Amino Acid Substitution in Trichophyton rubrum Squalene Epoxidase Associated with Resistance to Terbinafine

ABSTRACT There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.

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Intrinsic resistance to terbinafine among human and animal isolates of Trichophyton mentagrophytes related to amino acid substitution in the squalene epoxidase

Infection ◽

10.1007/s15010-020-01498-1 ◽

2020 ◽

Vol 48 (6) ◽

pp. 889-897 ◽

Cited By ~ 1

Author(s):

Dominik Łagowski ◽

Sebastian Gnat ◽

Aneta Nowakiewicz ◽

Marcelina Osińska ◽

Mariusz Dyląg

Keyword(s):

Amino Acid ◽

Fungal Infections ◽

Single Point ◽

Antifungal Susceptibility ◽

Point Mutations ◽

Trichophyton Mentagrophytes ◽

Single Amino Acid ◽

Squalene Epoxidase ◽

Intrinsic Resistance ◽

Resistant Strains

Abstract Background Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. Purpose Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. Methods We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. Results In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. Conclusion Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

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Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Virologica Sinica ◽

10.1007/s12250-014-3426-x ◽

2014 ◽

Vol 29 (2) ◽

pp. 103-111 ◽

Cited By ~ 13

Author(s):

Haiwei Wang ◽

Shanshan Song ◽

Jianxiong Zeng ◽

Guohui Zhou ◽

Decheng Yang ◽

...

Keyword(s):

Amino Acid ◽

Disease Virus ◽

Amino Acid Substitution ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Resistant Phenotype ◽

Mouth Disease Virus ◽

Foot And Mouth

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Biological, Biochemical, and Molecular Characterization of a New Clinical Trichophyton rubrum Isolate Resistant to Terbinafine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01600-05 ◽

2006 ◽

Vol 50 (6) ◽

pp. 2234-2236 ◽

Cited By ~ 37

Author(s):

Colin S. Osborne ◽

Ingrid Leitner ◽

Bettina Hofbauer ◽

Ceri A. Fielding ◽

Bertrand Favre ◽

...

Keyword(s):

Amino Acid ◽

Missense Mutation ◽

Molecular Characterization ◽

Amino Acid Substitution ◽

Trichophyton Rubrum ◽

Mechanisms Of Action ◽

Clinical Strain ◽

Squalene Epoxidase

ABSTRACT We have characterized a new clinical strain of Trichophyton rubrum highly resistant to terbinafine but exhibiting normal susceptibility to drugs with other mechanisms of action. Resistance to terbinafine in this strain is caused by a missense mutation in the squalene epoxidase gene leading to the amino acid substitution F397L.

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Contribution of Clinically Derived Mutations inERG11to Azole Resistance in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03470-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 450-460 ◽

Cited By ~ 96

Author(s):

Stephanie A. Flowers ◽

Brendan Colón ◽

Sarah G. Whaley ◽

Mary A. Schuler ◽

P. David Rogers

Keyword(s):

Candida Albicans ◽

Amino Acid ◽

Amino Acid Substitution ◽

Single Amino Acid Substitution ◽

Clinical Isolates ◽

Single Amino Acid ◽

Azole Resistance ◽

Amino Acid Substitutions ◽

Content Type ◽

Proximal Surface

ABSTRACTInCandida albicans, theERG11gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations inERG11that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. AlthoughERG11mutations have been observed in clinical isolates, the specific contributions of individualERG11mutations to azole resistance inC. albicanshave not been widely explored. We sequencedERG11in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation inERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinctERG11alleles were recovered, with 10ERG11alleles containing a single amino acid substitution. We then characterized 19 distinctERG11alleles by introducing them into the wild-type azole-susceptibleC. albicansSC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations inERG11are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.

