformans polysaccharide capsule was increased after exposure to 5% CO2 atmosphere. Also, changes were reported in C. albicans and C. glabrata which were drug-resistances due to exposure to 5% CO; expression of some virulence factors was also increased. Another study conducted by Yazdanparast and coworkers showed that certain conditions such as 10% CO2 in Trichophyton rubrum can cause the formation of arthroconidia from hyphal cells, so in some, dermatophytosis with this agent may trigger antifungal resistance [27]. Some studies have considered the general inhibitory effect of CO2 on fungal growth [28]. Some researches in 2004 announced that CO2 pressure could have detrimental effects on growth and metabolism of yeasts, such as inactivation of Saccharomyces cerevisiae cells. CO2 could change the C:N ratio and carbonic anhydrases (CAs) to affect the growth of fungal spores by protein production. Some reports indicated that some species of C. glabrata could produce pseudohyphal cells during nitrogen starvation. On other side, changes in CO2 pressure and oxygen can affect cell activity of S. cerevisiae which leads to cell inactivation [29].

Recent investigation demonstrates that C. glabrata had an ability to respond to CO2 pressure as a stimulus, and filamentation and pseudohyphal formation in C. glabrata under the effect of 5% CO2 atmosphere was seen [26]. Shimoda et al. showed CO2 pressure and temperature are applied for antimicrobial activity, also used for the preservation of foodstuffs by inactivation of cells such as Saccharomyces cerevisiae cells [30].

Another study has shown that MTBE and other ethers were used as a carbon source during the growth of propane by some microorganisms such as Mycobacterium spp. [31]. Peter Roslev and coworkers demonstrated the lack of androgenic response and weak estrogenic response in S. cerevisiae by exposing to MTBE [2].

Several species of Candida have infectious symptoms caused by their virulence factors, such as cell adhesion, biofilm formation, white-opaque (w-o), switching, and morphological transition that cause true and pseudohyphal forms in C. albicans. The importance of this commensal fungal reaction is due to resistance against antifungal drugs. Also, some main reasons for drug resistance are morphological transition and biofilm formation [32]. Treatments are often not possible, and many patients are unable to tolerate taking higher doses of antifungal drugs due to possible damages to some organs, including the liver and kidney. The Candida biofilm resistance phenomenon was for the first time established and demonstrated in 1995 for C. albicans by Hawser and Douglas. In Candida species knowledge of contribution of extracellular DNA to biofilm matrix and overall structure is scarce [33]. C. albicans biofilm matrix is composed of carbohydrate, protein, phosphorus, and hexosamine. Biofilm of C. glabrata consists of multilayers of blastospores with high connections among them and a high level of carbohydrate and protein [34]. The higher density of cells may be related to the high resistance of C. glabrata biofilm to antifungal azoles and amphotericin B [35], although some parameters such as PH, temperature, and oxygen availability are considered as inductors of biofilm architecture alternation and antifungal sensibility [36]. In 2014, Fonseca demonstrated the high level of proteins and carbohydrates in the matrix extract from biofilms in C. glabrata treated with fluconazole [35]. ERG11 is the most central point which increases the ergosterol production in C. glabrata cell membranes in response to azole. Probably during the early phase of biofilm growth efflux pumps in C. albicans and C. glabrata contribute to drug resistance [37].

Cell walls in C. glabrata have more mannoprotein that is linked to 1, 3β-glucan via 1,6-β glucan, and the largest group of mannoprotein is glycosylphosphatidylinositol (GPI). GPI-modified proteins are covalently bound to the wall by 1, 6-β glucan, which plays a key role in adhesion and biofilm formation. Cell wall protein and mannose/glucose ratio is higher in C. glabrata compared to C. albicans [6, 38]. Reducing drug resistance is considered the main goal of treatment for deep infection of candidiasis. In this study, we investigate the effect of a high concentration of CO2 (5%) and gasoline exhaust gases such as MTBE on C. albicans and C. glabrata isolates recovered from the patients.

Molecular proteins are produced by the aspartyl proteinase gene which has an important role in the virulence of Candida species. SAP expression is strain- and source-dependent and plays a significant and dramatic role in infections caused by Candida spp. [39]. SAPs are encoded by at least ten distinct highly regulated genes in a multigene family (SAP1 – SAP10). According to previous studies some members of this family are considered specific for some species of Candida, for instance, SAP 5-6 are exclusive to C. albicans but the lowest rate of proteinase production was found in C. glabrata and no specific SAP genes were detected in C. glabrata according to previous studies [40].

