Selecting Endogenous Promoters for Improving Biosynthesis of Squalene in Schizochytrium sp.

Squalene (C30H50) is an acyclic triterpenoid compound renowned for its myriad physiological functions, such as anticancer and antioxidative properties, rendering it invaluable in both the food and pharmaceutical sectors. Due to the natural resource constraints, microbial fermentation has emerged as a prominent trend. Schizochytrium sp., known to harbor the intact mevalonate acid (MVA) pathway, possesses the inherent capability to biosynthesize squalene. However, there is a dearth of reported key genes in both the MVA and the squalene synthesis pathways, along with the associated promoter elements for their modification. This study commenced by cloning and characterizing 13 endogenous promoters derived from transcriptome sequencing data. Subsequently, five promoters exhibiting varying expression intensities were chosen from the aforementioned pool to facilitate the overexpression of the squalene synthase gene squalene synthetase (SQS), pivotal in the MVA pathway. Ultimately, a transformed strain designated as SQS-3626, exhibiting squalene production 2.8 times greater than that of the wild-type strain, was identified. Finally, the optimization of nitrogen source concentrations and trace element contents in the fermentation medium was conducted. Following 120 h of fed-batch fermentation, the accumulated final squalene yield in the transformed strain SQS-3626 reached 2.2 g/L.

Enhancing vaccine efficacy: Evaluating the superiority of cationic liposome-embedded squalene adjuvant against PCV2 infection.

Cationic liposome-embedded squalene (CLS) is a promising adjuvant that enhances antigen stability and mobility and improves immune response. This study compares the efficacy of a CLS-adjuvant porcine circovirus type 2 (PCV2) vaccine (CSV) with a conventional vaccine against PCV2. The CSV vaccine showed superior stability and was effective against PCV2-induced growth decline. It significantly increased serum immunoglobulin and cytokine levels, reduced serum PCV2 DNA, shortened the duration of viremia, and provided robust protection. CSV outperformed conventional vaccines, highlighting its potential for innovative vaccine development.

Microbial Squalene: A Sustainable Alternative for the Cosmetics and Pharmaceutical Industry - A Review.

Squalene is a natural triterpenoid and a biosynthetic precursor of steroids and hopanoids in microorganisms, plants, humans, and other animals. Squalene has exceptional properties, such as its antioxidant activity, a high penetrability of the skin, and the ability to trigger the immune system, promoting its application in the cosmetic, sustenance, and pharmaceutical industries. Because sharks are the primary source of squalene, there is a need to identify low-cost, environment friendly, and sustainable alternatives for producing squalene commercially. This shift has prompted scientists to apply biotechnological advances to research microorganisms for synthesizing squalene. This review summarizes recent metabolic and bioprocess engineering strategies in various microorganisms for the biotechnological production of this valuable molecule.

Squalene monooxygenase (SQLE) protects ovarian cancer cells from ferroptosis.

Altered cholesterol metabolism has been linked to a poor prognosis in various types of cancer. Cholesterol oxidation can lead to lipid peroxidation, membrane damage, and cell death. Ferroptosis is a regulated form of cell death characterized by the accumulation of lipid peroxides, which significantly inhibits the growth of ovarian cancer cells. SQLE is the primary enzyme responsible for catalyzing cholesterol lipid synthesis and is notably expressed in ovarian cancer tissues and cells. This study aims to investigate the role of squalene monooxygenase (SQLE) in ferroptosis in ovarian cancer. The protein and mRNA expression of SQLE was assessed using qRT-PCR, Western Blot, and immunohistochemistry. The association between SQLE and ferroptosis was demonstrated through analysis of TCGA and GTEx databases, TMT protein sequencing, as well as validation by qRT-PCR, Western Blot, immunofluorescence, ROS detection, and lipid peroxide detection. Animal experiments further confirmed the relationship between SQLE and ferroptosis in ovarian cancer. The protein and mRNA expression of SQLE was found to be upregulated in both ovarian cancer tissues and cell lines. Decreased SQLE expression led to ferroptosis in ovarian cancer cells, thereby increasing their sensitivity to ferroptosis inducers. Our research demonstrates that SQLE is significantly upregulated in both ovarian cancer tissues and cells. The overexpression of SQLE in ovarian cancer may facilitate tumorigenesis by conferring resistance to ferroptosis, thus shedding light on potential novel therapeutic strategies for ovarian cancer.

