Invasive fungal infections (IFIs) due to Candida species are a frequent and life-threatening condition in hospital settings worldwide, and are often associated with high morbidity and mortality (Koehler et al., 2019). Candida albicans is the most isolated species, with close to a 30% mortality rate in candidemia (Pappas et al., 2018). However, non-albicans Candida species (NACS) such as C. glabrata and C. parapsilosis have emerged as a common cause, becoming the second or third most frequent species depending on geography, patient underlying condition, and age. For these species, the associated mortality rate is about 50% and 28%, respectively (Pemán et al., 2005; Lamoth et al., 2018). Another important NACS causing candidemia is C. auris, whose mortality rate ranges from 40% to 60% according to some studies, despite the fact its prevalence is unclear (Chen et al., 2020).

While current therapeutic options for IFIs are limited to only three classes of drugs (i.e., polyenes, azoles and echinocandins), the emergence of resistant strains to some of these molecules is even more concerning. For decades, azoles have been the most frequently used antifungal for treating Candida infections (Sheehan et al., 1999; Carradori et al., 2022). Although treatment with azoles can be effective, long-term use of fluconazole (FLC) has led to the emergence of Candida strains with decreased susceptibility. In the case of Candida spp, the molecular mechanisms behind FLC-resistance have been relatively well characterized (Morio et al., 2017; Carolus et al., 2021).

Unlike C. auris which is often resistant to FLC, C. albicans and C. parapsilosis isolates were thought to be universally susceptible to FLC, but recent studies show an increased resistance. For example, a multicenter laboratory-based survey of candidemia conducted in South Africa indicates that more than half of C. parapsilosis isolates 62% (332/531) are resistant to FLC (Govender et al., 2016). In addition, studies in Brazil, India, Kuwait, South Korea, Spain, Turkey, and the United States, as well as a recent global study, confirmed the emergence of FLC-resistance in C. parapsilosis (Berkow et al., 2015; Asadzadeh et al., 2017; Arastehfar et al., 2020; Castanheira et al., 2020; Díaz-García et al., 2022). Regarding C. albicans, this species exhibits lower levels of azole resistance. However, resistant isolates have been reported from many countries around the world, including Colombia (Costa-de-Oliveira and Rodrigues, 2020; Rojas et al., 2020; Ceballos-Garzon et al., 2020b).

The main FLC-resistance mechanisms are associated with i) up-regulation of drug transporters, ii) alteration or up-regulation of the gene encoding the enzyme being targeted, which decreases binding affinity for the drug and increases concentration of the enzyme target, iii) alterations in the ergosterol synthetic pathway and iv) activation of pathways involved in the stress response, such as the Ras/cAMP/PKA pathway, calmodulin/calcineurin pathway (CaM/CaL), and mitogen-activated protein kinase (MAPK) signaling pathways (Shapiro et al., 2011; Pristov and Ghannoum, 2019). However, the most studied of them are described below.

The ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) transporters are responsible for lowering the accumulation of azoles inside the yeast cell by translocating compounds actively across the cell membrane (Costa-de-Oliveira and Rodrigues, 2020). Overexpression of genes encoding drug transporters, e.g., Cdr1/2-ABC and Mdr1/Flu1-MFS, among resistant isolates of Candida species is predominantly due to gain-of-function (GOF) mutations in genes encoding zinc cluster transcription factors, such as TAC1 (transcriptional activator of CDR genes) and MRR1 (multidrug resistance regulator). For instance, GOFs in TAC1 (T225A, R693K, A736V, H741, N972D, G980E, N997D) and MRR1 (I283R, R479K, G583R, V854A, K873N), lead to overexpression of CDR1 and MDR1 in C. albicans and C. parapsilosis, respectively (Coste et al., 2006; Liu et al., 2020).

Overexpression of ERG11, the gene encoding lanosterol 14α-demethylase, the azole target, contributes directly to resistance as the increased abundance of the target requires higher drug doses for inhibition. Activating mutations in the gene encoding the transcription factor Upc2, which up-regulates most ergosterol biosynthesis genes, and the formation of an isochromosome with two copies of the left arm of chromosome 5 [i(5L)], or by duplication of the whole chromosome, on which ERG11 resides, are responsible for ERG11 overexpression (Cowen et al., 2014). Furthermore, point mutations in the ERG11 alter the 3D conformation of Erg11 and reduce its affinity for FLC. Some of the most frequent amino acid substitutions reported are Y132F and K143R substitutions, described in C. albicans, C. parapsilosis and C. auris (Berkow et al., 2015; Flowers et al., 2015; Chow et al., 2020).