Recent studies indicated some pathogenic changes in C. glabrata and C. albicans in growth under CO2 atmosphere; they also reported molecular changes by adenylyl cyclase Cyr1 that promote white-to-opaque switching and then affect mating changes in C. albicans [25, 26]. Our results indicate that MICs of itraconazole and fluconazole against C. albicans and C. glabrata increased after confronting with some 5% CO2 pressure and 5mg/ml MTBE. However, the mechanism of resistance remained inconspicuous. We used these environmental factors to induce drug resistance in two species of Candida. The final MICs were relatively higher than the initial ones.

Shabir Ahmad Lone and coworkers in 2019 had claimed that the new antifungal eugenol tosylate congeners (ETC-5, ETC-6, and ETC-7) have a fungicidal effect on Candida spp. They have also shown the downregulation of the ERG11 gene which is related to ergosterol synthesis [41].

In the present study, expression of secretory enzymes including aspartyl protease (SAP) and biofilm formation and study of drug sensitivity modification (MIC) are evaluated by real-time PCR. We evaluated the effect of CO2 and MTBE on drug susceptibility and some virulence factors of C. glabrata and C. albicans isolates that were gained from clinical samples. The obtained results indicated that CO2 atmosphere and MTBE as two air pollution elements could enhance some virulence factors such as SAP1-3 in C. glabrata and C. albicans. These interferences could cause drug resistance in some species which were susceptible before confronted with CO2 or MTBE. Most of the mentioned changes were observed after 2–4 weeks of incubation under a 5% CO2 atmosphere or 5mg/ml MTBE.

In addition to molecular studies of the aforementioned genes, the function of other genes along with the epigenetic and genetic investigation of these genes in molecular pathways is important.

Conclusions

Some genes associated with C. albicans and C. glabrata virulence factors were increased by gaining resistance against antifungal drugs and due to confronting with air pollutants such as CO2 and MTBE.

Fig. 1

Sensitivity pattern of C. albicans and C. glabrata
Sensitivity pattern of C. albicans and C. glabrata

Fig. 2

Total result of C. glabrata. genes expression (ERG11, EPA1, SAP3)
Total result of C. glabrata. genes expression (ERG11, EPA1, SAP3)

Fig. 3

Total result of C. albicans genes expression (CDR1, HWP1, SAP1-3)
Total result of C. albicans genes expression (CDR1, HWP1, SAP1-3)

Fig. 4

Result of biofilm formation for C. albicans according to the effects of CO2 and MTBE
Result of biofilm formation for C. albicans according to the effects of CO2 and MTBE

Fig. 5

Result of biofilm formation for C. glabrata according to the effects of CO2 and MTBE
Result of biofilm formation for C. glabrata according to the effects of CO2 and MTBE

Sequences of primers used in Real-time PCR reaction in C. albicans and C. glabrata

Gene Primer Sequence (5′->3′)
Sap1 Forward TGGGTTCCTGATGCTTCTGTT
Reverse TCGGCAAAGACTTGCTTTGTG
Sap2 Forward GGGGACATATGATCCAAGTGGT
Reverse CCACCGGCTTCATTGGTTTT
Sap3 Forward ATGTTACTGGTCCCCAAGGTG
Reverse CCTTGACCAGCTTGACATGAA
HWP1 Forward AATCATCAGCTCCTGCCACTG
Reverse GTCGTAGAGACGACAGCACTA
CDR1 Forward GGTGCTAATATCCAATGTTGG
Reverse GTAATGGTTCTCTTTCAGCTG
EPA1 Forward GGTCACTTACCCGCAAGCTA
Reverse CCAGATGGCGTAGGCTTGAT
ERG11 Forward GAGATTGCACCACCCATTGC
Reverse TGGAGATAGCACCGAAACCG
β-actin Forward ACGGTATTGTTTCCAACTGGGACG
Reverse TGGAGCTTCGGTCAACAAAACTGG

