title: Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy.
abstract: BACKGROUND
  Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known.
  
  
  OBJECTIVE
  In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes.
  
  
  METHODS
  Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes.
  
  
  RESULTS
  Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate.
  
  
  CONCLUSIONS
  The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. Electronic address: gpini@unifi.it.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Pini
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Faggi
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
authors.forename: Enza
authors.initials: E
authors.lastname: Campisi
mesh_terms: D000818:Animals; D001717:Birds / Q000382:microbiology; D003453:Cryptococcosis / Q000453:epidemiology / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification / Q000472:pathogenicity; D003470:Culture Media; D005243:Feces / Q000382:microbiology; D005656:Fungal Proteins / Q000032:analysis*; D006801:Humans; D007558:Italy / Q000453:epidemiology; D014442:Monophenol Monooxygenase / Q000032:analysis; D010740:Phospholipases / Q000032:analysis; D011933:Reagent Kits, Diagnostic; D012703:Serotyping; D012988:Soil Microbiology; D014774:Virulence
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D011933:Reagent Kits, Diagnostic; D014442:Monophenol Monooxygenase; D010740:Phospholipases
keywords: API ZYM; Aislamientos ambientales; Aislamientos clínicos; Clinical isolates; Cryptococcus neoformans; Environmental isolates; Fosfolipasa; Phospholipase