Physicochemical properties, profile of volatiles, fatty acids, lipids and concomitants from four Kadsura coccinea seed oils.

The Kadsura coccinea fruit is a wild fruit that may be eaten and used medicinally. Its seeds are rich in nutrients but are typically thrown away without processing.The physicochemical characterization, volatiles, fatty acids, lipids and concomitants of cold-processed seed oils from four kinds of K. coccinea were evaluated. The average kernel yield and oil yield of K. coccinea seeds were 68.21 % and 30.44 %, respectively. The seed oil contains a moderate level of total phenolics (368.99-503.99 mgGAE/100 g), total flavonoids (95.01-126.18 mg RE/100 g), and ß-sitosterol (1498.8-1712.7 mg/kg) with higher iodine value, lower acid value, saponification value and shorter induction time. GC analysis reveals appreciable amounts of linoleic acid (64.91-68.05 %) and squalene in seed oil. GC-MS analysis showed that the major volatile compounds were γ-muurolene (27.25-31.7 %), ß-himachalene (19.51-20.37 %) and ß-curcumene (15.78-16.78 %). Moreover, 16 terpenoids, 14 phenolics were identified by UPLC-QTOF-MS/MS. These results suggest that K. coccinea seed seems an promising alternative oilseed with biological ingredients for food, cosmetics and pharmaceutical industries.

Terbinafine Resistance in Trichophyton Strains Isolated from Humans and Animals: A Retrospective Cohort Study in Italy, 2016 to May 2024.

Background: In recent decades, globalization and international migration have increased the spread of infectious agents, including dermatophytes. Although considered minor infections, dermatophytoses are highly contagious, and they significantly reduce the quality of life, inducing itching, burning, sleep disturbances, and even depressive states. Moreover, the increasing resistance to antifungals threats the public health and burdens the costs for the healthcare system. Methods: DermaGenius® Resistance Multiplex real-time PCR assay allowed to analyze the terbinafine susceptibility/resistance of 172 Trichophyton strains, which were isolated from human and animal samples collected from 2016 to May 2024 and previously identified by Sanger sequencing. Results: All the 11 animal strains belonged to the T. interdigitale/T. mentagrophytes complex and tested terbinafine sensitive. Out of 161 human strains, 9 (5.6%) showed terbinafine resistance and 7 (4.3%) were identified as T. indotineae. Conclusions: This study provides preliminary data about behavior toward antifungals in animals and finalizes the scientific information currently available about human strains, highlighting the importance of the One Health concept. Moreover, it supports the relevant role of T. indotineae as an emerging dermatophyte with high proportion of terbinafine resistance.

New Sources of Resistance to Terbinafine Revealed and Squalene Epoxidase Modelled in the Dermatophyte Fungus Trichophyton interdigitale From Australia.

BACKGROUND: Terbinafine is widely used to treat onychomycosis caused by dermatophyte fungi. Terbinafine resistance in recent years is causing concern. Resistance has so far been associated with single-nucleotide substitutions in the DNA sequence of the enzyme squalene epoxidase (SQLE) but how this affects SQLE functionality is not understood. OBJECTIVES: The aim of this study was to understand newly discovered resistance in two Australian strains of Trichophyton interdigitale. PATIENTS/METHODS: Resistance to terbinafine was tested in four newly isolated strains. Three-dimensional SQLE models were prepared to investigate how the structure of their SQLE affected the binding of terbinafine. RESULTS: This study found the first Australian occurrences of terbinafine resistance in two T. interdigitale strains. Both strains had novel deletion mutations in erg1 and frameshifts during translation. Three-dimensional models had smaller SQLE proteins and open reading frames as well as fewer C-terminal α-helices than susceptible strains. In susceptible strains, the lipophilic tail of terbinafine was predicted to dock stably into a hydrophobic pocket in SQLE lined by over 20 hydrophobic amino acids. In resistant strains, molecular dynamics simulations showed that terbinafine docking was unstable and so terbinafine did not block squalene metabolism and ultimately ergosterol production. The resistant reference strain ATCC MYA-4438 T. rubrum showed a single erg1 mutation that resulted in frameshift during translation, leading to C-terminal helix deletion. CONCLUSIONS: Modelling their effects on their SQLE proteins will aid in the design of potential new treatments for these novel resistant strains, which pose clinical problems in treating dermatophyte infections with terbinafine.