MIC result after CO2 exposure and MTBE in C. albicans and C. glabrata

No Spp After 2 weeks with %5 CO2 After 4 weeks with %5 CO2 After 2 weeks with 5mg/dl MTBE After 4 weeks with 5mg/dl MTBE
Flu Itra Flu Itra Flu Itra Flu Itra
TMML1 C. glabrata 4 1 4 1 2 2 32 8
TMML2 C. glabrata 2 0.5 2 0.5 1 0.5 16 2
TMML3 C. glabrata 2 0.5 4 0.5 0.5 1 16 2
TMML4 C. glabrata 2 0.5 4 1 0.5 0.25 16 4
TMML5 C. glabrata 2 0.5 2 0.5 2 0.5 16 4
TMML6 C. glabrata 4 0.5 64 16 0.5 1 32 2
TMML7 C. glabrata 2 0.5 2 0.5 0.5 0.062 0.5 0.125
TMML8 C. glabrata 2 0.5 4 0.5 1 0.5 32 4
TMML9 C. glabrata 4 1 64 16 1 1 16 8
TMML10 C. glabrata 2 0.125 64 16 0.5 0.25 32 2
TMML11 C. albicans 0.125 0.062 0.5 0.125 1 0.4 16 4
TMML12 C. albicans 64 16 64 16 2 2 64 16
TMML13 C. albicans 64 16 64 16 0.125 0.125 0.125 0.25
TMML14 C. albicans 64 16 64 16 0.5 0.25 1 0.25
TMML15 C. albicans 64 16 64 16 1 0.25 16 8
TMML16 C. albicans 64 16 64 16 4 2 64 16
TMML17 C. albicans 64 16 64 16 2 2 64 16
TMML18 C. albicans 0.025 0.025 64 16 1 0.25 16 16
TMML19 C. albicans 64 16 64 16 1 0.25 2 0.5
TMML20 C. albicans 64 16 64 16 0.5 0.5 1 0.5
TMML21 C. albicans 64 16 64 16 0.5 0.5 1 0.5
TMML22 C. albicans 64 16 64 16 0.25 0.25 1 0.5
TMML23 C. albicans 64 16 64 16 2 0.125 64 16
TMML24 C. albicans 64 16 64 16 0.5 1 0.5 0.5
TMML25 C. albicans 1 0.5 2 0.5 0.25 0.125 2 0.5
TMML26 C. albicans 0.062 0.031 64 16 0.25 0.125 0.25 0.125
TMML27 C. albicans 1 0.25 2 1 0.25 0.5 1 0.5
TMML28 C. albicans 64 16 64 16 0.25 0.062 0.25 0.25
TMML29 C. albicans 64 16 64 16 0.5 1 16 4
TMML30 C. albicans 64 16 64 16 0.25 0.062 0.25 0.5

Isolates of C. albicans and C. glabrata which were sensitive for both drugs with PCR-RFLP

TMML no Source sample MIC (µg/ml) Itr MIC (µg/ml) Flu Candida spp
TMML1 BAL 0.016 0.125 albicans
TMML2 sputum 0.25 2 albicans
TMML3 sputum 0.125 1 albicans
TMML4 sputum 0.5 1 albicans
TMML5 mouth 0.016 1 albicans
TMML6 nail 0.5 1 albicans
TMML7 nail 1 2 albicans
TMML8 BAL 0.016 1 albicans
TMML9 nail 0.062 0.5 albicans
TMML10 groin 0.016 0.5 albicans
TMML11 BAL 0.016 0.5 albicans
TMML12 BAL 0.062 0.5 albicans
TMML13 sputum 0.125 0.25 albicans
TMML14 sputum 0.125 0.25 albicans
TMML15 sputum 0.016 0.5 albicans
TMML16 sputum 0.016 0.25 albicans
TMML17 skin 0.5 2 albicans
TMML18 sputum 0.125 0.5 albicans
TMML19 groin 0.016 1 albicans
TMML20 sputum 0.031 0.5 albicans
TMML21 stool 1 8 glabrata
TMML22 sputum 0.5 8 glabrata
TMML23 sputum 1 16 glabrata
TMML24 sputum 1 8 glabrata
TMML25 sputum 0.25 8 glabrata
TMML26 sputum 1 16 glabrata
TMML27 sputum 0.125 16 glabrata
TMML28 sputum 1 8 glabrata
TMML29 skin 1 8 glabrata
TMML30 BAL 0.062 1 glabrata

Dispersion the sources of Candida isolates

Nature of specimen C. albicans C. glabrata Other spices Number of Candida spp %
BAL 8 3 8 19 18.02
SPUTUM 13 10 31 54 51.42
NAIL 6 2 4 12 11.42
MOUTH 2 - 2 4 3.8
GROIN 3 - 4 7 6.68
SKIN 1 1 4 6 5.81
STOOL - 1 2 3 2.85
Total 33 17 60 105

