Non-sporulating molds in clinical specimens: agents of infections?

Abstract

Non-sporulating molds (NSMs) or mycelia sterilia isolated from clinical specimens are assumed to be environmental contaminants and their identification is not usually pursued. Knowing the identity of these isolates may help to determine their importance as pathogens. In this study, internal transcribed spacer (ITS) sequences were used to identify 15 non-sporulating fungi isolated as pure cultures from clinical specimens such as skin, nail, blood and other fluids. Fungal DNA was directly extracted from Whatman FTA® cards inoculated with fungal suspension, amplified using ITS 1 and 2 primers and sequenced. Sequences were matched using BLAST search, with published fungal identities. Of 15 NSMs, one isolate from blood taken from a lymphoma patient was identified as Schizophylum commune, a Basidiomycete fungus which has been reported as a human pathogen causing systemic disease. Four isolates were identified as plant pathogens; Penicillium ochrochloron from bronchoalveolar fluid, Phlebia sp. from pleural fluid, Phoma multirostrata and Inonotus rickii from skin specimens. Two others were identified as saprophytes or endophytes; Xylaria feejeensis from nail and Fomitopsis cf. meliae from skin. All other isolates, including three from blood and two from peritoneal & endotracheal fluid could not be conclusively identified based on the ITS sequences alone. To determine the presence of possible virulence factors in NSMs, the Phlebia sp. isolate was evaluated for keratinase activity in vitro, on human nail and was shown to have high keratin degrading activity which indicates potential pathogenicity. In conclusion, this study has found that non-sporulating fungi may have a role in causing infections in humans; further studies are needed to establish their involvement. Introduction When non-sporulating molds (NSM) are isolated as pure cultures from patients, it raises a question on their ability to cause infection. Although often dismissed as insignificant, environmental organisms, DNA based identification has revealed potential pathogens among NSMs (Pounder et al. 2007). Molecular methods for fungal identification are well established, with the ribosomal DNA internal transcribed spacer (ITS) sequence utilised for species determination. Pathogenic fungi are known to produce proteolytic enzymes, such as keratinase in dermatophytes, which is important for fungal invasion of cutaneous sites (Viani et al. 2001). The keratinase activity level correlates with severity of infection caused. This study aimed to identify clinical NSM isolates and determine their pathogenic capability. Results Figure 1 : NSM isolates identified based on ITS sequences ReferenceS Pounder, J.I., Simmon, K.E., Barton, C.A., Hohmann, S.L., Brandt, M.E. & Petti, C.A. 2007. Discovering Potential Pathogens among Fungi Identified as Nonsporulating Molds. Journal of Clinical Microbiology 45(2): 568-571. Viani, F.C., Santos, J.I.D., Paula, C.R., Larson, C.E. & Gambale, W. 2001. Production of extracellular enzymes by Microsporum canis and their role in its virulence. Medical Mycology 39: 463-468. Dentinger, B.T.M., Margaritescu, S. & Moncalvo J.M. 2010. Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms. Molecular Ecology Resources. 10: 628-633. METHODOLOGY UZ 177/10 Schizophylum commune UZ 517 Penicillium ochrochloron UZ 529/10 Phoma multirostrata UZ 586/10 Phlebia sp. UZ 661/10 Fomitopsis cf. meliae UZ 365/08 Inonotus rickii UZ 634 Xylaria feejeensis Fungal Molecular Identification (Dentinger et al. 2010) Fungal suspension prepared from PDA inoculated onto Whatman FTA® card (200 μL) DNA directly extracted from FTA card punches using Sigma Extract-N-AmpTM Plant PCR Kit Extracted DNA amplified using ITS 1 and ITS 2 primers PCR product purified and sequenced; sequences matched with published data Keratinase Activity Determination (Viani et al. 2001) JADUAL 1. Nilai purata dan sisihan piawai bagi kawalan negatif, kawalan positif dan fungus ujian Fungal isolate Keratinase Unit SD F. solani 5.340 2.097 T. mentagrophytes 4.000 1.572 Non-sporulating mold (Phlebia sp.) 3.060 1.501 F. oxysporum 2.273 1.258 Table 1: Keratinase activity of pathogenic fungi and NSM CONCLUSION Of fifteen NSMs, seven isolates were identified; one (S.commune) as a known pathogen. The Phlebia sp. produced keratinase enzyme at levels similar to dermatophyte fungi. Non-sporulationg molds have pathogenic potential and may be the cause of infections in humans. Fungal suspension incubated with human nail powder and keratin azure in a mineral medium for 21 days. Keratinase activity determined by quantitatively measuring dye release from keratin azure in mineral medium using a spectrophotometre. An increase of 0.01 in the absorbance measurement at 595 nm is equivalent to 1 Keratinase Unit (KU) of enzyme activity.