Sustainable (-)-Ambrox Production: Chemistry Meets Biocatalysis.

(-)-Ambrox, the most prominent olfactive component of ambergris, is one of the most widely used biodegradable fragrance ingredients. It is traditionally produced from the diterpene sclareol chemically modified and cyclized into (-)-ambrox. The availability of the new feedstock (E)-ß-farnesene produced by fermentation opened new routes to (E,E)-homofarnesol as a precursor to (-)-ambrox. Combining the chemical transformation of (E)-ß-farnesene to (E,E)-homofarnesol and its enzymatic cyclization with an engineered Squalene Hopene Cyclase provided a new sustainable route for the production of (-)-ambrox at industrial scale. Compared to the traditional synthesis from sclareol, the new and innovative route from (E)-ß-farnesene improves atom and step economy, reduces waste production, solvent and energy consumption.

Aurantiochytrium mutant strains exhibiting different colony colors altered the contents of squalene.

Aurantiochytrium sp. 18W-13a, a marine heterotrophic protist belonging to the genus thraustochytrid, is known to accumulate high levels of squalene and carotenoids. Nowadays, the mutagenesis breeding of microorganisms is still widely practiced because the induced mutations of DNA do not involve the permanent integration of heterologous DNA sequences. Therefore, in this study, we focused on the improvement of squalene yield by mutagenesis breeding using Aurantiochytrium sp. 18W-13a. To bypass the massively laborious screening, we propose to use colony colors as the first criterion to screen mutants with high squalene accumulation, since the carotenoid and squalene synthetic pathways share an intermediate. We selected pale (white)-colored mutants after carbon ion irradiation. The white mutants exhibited larger squalene yields than twice as much of the original strain. The results clearly indicate that the present screening method with colony colors promises to obtain productive strains of squalene.

SQLE-a promising prognostic biomarker in cervical cancer: implications for tumor malignant behavior, cholesterol synthesis, epithelial-mesenchymal transition, and immune infiltration.

BACKGROUND: Cervical cancer, encompassing squamous cell carcinoma and endocervical adenocarcinoma (CESC), presents a considerable risk to the well-being of women. Recent studies have reported that squalene epoxidase (SQLE) is overexpressed in several cancers, which contributes to cancer development. METHODS: RNA sequencing data for SQLE were obtained from The Cancer Genome Atlas. In vitro experiments, including colorimetry, colony formation, Transwell, RT-qPCR, and Western blotting were performed. Furthermore, a transplanted CESC nude mouse model was constructed to validate the tumorigenic activity of SQLE in vivo. Associations among the SQLE expression profiles, differentially expressed genes (DEGs), immune infiltration, and chemosensitivity were examined. The prognostic value of genetic changes and DNA methylation in SQLE were also assessed. RESULTS: SQLE mRNA expression was significantly increased in CESC. ROC analysis revealed the strong diagnostic ability of SQLE toward CESC. Patients with high SQLE expression experienced shorter overall survival. The promotional effects of SQLE on cancer cell proliferation, metastasis, cholesterol synthesis, and EMT were emphasized. DEGs functional enrichment analysis revealed the signaling pathways and biological processes. Notably, a connection existed between the SQLE expression and the presence of immune cells as well as the activation of immune checkpoints. Increased SQLE expressions exhibited increased chemotherapeutic responses. SQLE methylation status was significantly associated with CESC prognosis. CONCLUSION: SQLE significantly affects CESC prognosis, malignant behavior, cholesterol synthesis, EMT, and immune infiltration; thereby offering diagnostic and indicator roles in CESC. Thus, SQLE can be a novel therapeutic target in CESC treatment.

A novel amphiphilic squalene-based compound with open-chain polyethers reduces malignant melanoma metastasis in-vitro and in-vivo.

