Candida parapsilosis characterization in an outbreak setting

Abstract

Candida parapsilosis is an important non-albicans species which infects hospitalized patients. No studies have correlated outbreak infections of C. parapsilosis with multiple virulence factors. We used DNA fingerprinting to determine genetic variability among isolates from a C. parapsilosis outbreak and from our clinical database. We compared phenotypic markers of pathogenesis, including adherence, biofilm formation, and protein secretion (secretory aspartic protease [SAP] and phospholipase). Adherence was measured as colony counts on silicone elastomer disks immersed in agar. Biofilms formed on disks were quantified by dry weight. SAP expression was measured by hydrolysis of bovine albumin; a colorimetric assay was used to quantitate phospholipase. DNA fingerprinting indicated that the outbreak isolates were clonal and genetically distinct from our database. Biofilm expression by the outbreak clone was greater than that of sporadic isolates (p < or =0.0005). Adherence and protein secretion did not correlate with strain pathogenicity. These results suggest that biofilm production plays a role in C. parapsilosis outbreaks.

Figure 1

Genetic analysis of Candida parapsilosis clinical isolates. Southern blot hybridization patterns of the 14 C. parapsilosis test isolates were probed with the Cp3-13 DNA fingerprinting probe. The reference strain J940043 was run in the outer two lanes of the gel. Isolates associated with the hospital outbreak are indicated. Note that while isolates 167, 179, 177, 165, 173, and 317 displayed identical group I patterns, strains 313 and 385 showed patterns typical of non-group I strains with a lack of abundant intense bands. Molecular sizes are presented in kilobases to the left of the panel.

Figure 2

Relatedness of Candida parapsilosis clinical isolates. Dendrogram generated from S_ABs computed for pairwise comparisons of the 14 C. parapsilosis test isolates and the reference strain J940043 fingerprinted with Cp3-13. Note that with the exception of the six identical outbreak isolates, none of the dendrogram nodes exceed an S_AB value of 0.7 for the other test isolates.

Figure 3

Adherence properties of Candida parapsilosis clinical isolates. Graph shows adhesion ability of various C. parapsilosis strains, compared to strain 167 from the Centers for Disease Control and Prevention. Results were normalized to strain 167, which was taken as 100%. Each result is representative of at least two experiments. Error bars represent standard deviation. *p < 0.001 for comparison of values of strain 167 vs. strains 313 and 385; all other comparisons had p values > 0.05. (For details of methods used, see text.) Cath, catheter; Bld, bloodstream; Spt, sputum; Hnd, hand; Wnd, wound; Pdf, peritoneal dialysis fluid.

Figure 4

Relative dry weight of biofilms formed by Candida parapsilosis clinical isolates. Panel A shows relative dry weight of the C. parapsilosis strains from outbreak investigations by the Centers for Disease Control and Prevention from various culture sources. Results were normalized to control C. parapsilosis strain 167, which was taken as 100%. Each result is representative of at least two experiments. Error bars represent standard deviation. *p = 0.001 and p < 0.001 for dry weight comparison of 167 vs. 313 and 385, respectively. Panel B shows relative dry weight of the University Hospitals' C. parapsilosis strains from various culture sources, also compared to strain 167. p < 0.0005 for comparison of dry weight values of 167 vs. all others. (For details of methods used, see text.) Cath, catheter; Bld, bloodstream; Spt, sputum; Hnd, hand; Wnd, wound; Pdf, peritoneal dialysis fluid.

Figure 5

Secretory aspartic protease (SAP) expression by Candida parapsilosis clinical isolates. Panel A shows representative sodium dodecyl sulfate–polyacrylamide gel electrophoresis of various C. parapsilosis isolates. M, molecular weight marker lane; BSA, bovine serum albumin alone; other lanes show number of isolate; and +, supernatant plus protease inhibitor cocktail. Protease activity is evident from the appearance of lower molecular weight bands representing cleavage products. Thick arrow indicates the 20-kDa protein appearing after protease digestion. (For details of methods used see text.) Panel B shows densitometric scanning analysis of SAP activity. Strains 177 and 179 were included to demonstrate the heterogeneity in SAP production within the clonal strains. Cath, catheter; Bld, bloodstream; Spt, sputum; Hnd, hand; Wnd, wound; Pdf, peritoneal dialysis fluid.

Figure 6

Phospholipase expression by Candida parapsilosis clinical isolates. Phospholipase expression as determined by the colorimetric method is shown. C. albicans train M61 was included as it is a known phospholipase producer. (For details of the methods used, see text.) Cath, catheter; Bld, bloodstream; Spt, sputum; Hnd, hand; Pdf, peritoneal dialysis fluid.