Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors,

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Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors, and antifungal susceptibility S-J. Liaw, H-C. Wu Clinical Microbiology and Infection Volume 16, Issue 6, Pages 696-703 (June 2010) DOI: 10.1111/j.1469-0691.2009.02930.x Copyright © 2010 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1 (a) Agarose gel electrophoresis of the CAP59 gene restriction profiles of Cryptococcus neoformans serotypes. Upper panel, BsmFIrestricted profile; lower panel, HpaII-restricted profile. C1--C4, serotype A clinical isolates; E1-E4, environmental isolates; CB, serotype B clinical isolate; A, ATCC 90112, serotype A; B, ATCC 32269, serotype B; D, ATCC 34873, serotype D; M, 100-bp marker. (b) Amplified fragments of isolates obtained in multiplex PCR with the primers for LAC1 and CAP64. C1-C6, serotype A clinical isolates; A, B, D and M are as in (a) Clinical Microbiology and Infection 2010 16, 696-703DOI: (10.1111/j.1469-0691.2009.02930.x) Copyright © 2010 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 2 (a) Examples of URA5 gene restriction fragment profiles of clinical and environmental isolates of Cryptococcus neoformans. C6-C7, C9 and CB, clinical isolates; E5-E6, environmental isolates; A, B, D, and M are as in Fig. 1. (b) Examples of PCR fingerprinting patterns obtained with the primer M13. C9, C31-C32, clinical isolates of C. neoformans; A, B, D, and M are as in Fig. 1. (c) Examples of PCR fingerprinting patterns obtained with the primer (GACA)4. C31-32, clinical isolates; E1-E4, A, B, D, M are as in Fig.1; C, ATCC 32608, serotype C. Clinical Microbiology and Infection 2010 16, 696-703DOI: (10.1111/j.1469-0691.2009.02930.x) Copyright © 2010 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 3 Adhesion of phospholipase-producing Cryptococcus neoformans to A549 cells. (a) C24, 42, 45, low producers; C52, 59, 95, high producers; E7, 8, environmental isolates. The Pz value is shown above each bar. Results obtained for C59 cells were defined as 100%. The data are averages and standard deviations of three independent experiments. a, b significantly different from c (p <0.01, by Student's t test). (b) Lactate dehydrogenase (LDH) release of A549 cells after interaction with phospholipase-producing C. neoformans. C24, 42, 45, 52, 59, 95, E7-8, are as in (a). Supernatants, after incubation, were assayed for LDH release using colorimetry described in Materials and Methods. Data are expressed as a percentage of LDH release from unstimulated cells treated with Triton X-100 and represent the mean ± SD of triplicate measurements from two independent experiments. Control, medium only background. a vs. b, p <0.01 by Student's t test. (c) Light microscopy of C. neoformans adhering to A549 cells. A549 cells were stimulated for 6 h at 37°C with low (C24) or high (C59) phospholipase-producing isolates of C. neoformans, followed by washing, trypsinization, cytospin to slides and observation. a, no yeast control, 400×; b, A549 incubated with C59, 400×; c, A549 incubated with C24, 400×; d, A549 incubated with C59, 650×. Data are representative of three independent experiments. Clinical Microbiology and Infection 2010 16, 696-703DOI: (10.1111/j.1469-0691.2009.02930.x) Copyright © 2010 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 4 Amphotericin B killing assay of melanized and non-melanized Cryptococcus neoformans. The survival rates of melanized vs. non-melanized yeast cells (clinical isolates, C48 and C67) after exposure to 0.25 and 0.5 mg/L of amphotericin B for 2 h were compared with those of fungal cells incubated in phosphate-buffered saline. The data represent averages of four independent experiments with standard deviations. p <0.05 as determined by Student's t test for a comparison between non-melanized and melanized cells. Clinical Microbiology and Infection 2010 16, 696-703DOI: (10.1111/j.1469-0691.2009.02930.x) Copyright © 2010 European Society of Clinical Infectious Diseases Terms and Conditions