Squalene (SQ) is a well-known antioxidant and anti-inflammatory agent that provides promising anti-aging and UV-protective roles on human skin. However, its strong hydrophobic nature, accompanied by issues such as poor solubility and limited tissue permeation, has created challenges for scientists to investigate its untapped potential in more complex conditions, including cancer progression. The present study assessed the potent anti-metastatic properties of a newly synthesized amphiphilic ethylene glycol SQ derivative (SQ-diEG) in melanoma, the most fatal skin cancer. In vitro and in vivo experiments have discovered that SQ-diEG may exert its potential on melanoma malignancy through the mitochondria-mediated caspase activation apoptotic signaling pathway. The potent anti-metastatic effect of SQ-diEG was observed in vitro using highly proliferative and aggressive melanoma cells. Administration of SQ-diEG (25 mg/kg) significantly decreased the tumor burden on the lung and inhibited the metastasis-associated proteins and gene markers in B16F10 lung colonization mice model. Furthermore, global gene profiling also revealed a promising role of SQ-diEG in tumor microenvironment. We anticipated that the amphiphilic nature of the SQ compound bearing ethylene glycol oligomers could potentially augment its ability to reach the pathology site, thus enhancing its therapeutic potential in melanoma.

Preliminary Evaluation of the Effect of Domestication on the Marketable and Nutritional Quality of B. aegyptiaca (L.) Delile Oil from Algeria.

Balanites aegyptiaca is a multipurpose fruit tree that grows wild in many arid and semi-arid African areas; however, recent domestication efforts have been undertaken to protect the species from the threat of urbanization and climate change. Within this context, the impact of the domestication of Algerian B. aegyptiaca was evaluated on its seed oil, which is already valued as food. Hence, oils from wild and domesticated trees were comparatively investigated for their physicochemical and compositional quality. Both oil types had a good oxidative stability and met the requirements for human consumption in terms of the saponification index, the free acidity, and the peroxide value. Moreover, they showed a comparable FA composition, with high levels of oleic and linoleic acids, which are beneficial for the consumer's health. Domestication led to a statistically significant decrease in the tocopherols and polyphenols in the oil. The phytosterols and squalene were slightly lower in the domesticated oil than in the wild relative, although no statistically significant differences were observed. A comparable mineral profile was revealed and the minimal variations in the trace elements between the oils could be related to the natural variability in the seeds. Hopefully, this study will encourage the domestication of B. aegyptiaca as a sustainable strategy for enhancing its socioeconomic value in Algerian rural areas.

Lipid Quality Changes in French Fries, Chicken Croquettes, and Chicken Nuggets Fried with High-Linoleic and High-Oleic Sunflower Oils in Domestic Deep Fryers.

The quality of fried products greatly depends on the changes occurring during frying. The purpose of this work was to study the lipid quality changes taking place in selected frozen foods after domestic deep-frying. Conventional, high-linoleic sunflower oil (HLSO) and high-oleic sunflower oil (HOSO) were used, and the frozen foods selected were French fries, croquettes, and nuggets. The foods were fried in domestic fryers under discontinuous conditions. Analyses included fatty acid composition, sterols, tocopherols, squalene, and lipid alteration levels. In all fried foods, the content of lipids increased after frying, which is consistent with previous findings. However, the lipid exchange between the food and the oil greatly depended on the food characteristics. Specifically, the levels of frying oil in the food lipids were about 90, 40, and 58% for French fries, croquettes, and nuggets, respectively. The main results obtained showed that lipid alteration levels considerably decreased and amounts of sterols and tocopherols significantly increased in French fries' lipids after frying. In both chicken products, croquettes and nuggets, the best quality improvement observed was a significant decrease in cholesterol in food lipids due to the lipid exchange. Overall, frying with HLSO and HOSO improved the quality and nutritional properties of all products tested.

Investigation of bioactive phytochemical compounds of the Ethiopian medicinal plant using GC-MS and FTIR.

Medicinal plants Highly aromatic crude materials are utilized for treating warts as an alternative medicine to surgical treatment because they can be permanently removed from the body. Thus, this investigation aimed to extract plant material from Calotropis procera leaves, describe the phytochemical screening, analyze anti-microbial activities, determine the functional groups in FTIR, and identify the chemical compounds in GC-MS. The PH, specific gravity, and viscosity of the crude extracts of Calotropis procera were determined at 4.5, 0.79, and 0.49, respectively. Analyze the solubility of crude extracts; ethanol can dissolve while water does not. Flavonoids, alkaloids, phenols, tannins, and saponins were also present in the phytochemical screening tests of the Calotropis procera extracts, triterpenoids, terpenoids, and steroids were not present in the crude extract. Flavonoids, alkaloids, phenols, tannins, and saponins are the primary phytochemical components found in therapeutic plant material. The Calotropis procera crude extracts analyzed for functional groups by FT-IR contained a hydroxyl group, alkane, carbonyl, aldehyde, ketone, phenols, ester, alcohol, and methylene. The chemical compounds analyzed by GC-MS of Calotropis procera crude material were found to have 22 main compounds. Of 22 compounds, 5 compounds are active ingredients for the applications of medical purposes. The bioactive compounds found in the Calotropis procera plant extract are neophytadiene, hexahydrofarnesyl, lanosterol, 2,4-dimethylbenzo [H]quinolone, and squalene. Those bioactive compounds have anti-bacterial, analgesic, antipyretic, anti-inflammatory, antimicrobial, antioxidant, antiviral, and anti-cancer properties. In an in vitro antimicrobial activity test, the crude extract effectively inhibited more gram-positive bacteria than gram-negative bacteria. This collective reason is why the traditional therapist uses this Calotropis procera plant for the treatment of warts.