Expression of CDR1, HWP1, SAP1-3 genes in comparison with β-actin in C. albicans

Gene Type Reaction Efficiency Expression Std. Error 95% C.I. P(H1) Result
CDR1 TRG 1.0 1.623 0.160 - 20.966 0.035 - 68.781 0.342
HWP1 TRG 1.0 1.300 0.069 - 21.856 0.009 - 533.742 0.702
SAp3 TRG 1.0 2.298 0.471 - 20.190 0.077 - 48.176 0.042 UP
Ssp2 TRG 1.0 4.547 0.388 - 86.223 0.007 - 849.223 0.024 UP
Sap1 TRG 1.0 2.243 0.025 - 763.031 0.000 - 12,429.932 0.491
B-act REF 1.0 1.000

Expression of ERG11, EPA1, SAP3 genes in comparison with β-actin in C. glabrata

Gene Type Reaction Efficiency Expression Std. Error 95% C.I. P(H1) Result
ERG11 TRG 1.0 0.876 0.045 - 29.445 0.000 - 123.640 0.894
EPA1 TRG 1.0 64.669 1.893 - 866.949 0.438 - 8,060.454 0.000 UP
SAP3 TRG 1.0 0.745 0.009 - 17.387 0.000 - 434.218 0.784
B-act REF 1.0 1.000

Wei K, Qiu M, Zhang R, Zhou L, Zhang T, Yao M, Luo C. Single Living yEast PM Toxicity Sensor (SLEPTor) system. J Aerosol Sci. 2017; 107: 65–73. WeiK QiuM ZhangR ZhouL ZhangT YaoM LuoC Single Living yEast PM Toxicity Sensor (SLEPTor) system J Aerosol Sci. 2017 107 65 73 10.1016/j.jaerosci.2017.02.006 Search in Google Scholar

Roslev P, Lentz T, Hesselsoe M. Microbial toxicity of methyl tert-butyl ether (MTBE) determined with fluorescent and luminescent bioassays. Chemosphere. 2015; 120: 284–291. RoslevP LentzT HesselsoeM Microbial toxicity of methyl tert-butyl ether (MTBE) determined with fluorescent and luminescent bioassays Chemosphere. 2015 120 284 291 10.1016/j.chemosphere.2014.07.003 Search in Google Scholar

Hani U, Shivakumar HG, Vaghela R, Osmani RA, Shrivastava A. Candidiasis: A fungal infection-current challenges and progress in prevention and treatment. Infect Disord Drug Targets. 2015; 15: 42–52. HaniU ShivakumarHG VaghelaR OsmaniRA ShrivastavaA Candidiasis: A fungal infection-current challenges and progress in prevention and treatment Infect Disord Drug Targets. 2015 15 42 52 10.2174/1871526515666150320162036 Search in Google Scholar

Krcmery V, Barnes AJ. Non-albicans Candida spp. causing fungaemia: Pathogenicity and antifungal resistance. J Hosp Infect. 2002; 50: 243–260. KrcmeryV BarnesAJ Non-albicans Candida spp. causing fungaemia: Pathogenicity and antifungal resistance J Hosp Infect. 2002 50 243 260 10.1053/jhin.2001.1151 Search in Google Scholar

Singh A, Healey KR, Yadav P, Upadhyaya G, Sachdeva N, Sarma S, Kumar A, Tarai B, Perlin DS, Chowdhary A. Absence of azole or echinocandin resistance in Candida glabrata isolates in India despite background prevalence of strains with defects in the DNA mismatch repair pathway. Antimicrob Agents Chemother. 2018; 62: e00195–18. SinghA HealeyKR YadavP UpadhyayaG SachdevaN SarmaS KumarA TaraiB PerlinDS ChowdharyA Absence of azole or echinocandin resistance in Candida glabrata isolates in India despite background prevalence of strains with defects in the DNA mismatch repair pathway Antimicrob Agents Chemother. 2018 62 e00195 18 10.1128/AAC.00195-18 Search in Google Scholar

de Groot PW, Kraneveld EA, Yin QY, Dekker HL, Groß U, Crielaard W, de Koster CG, Bader O, Klis FM, Weig M. The cell wall of the human pathogen Candida glabrata: Differential incorporation of novel adhesin-like wall proteins. Eukaryot Cell. 2008; 7: 1951–1964. de GrootPW KraneveldEA YinQY DekkerHL GroßU CrielaardW de KosterCG BaderO KlisFM WeigM The cell wall of the human pathogen Candida glabrata: Differential incorporation of novel adhesin-like wall proteins Eukaryot Cell. 2008 7 1951 1964 10.1128/EC.00284-08 Search in Google Scholar

