Epidemiological trends, antifungal drug susceptibility and SQLE point mutations in etiologic species of human dermatophytosis in Al-Diwaneyah, Iraq

Abstract

Dermatophytes show a wide geographic distribution and are the main causative agents of skin fungal infections in many regions of the world. Recently, their resistance to antifungal drugs has led to an obstacle to effective treatment. To address the lack of dermatophytosis data in Iraq, this study was designed to investigate the distribution and prevalence of dermatophytes in the human population and single point mutations in squalene epoxidase gene (SQLE) of terbinafine resistant isolates. The identification of 102 dermatophytes isolated from clinical human dermatophytosis was performed through morphological and microscopic characteristics followed by molecular analysis based on ITS and TEF-1α sequencing. Phylogeny was achieved through RAxML analysis. CLSI M38-A2 protocol was used to assess antifungal susceptibility of the isolates to four major antifungal drugs. Additionally, the presence of point mutations in SQLE gene, which are responsible for terbinafine resistance was investigated. Tinea corporis was the most prevalent clinical manifestation accounting for 37.24% of examined cases of dermatophytosis. Based on ITS, T. indotineae (50.98%), T. mentagrophytes (19.61%), and M. canis (29.41%) was identified as an etiologic species. T. indotineae and T. mentagrophytes strains were identified as T. interdigitale based on TEF-1α. Terbinafine showed the highest efficacy among the tested antifungal drugs. T. indotineae and T. mentagrophytes showed the highest resistance to antifungal drugs with MICs of 2-4 and 4 μg/mL, while M. canis was the most susceptible species. Three of T. indotineae isolates showed mutations in SQLE gene Phe³⁹⁷Leu substitution. A non-previously described point mutation, Phe³¹¹Leu was identified in T. indotineae and mutations Lys²⁷⁶Asn, Phe³⁹⁷Leu and Leu⁴¹⁹Phe were diagnosed in T. mentagrophytes XVII. The results of mutation analysis showed that Phe³⁹⁷Leu was a destabilizing mutation; protein stability has decreased with variations in pH, and point mutations affected the interatomic interaction, resulting in bond disruption. These results could help to control the progression of disease effectively and make decisions regarding the selection of appropriate drugs for dermatophyte infections.

Keywords: SQLE mutation; Antifungal susceptibility; Dermatophytes; Dermatophytosis; Iraq; Terbinafine resistance.

Figure 1

Clinical manifestations of dermatophytosis in patients referred to Al-Diwaneyah Teaching Hospital. Tinea capitis (a), tinea corporis (b), tinea pedis (c) and tinea cruris (d).

Figure 2

ClustalW multiple sequence alignment of M. canis isolates in the current study compared to CBS isolate from GenBank (blue underlined). M. canis type I green underlined and M. canis type II without underlined. The alignment created by Unipro UGENE 47.0 software. SNPS showed nucleotide substitution at positions T¹⁵⁷C, C¹⁹⁴T, T²³⁷C, G⁵⁰⁵A and T⁶¹¹C.

Figure 3

Maximum likelihood phylogenetic tree of dermatophytes included in the current study is based on ITS and TEF-1α sequences constructed by RAxML through CIPRES Science Gateway and edited by iTOL software. Values at the nodes indicate bootstrap percentages according to 1000 replicates, and branches with bootstrap values above 76% are shown (different clades are highlighted using different colors).

Figure 4

T. mentagrophytes and T. indotineae: Phylogenetic tree of Iraqi (designated by bold font) and international genotypes deposited in the GenBank database based on sequencing of the ITS rDNA region. Tree created through CIPRES Science Gateway and edited by iTOL software.

Figure 5

RAxML phylogenetic tree of M. canis of the present study and global strains: yellow background accession numbers refer to M. canis type I and green background accession numbers refer to M. canis type II. Simple bar annotations point to the sequence length (nts). The tree was created through CIPRES Science Gateway and edited by iTOL software.

Figure 6

Amino acid substitutions of squalene epoxidase are marked by a black square. UYO77308 represents the wild type from GenBank, WNA16468 is the sensitive isolate (this study) and the rest of the isolates are terbinafine resistant. Multiple sequence alignment (MSA) of amino acids was performed by PSI-BLAST (http://www.ibi.vu.nl/programs/pralinewww/).

Figure 6

Amino acid substitutions of squalene epoxidase are marked by a black square. UYO77308 represents the wild type from GenBank, WNA16468 is the sensitive isolate (this study) and the rest of the isolates are terbinafine resistant. Multiple sequence alignment (MSA) of amino acids was performed by PSI-BLAST (http://www.ibi.vu.nl/programs/pralinewww/).

Figure 7

3D homology model of sqle mutant. Wild types are designated by a red color while mutant are designated by a blue color. Based on the ΔΔG results, the amino acid changes distort the protein structure. The model was predicted and assessed by SWISS-MODEL (https://swissmodel.expasy.org/) and visualized by Pymol software.

Figure 8

Analyzing point mutations in a range of pH (5.5–8.5) using the MAESTRO web server (https://pbwww.services.came.sbg.ac.at/maestro/web) shows that as the pH increases, the protein structure confirmation changes, and the protein stability deteriorates.

Figure 9

The figure illustrates the interatomic interaction between wild type and mutant, which shows bond disruptions in four residues by the Dynamut server (https://biosig.lab.uq.edu.au/dynamut/).