Characterization of Lichtheimia hongkongensis, a novel fungal species that causes mucormycosis

Abstract

Three thermotolerant “Lichtheimia-like” isolates from patients suffering from mucormycosis (nasopharyngeal swab of a liver transplant recipient, gastric biopsy of a renal transplant recipient, and skin biopsy of a man with burn, respectively) were characterized. Phenotypic characteristics, including colony morphology, microscopic morphology and thiamine dependency, were determined. Microscopic examination of agar block smear preparations showed that most side branches of the three “Lichtheimia-like” isolates and Lichtheimia ramosa were circinate, with abundant pleomorphic giant cells with fingerlike projections commonly observed. Sequence analysis of four loci (internal transcribed spacer region of the ribosomal DNA, partial elongation factor 1-α gene, partial β-actin gene, and the D1-D2 region of 28S rRNA) showed that the three “Lichtheimia-like” isolates and L. ramosa formed a distinct cluster, and are closely related to, but distinct from, other Lichtheimia sp.. A new species, Lichtheimia hongkongensis (synonym of L. ramosa), was proposed to describe this fungus.

It was also hypothesized that a proportion of “A. corymbifera (L. corymbifera)” reported in the literature could be L. hongkongensis. In this study, 13 fungal strains that were reported as “A. corymbifera (L. corymbifera)” in the literature in an 11-year period were collected. Microscopic examination of agar block smear preparations of these 13 strains revealed unique characteristics of L. hongkongensis, including circinate side branches and pleomorphic giant cell with finger-like projections. Phylogenetic analysis also showed that all 13 strains are closely related with L. hongkongensis. It was confirmed that a significant number of reported A. corymbifera (L. corymbifera) infections are L. hongkongensis infections which are of global distribution.

In order to confirm the distinct phylogenetic position of L. hongkongensis, the mitochondrial genomes of three L. hongkongensis strains and a strain of L. corymbifera were sequenced. L. hongkongensis HKU21, HKU22 and HKU23 have circular mitochondrial genomes with sizes ranging from 31830 bp to 32167 bp, which contain 39 to 41 genes transcribed by both strands. L. corymbifera HKU25 has a circular mitochondrial genome of size 37262 bp, which contains 41 genes that are transcribed by both strands. All the four mitochondrial genomes contain a complete set of tRNAs, the small and the large rRNAs, as well as the basic 14 protein-coding genes. Analysis of gene order showed that the three L. hongkongensis strains are clustered together. It is also shown that the Lichtheimia group is also closely related to Rhizopus oryzae, which is a member of Mucorales. A mouse model was used to determine if there is a difference in virulence between L. hongkongensis and L. corymbifera. The groups of mice challenged with L. hongkongensis have a higher survival rate than those challenged with L. corymbifera. Intravenous administration of Lichtheimia spores resulted in an infection in livers and spleens as indicated by positive cultures. The number of spores that could be recovered from these organs was significantly lower in mice challenged with L. hongkongensis than those challenged with L. corymbifera, indicating that L. hongkongensis could be less virulent than L. corymbifera.