The association of air pollutants (CO₂, MTBE) on Candida albicans and Candida glabrata drug resistance

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.

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.

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

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).

Fig. 2

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).

Fig. 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

Fig. 5

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.

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.