Fox EP, Nobile CJ. A sticky situation: Untangling the transcriptional network controlling biofilm development in Candida albicans. Transcription. 2012; 3: 315–322. FoxEP NobileCJ A sticky situation: Untangling the transcriptional network controlling biofilm development in Candida albicans Transcription. 2012 3 315 322 10.4161/trns.22281 Search in Google Scholar

Cornely OA, Bassetti M, Calandra T, Garbino J, Kullberg BJ, Lortholary O, Meersseman W, Akova M, Arendrup MC, Arikan-Akdagli S, et al. ESCMID* guideline for the diagnosis and management of Candida diseases 2012: Non-neutropenic adult patients. Clin Microbiol Infect. 2012; 18: 19–37. CornelyOA BassettiM CalandraT GarbinoJ KullbergBJ LortholaryO MeerssemanW AkovaM ArendrupMC Arikan-AkdagliS ESCMID* guideline for the diagnosis and management of Candida diseases 2012: Non-neutropenic adult patients Clin Microbiol Infect. 2012 18 19 37 10.1111/1469-0691.12039 Search in Google Scholar

Calderone RA, Fonzi WA. Virulence factors of Candida albicans. Trends Microbiol. 2001; 9: 327–335. CalderoneRA FonziWA Virulence factors of Candida albicans Trends Microbiol. 2001 9 327 335 10.1016/S0966-842X(01)02094-7 Search in Google Scholar

Naglik J, Albrecht A, Bader O, Hube B. Candida albicans proteinases and host/pathogen interactions. Cel. Microbiol. 2004; 6: 915–926. NaglikJ AlbrechtA BaderO HubeB Candida albicans proteinases and host/pathogen interactions Cel. Microbiol. 2004 6 915 926 10.1111/j.1462-5822.2004.00439.x15339267 Search in Google Scholar

Kadry AA, El-Ganiny AM, El-Baz AM. Relationship between Sap prevalence and biofilm formation among resistant clinical isolates of Candida albicans. Afr Health Sci. 2018; 18: 1166–1174. KadryAA El-GaninyAM El-BazAM Relationship between Sap prevalence and biofilm formation among resistant clinical isolates of Candida albicans Afr Health Sci. 2018 18 1166 1174 10.4314/ahs.v18i4.37635488830766582 Search in Google Scholar

Alfonso-Gordillo G, Flores-Ortiz CM, Morales-Barrera L, Cristiani-Urbina E. Biodegradation of methyl tertiary butyl ether (MTBE) by a microbial consortium in a continuous up-flow packed-bed biofilm reactor: Kinetic study, metabolite identification and toxicity bioassays. PLoS One. 2016; 11: e0167494. Alfonso-GordilloG Flores-OrtizCM Morales-BarreraL Cristiani-UrbinaE Biodegradation of methyl tertiary butyl ether (MTBE) by a microbial consortium in a continuous up-flow packed-bed biofilm reactor: Kinetic study, metabolite identification and toxicity bioassays PLoS One. 2016 11 e0167494 10.1371/journal.pone.0167494513233227907122 Search in Google Scholar

Salimi A, Vaghar-Moussavi M, Seydi E, Pourahmad J. Toxicity of methyl tertiary-butyl ether on human blood lymphocytes. Environ Sci Pollut Res Int. 2016; 23: 8556–8564. SalimiA Vaghar-MoussaviM SeydiE PourahmadJ Toxicity of methyl tertiary-butyl ether on human blood lymphocytes Environ Sci Pollut Res Int. 2016 23 8556 8564 10.1007/s11356-016-6090-x26797945 Search in Google Scholar

Juwono H, Yamin A, Alfian R, Ni’mah YL, Harmami H. Production of liquid fuel from plastic waste with co-reactan nyamplung oil (callophyllum inophyllum) and its performance in gasoline machine by adding MTBE additive. AIP Conf Proc. 2018; 2049: 020081 JuwonoH YaminA AlfianR Ni’mahYL HarmamiH Production of liquid fuel from plastic waste with co-reactan nyamplung oil (callophyllum inophyllum) and its performance in gasoline machine by adding MTBE additive AIP Conf Proc. 2018 2049 020081 10.1063/1.5082486 Search in Google Scholar