Although Candida isolates from individual institutions may not be representative of the data of a country, such studies can provide a useful baseline snapshot of species distribution and antifungal susceptibility for candidemia in resource-limited settings (Govender et al., 2016). In Colombia, there is a lack of data about antifungal resistance and its molecular mechanisms in Candida spp. Therefore, this study aimed to investigate the prevalence of resistance and to describe the mechanisms behind FLC-resistance in a collection of bloodstream isolates.

In the region, a study that evaluated susceptibility of Candida species identified in Colombia, Ecuador, and Venezuela, found a percentage of resistance to FLC of 6.8% (De bedout et al., 2003). This shows that there is an important change in the rate of resistance, which in our study reached 18%. Nevertheless, the majority of C. albicans isolates were azole susceptible, thus the observed resistance percentage is mainly attributed to the presence of C. auris, which agrees with previous studies (Pfaller et al., 2019; Sathi et al., 2022). Furthermore, it is noteworthy that 25% of the C. parapsilosis isolates were resistant to FLC, which confirms the increase of resistance in this species (Escribano and Guinea, 2022) (see Figure 5 ). High azole resistance rates have been reported for this species in other single-center studies conducted in Brazil (67.9%), Italy (33%), France (9.2%), Mexico (54%), Saudi Arabia (33%), Spain (13.6%), South Africa (78%) or Turkey (26.4%) (Arastehfar et al., 2020; Magobo et al., 2020; Mesini et al., 2020; Corzo-Leon et al., 2021; Fekkar et al., 2021; Thomaz et al., 2021; Díaz-García et al., 2022).

Overall, the global data indicate that C. albicans remains the predominant species identified in Candida infections. In this study, C. albicans was the most predominant species, nevertheless, NACS accounted for a high proportion (62.6%). Although reported resistance rates vary from study to study, the surveillance data collected suggest that azole resistance rates for C. albicans remain low (Pfaller et al., 2019; Nishimoto et al., 2020). However, C. albicans infections in the bloodstream pose a considerable threat in immunocompromised populations and the associated high mortality remains a major problem in the clinical setting. Therefore, C. albicans should not be overlooked as a serious public health threat. Remarkably, in this study, the percentage of FLC resistance in C. albicans was 13%, which is relatively high.

The acquisition of azole resistance is a serious concern given the limited number of molecules available for the treatment of IFIs. Moreover, this is much more worrying in resource-limited regions where FLC is the only available therapy (Kneale et al., 2016). As previously described, azole resistance is mainly conferred by mutations in the ERG11 gene and by the activity of efflux pumps. The ERG11 gene is highly polymorphic and more than 140 amino acid substitutions have been reported, indicating that this protein is very permissive to conformational changes. Most substitutions occur in three amino acid hotspot regions (105-165, 266-287 and 405-488), although mutations outside these regions can also be found (Debnath and Addya, 2014; Oliveira-Carvalho and Del Negro, 2014).

In the present study, 11 of the 19 FLC-resistant isolates harbored mutations which have been previously described in resistant isolates (T220L, Y132F, K143R) (Healey et al., 2018; Arastehfar et al., 2020). Notably, the T220L substitution was observed in one C. albicans isolate. In C. auris, five isolates carried the Y132F, and in the case of C. parapsilosis four isolates harbored the Y132F and one the K143R substitution.

The substitution of lysine for threonine at position 128 (K128T) was found in CAAL-A, CAAL-B and CAAL-D isolates, which is consistent with the findings of Cernicka et al., and Peron et al., who identified this amino acid substitution in FLC-resistant strains (Cernicka and Subik, 2006; Peron et al., 2016). However, several studies refute this association because K128T substitution has been found in multiple FLC-susceptible isolates, hence, in this study, we do not consider it as a mutation associated with resistance (Morio et al., 2010; Flowers et al., 2015). Nevertheless, it might influence translation efficiency, leading to alterations in protein production as it occurs with nearby mutations such as G129R and Y132F (Kumar et al., 2020).

