Page 54 - Biennial Report 2018-20 Jun 2021

INVESTIGATION OF MECHANISMS OF EMERGING DRUG RESISTANCE IN
TRICHOPHYTON SP., THE MAJOR CAUSATIVE AGENTS OF FUNGAL SKIN
INFECTIONS

Fungal skin infections can be notoriously difficult to treat because of the similarity of these
eukaryotic cells to our own cells. Trichophyton species, the dermatophytic fungus that colonizes
the outer layers of our skin, is the leading cause of skin and nail infections including ringworm
and athlete’s foot. To study emergence of drug resistance in this fungus, Bhupesh Taneja’s group
collected nearly 100 skin/nail samples from GTB Hospital, Delhi, SP Medical College, Bikaner and
SMS Medical College, Jaipur from patients suffering from dermatophytosis. Identification of
clinical isolates was done by direct microscopic examination, as well as molecular phylogeny and
T. interdigitale was found as the predominant species amongst the isolates followed by T.
rubrum and T. violaceum. Antifungal susceptibility testing was performed by broth microdilution
methods for terbinafine, fluconazole and griseofulvin for 21 isolates. Among the tested isolates,
high MIC was seen in 5 isolates for Fluconazole, 8 isolates for Terbinafine, 5 isolates for
Terbinafine while four isolates showed high MIC for more than one antifungal agent. As
terbinafine resistant cases were predominant and change in MIC levels was very high, isolates
with clinical resistance to terbinafine were first selected for genome sequencing and further
analysis. Whole genome sequencing was carried out for fourteen T. interdigitale isolates
exhibiting variable levels of resistance in lab MICs to terbinafine. Comparative genomics of T.
rubrum IGIB-SBL-CI1 with nine other genome sequences of T. rubrum available at NCBI indicated
that T. rubrum IGIB-SBL-CI1 strain harbors nearly 2-fold more single nucleotide variations (SNV)
compared to the NCBI strains.

In the lab’s cross-sectional observational study, an alarming rise in dermatophytic cases was
found with >50% of patients presenting as reinfection and nearly 40% as recalcitrant
dermatophytosis with T. interdigitale as the predominant species identified in isolates.
Antifungal susceptibility testing was performed by broth microdilution methods for terbinafine,
fluconazole and griseofulvin according to NCCLS/CLSI-M38-A2 guidelines suggested for
filamentous fungi and compared with drug sensitive T. mentagrophytes interdigitale ATCC 18748
or T. rubrum ATCC 28188 as control strains before further analysis. The antifungal susceptibility
demonstrated increased MIC to terbinafine in several cases. ErgA was amplified to identify any
mutations that may lead to low drug sensitivity in lab MICs in clinical isolates exhibiting altered
drug sensitivity. The strains having high MICs revealed mutations in ergA in T. rubrum or T.
interdigitale.

Whole genome sequencing of (terbinafine) drug resistant strains was done next. The non-
responder strains (to terbinafine) were grown on SDA at 30°C for 14-21 days. Genomic DNA was
isolated, and the library prepared for genome sequencing as per manufacturer supplied
protocols. The selected isolates included 16 T. interdigitale strains, where an ergA mutation was
also identified and 3 T. interdigitale where no mutation was identified. Among T. rubrum, 4
different isolates encompassing a unique ergA mutation were also shortlisted. Finally, a total of
23 different isolates were shortlisted for whole genome sequencing and comparative genomics
analysis. Assembly of the genomes was completed, and gene prediction carried out using
Augustus and annotation using COG database. Comparative genomics analysis of the sequenced
dermatophyte genomes with the Refseq genomes is ongoing in the lab. As none of the previously
completed genomes have reported clinical resistance to any antifungal agent, SNV analysis of

53