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Activities of Newer Fluoroquinolones against Streptococcus pneumoniae Clinical Isolates Including Those with Mutations in the gyrA, parC, and parELoci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.2.329 ◽

1999 ◽

Vol 43 (2) ◽

pp. 329-334 ◽

Cited By ~ 79

Author(s):

J. H. Jorgensen ◽

L. M. Weigel ◽

M. J. Ferraro ◽

J. M. Swenson ◽

F. C. Tenover

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Amino Acid Sequences ◽

Clinical Isolates ◽

Single Amino Acid ◽

Single Mutation ◽

Topoisomerase Iv ◽

In Vitro Susceptibility ◽

Resistant Strains ◽

Level Resistance

ABSTRACT Resistance to fluoroquinolone (FQ) antibiotics inStreptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA andgyrB) and topoisomerase IV (parC andparE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) ofgyrA, gyrB, parC, andparE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation inparC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA,parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

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Spread of Terbinafine-Resistant Trichophyton mentagrophytes Type VIII (India) in Germany–“The Tip of the Iceberg?”

Journal of Fungi ◽

10.3390/jof6040207 ◽

2020 ◽

Vol 6 (4) ◽

pp. 207

Author(s):

Pietro Nenoff ◽

Shyam B. Verma ◽

Andreas Ebert ◽

Anke Süß ◽

Eleni Fischer ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Indian Subcontinent ◽

Resistance Testing ◽

Public Health Issue ◽

Squalene Epoxidase ◽

Amino Acid Substitutions ◽

Type Viii ◽

Resistant Strains

Chronic recalcitrant dermatophytoses, due to Trichophyton (T.) mentagrophytes Type VIII are on the rise in India and are noteworthy for their predominance. It would not be wrong to assume that travel and migration would be responsible for the spread of T. mentagrophytes Type VIII from India, with many strains resistant to terbinafine, to other parts of the world. From September 2016 until March 2020, a total of 29 strains of T. mentagrophytes Type VIII (India) were isolated. All patients were residents of Germany: 12 females, 15 males and the gender of the remaining two was not assignable. Patients originated from India (11), Pakistan (two), Bangladesh (one), Iraq (two), Bahrain (one), Libya (one) and other unspecified countries (10). At least two patients were German-born residents. Most samples (21) were collected in 2019 and 2020. All 29 T. mentagrophytes isolates were sequenced (internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1-α)). All were identified as genotype VIII (India) of T. mentagrophytes. In vitro resistance testing revealed 13/29 strains (45%) to be terbinafine-resistant with minimum inhibitory concentration (MIC) breakpoints ≥0.2 µg/mL. The remaining 16 strains (55%) were terbinafine-sensitive. Point mutation analysis revealed that 10/13 resistant strains exhibited Phe397Leu amino acid substitution of squalene epoxidase (SQLE), indicative for in vitro resistance to terbinafine. Two resistant strains showed combined Phe397Leu and Ala448Thr amino acid substitutions, and one strain a single Leu393Phe amino acid substitution. Out of 16 terbinafine-sensitive strains, in eight Ala448Thr, and in one Ala448Thr +, new Val444 Ile amino acid substitutions were detected. Resistance to both itraconazole and voriconazole was observed in three out of 13 analyzed strains. Treatment included topical ciclopirox olamine plus topical miconazole or sertaconazole. Oral itraconazole 200 mg twice daily for four to eight weeks was found to be adequate. Terbinafine-resistant T. mentagrophytes Type VIII are being increasingly isolated. In Germany, transmission of T. mentagrophytes Type VIII from the Indian subcontinent to Europe should be viewed as a significant public health issue.

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Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00115-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 80

Author(s):

Tsuyoshi Yamada ◽

Mari Maeda ◽

Mohamed Mahdi Alshahni ◽

Reiko Tanaka ◽

Takashi Yaguchi ◽

...

Keyword(s):

Amino Acid ◽

Single Point ◽

Point Mutations ◽

Trichophyton Mentagrophytes ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Single Amino Acid ◽

Squalene Epoxidase ◽

Amino Acid Substitutions ◽

Content Type

ABSTRACT Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

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