A practical clinical device for evaluating changes to the skin's sebum from ozone.

BACKGROUND: It is known that tropospheric ozone (O3) generated from pollutants reacting with UV forms lipid peroxidation products and induces oxidative stress to the skin. With the ever-increasing consumer awareness of the effects pollution has on skin, more testing methods will be needed to evaluate cosmetic ingredients. Recently, others have shown how antioxidants are able to reduce the effects from ozone on skin through in vitro, ex vivo and clinical studies where human subjects place their arms into large stationary chambers. AIMS: To develop a small, easy to use ozone exposure module (OEM) that can be used on various sites of the body and to validate this device for use in testing the ability of topical products to mitigate the effects of ozone exposure on the skin. MATERIALS AND METHODS: We have produced an OEM which can generate levels of ozone in excess of 1000 ppb and can be set to achieve the equivalent exposure to what is found in polluted environmental conditions. After exposure we used D-squame discs to remove the sebum and analytically quantitate squalene depletion. Squalene, which is very sensitive to ROS, easily oxidizes into early metabolite squalene monohydroperoxide (SQOOH) with ozone exposure. RESULTS: We were able to show decreases in squalene levels after exposure and protective effects from a topical formulation. CONCLUSION: This generator will be a useful tool for researchers to easily create a small and safe exposure from ozone for clinical testing.

CONTEXTE: On sait que l'ozone troposphérique (O3) générée par les polluants réagissant avec les UV forme des produits de peroxydation lipidique et induit un stress oxydatif pour la peau. Avec la sensibilisation croissante des consommateurs aux effets de la pollution sur la peau, plus de méthodes d'analyse seront nécessaires pour évaluer les ingrédients cosmétiques. Récemment, d'autres méthodes ont montré la capacité des antioxydants à réduire les effets de l'ozone sur la peau grâce à des études in vitro, ex vivo et cliniques où les sujets humains placent leurs bras dans de grandes chambres stationnaires. OBJECTIFS: Développer un petit module d'exposition à l'ozone (MEO) facile à utiliser qui peut être utilisé sur divers sites corporels afin de valider ce dispositif pour utilisation dans des analyses de capacité des produits topiques à atténuer les effets de l'exposition à l'ozone sur la peau MATÉRIELS ET MÉTHODES: Nous avons produit un MEO qui peut générer des niveaux d'ozone supérieurs à 1 000 ppb et qui peut être réglé pour atteindre l'exposition équivalente aux niveaux présents dans des conditions d'environnement pollué. Après exposition, nous avons utilisé des disques D­squame pour éliminer le sébum et quantifier analytiquement la déplétion en squalène. Le squalène, qui est très sensible au ROS, s'oxyde facilement en métabolite précoce monohydroperoxyde de squalène (SQOOH) avec exposition à l'ozone RÉSULTATS: Nous avons pu montrer des diminutions des taux de squalène après exposition et les effets protecteurs d'une formulation topique. CONCLUSION: Ce générateur sera un outil utile pour que les chercheurs puissent facilement créer une petite exposition sûre à l'ozone pour les analyses cliniques.

Use of Adjuvant Compositions Based on Squalene Ensures Induction of Neutralizing Antibodies against SARS-CoV-2.

Squalene-based adjuvant compositions that can provide effective induction of specific humoral immune response have been developed. Recombinant receptor-binding domain (RBD) of surface S-protein of SARS-CoV-2 was used to evaluate the properties of the composition. Immunization of mice with the developed squalene-based compositions in combination with RBD allows obtaining high titers of specific antibodies: from 105 to 2×106. The blood sera from immunized mice exhibit neutralizing activity against SARS-CoV-2 Delta variant (B.1.617.2) with a titer up to 1:2000.

