Molecular identification of zygomycetes from culture and experimentally infected tissues

Abstract

Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.

FIG. 1.

Intraspecies sequence variability within the ITS1-5.8S-ITS2 region for strains of R. oryzae and M. circinelloides. Among the 17 isolates of R. oryzae, 3 types were distinguished. Five types were found for the 5 isolates of M. circinelloides. Different types are represented by Roman numerals. Numbers represent positions on the sequence alignment (position 0 corresponds to the 5′ end of the universal fungal primer ITS 1).

FIG. 2.

Alignment of Zygomycetes ITS1 sequences, including the 3′ end of 18S rRNA gene: R. oryz, R. oryzae CBS 112.07^T; A cory, A. corymbifera IP 1129.75; C. recu, C. recurvatus CBS 158.50^T; M. circ, M. circinelloides CBS 195.68^NT; M. hiem, M. hiemalis CBS 201.64^NT; M. indi, M. indicus CBS 226.29^T; M. race, M. racemosus CBS 260.68 ^T; M. ramosissimus CBS 135.65^NT; M. roux, M. rouxii CBS 416.77; R. mieh, R. miehei CBS 182.67^T; R. pusi, R. pusillus CBS 354.68^NT; R. vari, R. variabilis CBS 103.93^T; R. vare, R. variabilis var. regularior CBS 384.95^T; R. azyg, R. azygosporus CBS 357.93^T; R. micr, R. microsporus var. rhizopodiformis IP 676.72; R. micc, R. microsporus var. chinensis CBS 631.82^T; R. micm, R. microsporus var. microsporus IP 1124.75; R. mico, R. microsporus var. oligosporus CBS 339.62, R. schi, R. schippereae CBS 138.95^T; S. race, S. racemosum CNRMA 03.414. ^T, type strain; ^NT, neotype strain. Alignment was made with the type strain of the species or with a reference strain from an international collection when no type strain was available, except for S. racemosum.

FIG. 3.

Alignment of Zygomycetes ITS2 sequences, including the 5′ end of 28S rRNA gene. Strain identification is the same as indicated in the legend to Fig. 2. Alignment was made with the type strain of the species or with a reference strain from an international collection when no type strain was available, except for S. racemosum.