Hmg1 Gene Mutation Prevalence in Triazole-Resistant Aspergillus fumigatus Clinical Isolates

Abstract

Recently, mutations in the 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase-encoding gene (hmg1), a gene involved in ergosterol production, were associated with triazole-resistance in Aspergillus fumigatus. In this study, we determined the prevalence and characteristics of hmg1 mutations in a collection of clinical triazole-resistant A. fumigatus isolates collected during 2001–2019 from two international mycology reference centers: the Belgian National Reference Center for Mycosis and the Center of Expertise in Mycology Radboudumc/CWZ. Clinical isolates with and without cyp51A gene mutations and randomly selected wild-type (WT) controls were included. Isolates were characterized by in vitro susceptibility testing, cyp51A and hmg1 sequencing, and short tandem repeat typing. Available clinical records were analyzed for previous triazole exposure. In 23 isolates (24%) of the 95 triazole-resistant A. fumigatus isolates, hmg1 gene mutations were observed; including 5/23 (22%) isolates without cyp51A gene mutations and 18/72 (25%) with cyp51A mutations. Four previously described hmg1 gene mutations (E105K, G307R/D, G466V, and S541G) and two novel mutations (W273S and L304P) were found; 4/23 (17%) in the sterol-sensing-domain region. No triazole-antifungal exposure was reported in 75% (9/12) of patients harboring an isolate with hmg1 gene mutations. Three of 39 WT isolates (8%) contained a hmg1 gene mutation; E105K (2-isolates) and S541G. Hmg1 gene mutations were predominantly found in A. fumigatus with cyp51A mutations with voriconazole MICs ≥ 8 mg/L.

A Cyp51B Mutation Contributes to Azole Resistance in Aspergillus fumigatus

“…However, azole resistant strains lacking cyp51A -related resistance mechanisms have always been left behind due to the lower frequency of these isolates [ 9 ]. More recently, the incidence of these cyp51A wild-type resistant isolates is increasing and, with it, the number of studies investigating other azole resistance mechanisms Cyp51A-independent [ 11 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 24 , 25 , 55 ].…”

“…However, we did not find any modification related to the cyp51A gene. In order to further investigate the resistance mechanism present in this strain, we looked for mutations in Cyp51B and also in Hmg1, an enzyme involved in the ergosterol biosynthetic pathway that has recently been implicated in azole resistance [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Interestingly, we found that the strain CM9640 has two amino acid mutations: a G457S in Cyp51B and a F390L in Hmg1.…”

“…In A. fumigatus, hmg1 encodes an HMG-CoA reductase (HMGR), an enzyme that participates in one of the first steps of the ergosterol pathway catalyzing the conversion of 3-hydroxy-3-methylglutaryl-CoA in mevalonic acid [ 79 ]. Mutations in Hmg1, more specifically in the sterol-sensing domain (SSD) of the protein, are one of the recently reported azole resistance mechanisms in strains with a wild-type cyp51A , although the underlying reason for these mechanisms has not been elucidated yet [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 80 ]. In addition, most of the strains that had resistant MICs profiles and hmg1 mutations also had modifications in cyp51A in combination [ 13 , 14 , 16 , 17 , 18 , 19 ].…”

“…More recently, the reports of A. fumigatus azole resistant isolates, without Cyp51A modifications, indicate that there are other novel mechanisms mediating resistance to azole drugs [ 11 , 12 ]. These studies have reported roles in azole resistance of other genes from the ergosterol biosynthesis pathway, including hmg1 [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ], erg6 [ 15 , 17 ], some transcriptional factors [ 20 , 21 , 22 ], hapE [ 23 ] or overexpression of drug efflux pumps [ 24 , 25 ].…”

Triazole-Resistance in Environmental Aspergillus fumigatus in Latin American and African Countries

“…Genetic relatedness between isolates was determined using two separate multiplex PCRs specific for A. fumigatus (M3, M4), each amplifying three short tandem repeats (STRs) of three trinucleotide loci (M3; STRAf3-A, B, C primers) and three tetranucleotide loci (M4; STRAf4-A, B, C primers) as indicated earlier [23,24]. Briefly, 4 µL of cleaned PCR product was combined with 20 µL mix of HiDi Formamide and GeneScan 500 LIZ size standard (Applied Biosystems, Foster City, CA, USA) for fragment detection on a 96-capillary array ABI3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).…”