Hube B, Sanglard D, Odds FC, Hess D, Monod M, Schäfer W, Brown AJ, Gow NA. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect Immun. 1997; 65: 3529–3538. HubeB SanglardD OddsFC HessD MonodM SchäferW BrownAJ GowNA Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence Infect Immun. 1997 65 3529 3538 10.1128/iai.65.9.3529-3538.19971755039284116 Search in Google Scholar

Modrzewska B, Kurnatowski P, Khalid K. Comparison of proteolytic activity of Candida sp. strains depending on their origin. J Mycol Med. 2016; 26: 138–147. ModrzewskaB KurnatowskiP KhalidK Comparison of proteolytic activity of Candida sp. strains depending on their origin J Mycol Med. 2016 26 138 147 10.1016/j.mycmed.2016.01.00526922385 Search in Google Scholar

de Barros PP, Freire F, Rossoni RD, Junqueira JC, Jorge AO. Candida krusei and Candida glabrata reduce the filamentation of Candida albicans by downregulating expression of HWP1 gene. Folia Microbiol. 2017; 62: 317–323. de BarrosPP FreireF RossoniRD JunqueiraJC JorgeAO Candida krusei and Candida glabrata reduce the filamentation of Candida albicans by downregulating expression of HWP1 gene Folia Microbiol. 2017 62 317 323 10.1007/s12223-017-0500-428164244 Search in Google Scholar

Feng W, Yang J, Wang Y, Chen J, Xi Z, Qiao Z. ERG11 mutations and up-regulation in clinical itraconazole-resistant isolates of Candida krusei. Can J Microbiol. 2016; 62: 938–943. FengW YangJ WangY ChenJ XiZ QiaoZ ERG11 mutations and up-regulation in clinical itraconazole-resistant isolates of Candida krusei Can J Microbiol. 2016 62 938 943 10.1139/cjm-2016-005527622981 Search in Google Scholar

Gallegos-García V, Pan SJ, Juárez-Cepeda J, Ramírez-Zavaleta CY, Martin-del-Campo MB, Martínez-Jiménez V, Castaño I, Cormack B, De Las Peñas A. A novel downstream regulatory element cooperates with the silencing machinery to repress EPA1 expression in Candida glabrata. Genetics. 2012; 190: 1285–1297. Gallegos-GarcíaV PanSJ Juárez-CepedaJ Ramírez-ZavaletaCY Martin-del-CampoMB Martínez-JiménezV CastañoI CormackB De Las PeñasA A novel downstream regulatory element cooperates with the silencing machinery to repress EPA1 expression in Candida glabrata Genetics. 2012 190 1285 1297 10.1534/genetics.111.138099331664322234857 Search in Google Scholar

Zhu SL, Yan L, Zhang YX, Jiang ZH, Gao PH, Qiu Y, Wang L, Zhao MZ, Ni TJ, Cai Z, et al.: Berberine inhibits fluphenazine-induced up-regulation of CDR1 in Candida albicans. Biol Pharm Bull. 2014; 37: 268–273. ZhuSL YanL ZhangYX JiangZH GaoPH QiuY WangL ZhaoMZ NiTJ CaiZ Berberine inhibits fluphenazine-induced up-regulation of CDR1 in Candida albicans Biol Pharm Bull. 2014 37 268 273 10.1248/bpb.b13-0073424492724 Search in Google Scholar

Tobal JM, da Silva Ferreina Balieiro ME. Role of carbonic anhydrases in pathogenic micro-organisms: A focus on Aspergillus fumigatus. J Med Microbiol. 2014; 63: 15–27. TobalJM da Silva Ferreina BalieiroME Role of carbonic anhydrases in pathogenic micro-organisms: A focus on Aspergillus fumigatus J Med Microbiol. 2014 63 15 27 10.1099/jmm.0.064444-024149624 Search in Google Scholar

Levin DE. Regulation of cell wall biogenesis in Saccharomyces cerevisiae: The cell wall integrity signaling pathway. Genetics. 2011; 189: 1145–1175. LevinDE Regulation of cell wall biogenesis in Saccharomyces cerevisiae: The cell wall integrity signaling pathway Genetics. 2011 189 1145 1175 10.1534/genetics.111.128264324142222174182 Search in Google Scholar