Sustainable biosynthesis of squalene from waste cooking oil by the yeast Yarrowia lipolytica.

Squalene is a highly sought-after triterpene compound in growing demand, and its production offers a promising avenue for circular economy practices. In this study, we applied metabolic engineering principles to enhance squalene production in the nonconventional yeast Yarrowia lipolytica, using waste cooking oil as a substrate. By overexpressing key enzymes in the mevalonate pathway - specifically ERG9 encoding squalene synthase, ERG20 encoding farnesyl diphosphate synthase, and HMGR encoding hydroxy-methyl-glutaryl-CoA reductase - we achieved a yield of 779.9 mg/L of squalene. Further co-overexpression of DGA1, encoding diacylglycerol acyltransferase, and CAT2, encoding carnitine acetyltransferase, in combination with prior metabolic enhancements, boosted squalene production to 1381.4 mg/L in the engineered strain Po1g17. To enhance the supply of the precursor acetyl-CoA and inhibit downstream squalene conversion, we supplemented with 6 g/L pyruvic acid and 0.7 mg/L terbinafine, resulting in an overall squalene titer of 2594.1 mg/L. These advancements underscore the potential for sustainable, large-scale squalene production using Y. lipolytica cell factories, contributing to circular economy initiatives by valorizing waste materials.

Kinetics of Squalene Quenching Singlet Oxygen and the Thermal Degradation Products Identification.

Squalene has been proven to possess various bioactive functions that are widely present in vegetable oils. A more comprehensive understanding of the reaction behavior of squalene under oxidative conditions was achieved by studying its antioxidant capacity and thermal degradation products. The total singlet oxygen quenching rate constant (kr + kq) of squalene was 3.8 × 107 M-1 s-1, and both physical and chemical quenching mechanisms equally contribute to the overall singlet oxygen quenching. Fourteen degradation products of squalene were identified at 180 °C by using gas chromatography-mass spectrometry (GC-MS). Combining with DFT calculations, the thermal degradation pathway of squalene was proposed: the aldehydes, ketones, and alcohols, and epoxy compounds were formed by the homolytic cleavage of squalene hydroperoxides to form alkoxy radicals, followed by ß-scission of the alkoxyl radicals at adjacent C-C bonds or intramolecular cyclization.

[Cloning and functional characterization of oxidosqualene cyclase gene for α-amyrin synthesis].

Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, ß-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.

Potential emergence of terbinafine resistance by squalene epoxidase gene mutations: An 18-month cohort study of onychomycosis patients in the United States.

BACKGROUND: There is a concerning rise in antifungal-resistant dermatophytosis globally, with resistance to terbinafine conferred by point mutations in the squalene epoxidase (SQLE) gene. OBJECTIVES: Report changes in the prevalence and profile of SQLE mutations in onychomycosis patients in the United States. METHODS: A longitudinal cohort study of toenail samples was collected from suspected onychomycosis patients over an 18-month period from 2022 to 2023. Samples were submitted from across the United States and subjected to multiplex real-time polymerase chain reactions for dermatophyte detection, with further screening of SQLE mutations at four known hotspots (393Leu, 397Phe, 415Phe and 440His). RESULTS: A total of 62,056 samples were submitted (mean age: 57.5 years; female: 60.4%). Dermatophytes were detected in 38.5% of samples, primarily Trichophyton rubrum complex (83.6%) and T. mentagrophytes complex (10.7%). A survey of SQLE mutations was carried out in 22,610 dermatophyte samples; there was a significant increase in the prevalence of SQLE mutations between the first quarter of 2022 and the second quarter of 2023 (29.0 to 61.9 per 1000 persons). The Phe397Leu substitution was the predominant mutation; Phe415Ser and His440Tyr have also emerged which were previously reported as minor mutations in skin samples. The temporal change in mutation rates can be primarily attributed to the Phe415Ser substitution. Samples from elderly patients (>70 years) are more likely to be infected with the T. mentagrophytes complex including strains harbouring the Phe415Ser substitution. CONCLUSION: The prevalence of SQLE mutations among onychomycosis patients with Trichophyton infections may be underestimated. Older individuals may have a higher risk.