According to Chow and coworkers, mutations contributing to FLC resistance are clade-specific in C. auris, being Y132F and K143R the most predominant in clade I, F126L in clade III, and Y132F in clade IV. However, Y132F in Erg11 is the most prevalent one (Chow et al., 2020). As described for isolates of clade IV (South America), we found only one mutation (i.e., Y132F). Concerning C. parapsilosis, high prevalence of the Y132F substitution was also noted in previous studies (Grossman et al., 2015; Choi et al., 2018). In addition, the latter suggested that isolates harbouring this mutation may have a higher propensity to cause clonal transmission and to persist in nosocomial settings (Thomaz et al., 2018). Regarding our isolates, the Y132F substitution was detected in four out of six C. parapsilosis isolates. However, considering the dates of collection of the isolates, It does not seem to be a clonal spread ( Table S3 ). Although this report is due in 2023, some resistant isolates were obtained in 2015. Therefore, prior to the reports made in some parts of the globe ( Figure 5 ).

Although previous reports indicate that the Mrr1 substitutions I283R, R479K, G583R, A854V, K873N and L986P are associated with FLC and/or VRC resistance, none of our isolates harbored them (Branco et al., 2015). Similar to Tac1, residues located near the C terminus (760, 761, 803, 956, and 966) might contribute to azole resistance (Nishimoto et al., 2020).

This study also illustrates that acquired azole resistance commonly relies on combined molecular mechanisms in clinical isolates (Morio et al., 2013). In addition to amino acid substitutions in Erg11, six of the 19 isolates also displayed active efflux activity. Interestingly, in two isolates lacking resistance-related mutations, efflux pumps activity was observed. Considering that efflux pumps play a key role in azole resistance, this might be the explanation for the observed phenotype. However, a further study evaluating the expression of all genes involved in efflux pump activity is required.

Regarding the isolates in which no mechanism for resistance was found, namely CAAL-A,B,D, CAAU-D, CAAU-D,E and CAPA-A, six novel Tac1 or Mrr1 substitutions (G52R, V366del, F682T, F683L, T695S and P229A) were identified in three of the six isolates. Moreover, four novel Erg11 substitutions (K22E, Q38T, F72V, Q77S) in the FLC resistant C. albicans (CAAL-B, CAAL-C) and C. auris (CAAU-B) were found. The putative role of these substitutions remains to be investigated. In isolates CAAL-A-D and CAAU-E, a different mechanism to those evaluated here should confer resistance; further analysis is ongoing.

Our study has some limitations. For instance, the antifungal susceptibility data obtained for antifungals other than FLC are not complete and were not performed by broth microdilution. The molecular basis of FLC resistance was investigated only by analysis of the ERG11, TAC1, and MRR1 genes, while other mechanisms conferring resistance, such as sterol composition and gene expression, were not investigated due to lack of funds. Finally, the clinical description of the patients was missing and will be reported alongside other patients infected by Candida antifungal resistant strains elsewhere.

Recently, the WHO released the fungal priority pathogens list (WHO FPPL) which includes C. albicans, C auris (Critical group), and C. parapsilosis (high group). The WHO describes that to overcome the lack of knowledge on infections caused by these fungi, more data and evidence on fungal infections and antifungal resistance to inform and improve response to FPP is needed (World Health Organization (WHO), 2022). Although our study does not have a large number of isolates and does not include other healthcare institutions, we provide evidence of Colombian isolates harboring resistance mutations to one of the most used molecules in the hospital setting.

In conclusion, we describe for the first time the presence of Candida spp isolates harboring Erg11 FLC resistant-related substitutions (Y132F, K143R and T220L) in patients admitted to a Colombian hospital. Although FLC-resistance rates differ significantly between countries and individual health facilities, the resistance rate in our study is relatively high, emphasizing the need for active surveillance to prevent further expansion of FLC-resistant Candida spp isolates in the clinical setting.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

AP carried out the experiments. YV-C contributed to the development of efflux pumps protocol. AC-G, and AP analyzed the data. AC-G wrote the main manuscript. AP, SV-B, BA, and CP-G review and editing. and CP-G conceived the experiments and managed the resources. All authors contributed to the article and approved the submitted version.