Ikezaki S., Cho T, Nagao JI, Tasaki S, Yamaguchi M, Arita-Morioka KI, Yasumatsu K, Chibana H, Ikebe T, Tanaka Y. Mild heat stress affects on the cell wall structure in Candida albicans biofilm. Med Mycol J. 2019; 60: 29–37. IkezakiS. ChoT NagaoJI TasakiS YamaguchiM Arita-MoriokaKI YasumatsuK ChibanaH IkebeT TanakaY Mild heat stress affects on the cell wall structure in Candida albicans biofilm Med Mycol J. 2019 60 29 37 10.3314/mmj.19-0000131155569 Search in Google Scholar

Abu El-Asrar AM, Missotten L, Geboes K. Expression of hypoxiainducible factor-1α and the protein products of its target genes in diabetic fibrovascular epiretinal membranes. Br J Ophthalmol. 2007; 91: 822–826. Abu El-AsrarAM MissottenL GeboesK Expression of hypoxiainducible factor-1α and the protein products of its target genes in diabetic fibrovascular epiretinal membranes Br J Ophthalmol. 2007 91 822 826 10.1136/bjo.2006.109876195557117229797 Search in Google Scholar

Du H, Guan G, Xie J, Cottier F, Sun Y, Jia W, Mühlschlegel FA, Huang G. The transcription factor Flo8 mediates CO2 sensing in the human fungal pathogen Candida albicans. Mol Biol Cell. 2012; 23: 2692–2701. DuH GuanG XieJ CottierF SunY JiaW MühlschlegelFA HuangG The transcription factor Flo8 mediates CO2 sensing in the human fungal pathogen Candida albicans Mol Biol Cell. 2012 23 2692 2701 10.1091/mbc.e12-02-0094 Search in Google Scholar

Sasani E, Khodavaisy S, Agha Kuchak Afshari S, Darabian S, Aala F, Rezaie S. Pseudohyphae formation in Candida glabrata due to CO2 exposure. Curr Med Mycol. 2016; 2: 49–52. SasaniE KhodavaisyS Agha Kuchak AfshariS DarabianS AalaF RezaieS Pseudohyphae formation in Candida glabrata due to CO2 exposure Curr Med Mycol. 2016 2 49 52 10.18869/acadpub.cmm.2.4.49561169728959796 Search in Google Scholar

Yazdanparast SA, Barton RC. Arthroconidia production in Trichophyton rubrum and a new ex vivo model of onychomycosis. J Med Microbiol. 2006; 55: 1577–1581. YazdanparastSA BartonRC Arthroconidia production in Trichophyton rubrum and a new ex vivo model of onychomycosis J Med Microbiol. 2006 55 1577 1581 10.1099/jmm.0.46474-017030919 Search in Google Scholar

Papagianni M. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv. 2004; 22: 189–259. PapagianniM Fungal morphology and metabolite production in submerged mycelial processes Biotechnol Adv. 2004 22 189 259 10.1016/j.biotechadv.2003.09.00514665401 Search in Google Scholar

Coelho M, Belo I, Pinheiro R, Amaral A, Mota M, Coutinho J, Ferreira E. Effect of hyperbaric stress on yeast morphology: Study by automated image analysis. Appl Microbiol Biotechnol. 2004; 66: 318–324. CoelhoM BeloI PinheiroR AmaralA MotaM CoutinhoJ FerreiraE Effect of hyperbaric stress on yeast morphology: Study by automated image analysis Appl Microbiol Biotechnol. 2004 66 318 324 10.1007/s00253-004-1648-915257421 Search in Google Scholar

Shimoda M, Cocunubo-Castellanos J, Kago H, Miyake M, Osajima Y, Hayakawa I. The influence of dissolved CO2 concentration on the death kinetics of Saccharomyces cerevisiae. J Appl Microbiol. 2001; 91: 306–311. ShimodaM Cocunubo-CastellanosJ KagoH MiyakeM OsajimaY HayakawaI The influence of dissolved CO2 concentration on the death kinetics of Saccharomyces cerevisiae J Appl Microbiol. 2001 91 306 311 10.1046/j.1365-2672.2001.01386.x11473595 Search in Google Scholar

Tupa PR, Masuda H. Genomic analysis of propane metabolism in methyl tert-butyl ether-degrading Mycobacterium sp. strain ENV421. J Genomics. 2018; 6: 24–29. TupaPR MasudaH Genomic analysis of propane metabolism in methyl tert-butyl ether-degrading Mycobacterium sp. strain ENV421 J Genomics. 2018 6 24 29 10.7150/jgen.24929 Search in Google Scholar

Graybill JR. The long and the short of antifungal therapy. Infect Dis Clin North Am. 1988; 2: 805–825. GraybillJR The long and the short of antifungal therapy Infect Dis Clin North Am. 1988 2 805 825 10.1016/S0891-5520(20)30229-4 Search in Google Scholar

Harvey RJ, Lund VJ. Biofilms and chronic rhinosinusitis: Systematic review of evidence, current concepts and directions for research. Rhinology. 2007; 45: 3–13. HarveyRJ LundVJ Biofilms and chronic rhinosinusitis: Systematic review of evidence, current concepts and directions for research Rhinology. 2007 45 3 13 Search in Google Scholar

Silva S, Henriques M, Martins A, Oliveira R, Williams D, Azeredo J. Bio-films of non-Candida albicans Candida species: Quantification, structure and matrix composition. Med Mycol. 2009; 47: 681–689. SilvaS HenriquesM MartinsA OliveiraR WilliamsD AzeredoJ Bio-films of non-Candida albicans Candida species: Quantification, structure and matrix composition Med Mycol. 2009 47 681 689 10.3109/13693780802549594 Search in Google Scholar

Fonseca E, Silva S, Rodrigues CF, Alves CT, Azeredo J, Henriques M. Effects of fluconazole on Candida glabrata biofilms and its relationship with ABC transporter gene expression. Biofouling. 2014; 30: 447–457. FonsecaE SilvaS RodriguesCF AlvesCT AzeredoJ HenriquesM Effects of fluconazole on Candida glabrata biofilms and its relationship with ABC transporter gene expression Biofouling. 2014 30 447 457 10.1080/08927014.2014.886108 Search in Google Scholar

Pettit RK, Repp KK, Hazen KC. Temperature affects the susceptibility of Cryptococcus neoformans biofilms to antifungal agents. Med Mycol J. 2010; 48: 421–426. PettitRK ReppKK HazenKC Temperature affects the susceptibility of Cryptococcus neoformans biofilms to antifungal agents Med Mycol J. 2010 48 421 426 10.1080/13693780903136879 Search in Google Scholar

Akins RA. An update on antifungal targets and mechanisms of resistance in Candida albicans. Med Mycol J. 2005; 43: 285–318. AkinsRA An update on antifungal targets and mechanisms of resistance in Candida albicans Med Mycol J. 2005 43 285 318 10.1080/13693780500138971 Search in Google Scholar

Klis FM, De Groot P, Brul S. 13 identification, characterization, and phenotypic analysis of covalently linked cell wall proteins. Methods Microbiol. 2007; 36: 281–301. KlisFM De GrootP BrulS 13 identification, characterization, and phenotypic analysis of covalently linked cell wall proteins Methods Microbiol. 2007 36 281 301 10.1016/S0580-9517(06)36013-8 Search in Google Scholar

Newport G, Agabian N. KEX2 influences Candida albicans proteinase secretion and hyphal formation. J Biol Chem. 1997; 272: 28954–28961. NewportG AgabianN KEX2 influences Candida albicans proteinase secretion and hyphal formation J Biol Chem. 1997 272 28954 28961 10.1074/jbc.272.46.289549360967 Search in Google Scholar

Dabiri S, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M. SAP (1-3) gene expression in high proteinase producer Candida species strains isolated from Iranian patients with different Candidosis. J Pure Appl Microbiol. 2016; 10: 1891–1896. DabiriS Shams-GhahfarokhiM Razzaghi-AbyanehM SAP (1-3) gene expression in high proteinase producer Candida species strains isolated from Iranian patients with different Candidosis J Pure Appl Microbiol. 2016 10 1891 1896 Search in Google Scholar

Lone SA, Khan S, Ahmad A. Inhibition of ergosterol synthesis in Candida albicans by novel eugenol tosylate congeners targeting sterol 14α-demethylase (CYP51) enzyme. Arch Microbiol. 2020; 202: 711–726. LoneSA KhanS AhmadA Inhibition of ergosterol synthesis in Candida albicans by novel eugenol tosylate congeners targeting sterol 14α-demethylase (CYP51) enzyme Arch Microbiol. 2020 202 711 726 10.1007/s00203-019-01781-231786635 Search in Google Scholar

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