Biofilm formation, an important virulence trait of Candida species was measured in 107 Candida isolates from 32 candidemic patients by XTT [2,3-bis (2-methoxy-4nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] activity and compared to biofilm formation of Candida isolates from oropharyngeal lesions of 19 AIDS patients. Biofilm formation by XTT varied among species and C. albicans; C. lusitaniae and C. krusei produced more biofilm than the other Candida species. C. tropicalis was the most dominant species isolated from blood followed by C. albicans, and other non-albicans species whereas only C. albicans was recovered from oral lesions. Importantly, though Biofilm formation was variable within a species it was stable in sequential isolates during chronic infection. Sequential isolates exhibited identical Karyotype pattern or RAPD patterns unless patients were co-infected with more than one strain. High biofilm formation was associated with slow growth rate but not with adherence. Murine infection studies demonstrated that in mice, degree of biofilm formation was associated with enhanced virulence as mice infected both with no and low biofilm formers survived longer than mice infected with high biofilm formers (p≤0.001). We conclude that biofilm formation is a stable but strain specific characteristic that can greatly vary among C. albicans and non-albicans strains, and plays an important role in persistence of infection.

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillinresistant and methicillin-sensitive S. aureus isolates, as well as wound-and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.As a commensal, Staphylococcus aureus colonizes the nasal mucosa of 20 to 40% of humans (54), and as a pathogen it causes pyogenic diseases and toxin-mediated diseases (38). S. aureus produces many different virulence factors, including enterotoxins (SEs), which can cause defined toxic shock syndromes (4). The characterization of some of these toxins led to the discovery of superantigens (41), which bind to major histocompatibility complex class II molecules and V␤ chains of T-cell receptors, resulting in the activation of large numbers of T cells (20 to 30%) and massive cytokine production (10, 18). These superantigen-induced "cytokine storms" are responsible for the toxic effects seen in staphylococcal entertoxin B (SEB)-and toxic shock syndrome toxin (TSST)-associated shock syndromes in S. aureus infections (13, 40, 47). To date, 19 SEs have been identified based on sequence homologies, and studies have reported enterotoxin genes in up to 80% of all S. aureus strains (4, 21). Although many new enterotoxins have been identified, i.e., seg ser and seu (33,37,44,49), their precise functions have not been characterized yet. The majority of experimental work with SEs is still done with SEB, toxic shock syndrome toxin 1, and SEA (27, 31), because these toxins are commercially available. Most SEs are located on mobile elements in bacterial genomes such as plasmids or pathogenicity islands and can thu...

Caveolins are the principal protein components of caveolae, invaginations of the plasma membrane involved in cell signaling and trafficking. Caveolin-3 (Cav-3) is the muscle-specific isoform of the caveolin family and mutations in the CAV3 gene lead to a large group of neuromuscular disorders. In unrelated patients, we identified two distinct CAV3 mutations involving the same codon 78. Patient 1, affected by dilated cardiomyopathy and limb girdle muscular dystrophy (LGMD)-1C, shows an autosomal recessive mutation converting threonine to methionine (T78M). Patient 2, affected by isolated familiar hyperCKemia, shows an autosomal dominant mutation converting threonine to lysine (T78K). Cav-3 wild type (WT) and Cav-3 mutations were transiently transfected into Cos-7 cells. Cav-3 WT and Cav-3 T78M mutant localized at the plasma membrane, whereas Cav-3 T78K was retained in a perinuclear compartment. Cav-3 T78K expression was decreased by 87% when compared with Cav-3 WT, whereas Cav-3 T78M protein levels were unchanged. To evaluate whether Cav-3 T78K and Cav-3 T78M mutants behaved with a dominant negative pattern, Cos-7 cells were cotransfected with green fluorescent protein (GFP)-Cav-3 WT in combination with either mutant or WT Cav-3. When cotransfected with Cav-3 WT or Cav-3 T78M, GFP-Cav-3 WT was localized at the plasma membrane, as expected. However, when cotransfected with Cav-3 T78K, GFP-Cav-3 WT was retained in a perinuclear compartment, and its protein levels were reduced by 60%, suggesting a dominant negative action. Accordingly, Cav-3 protein levels in muscles from a biopsy of patient 2 (T78K mutation) were reduced by 80%. In conclusion, CAV3 T78M and T78K mutations lead to distinct disorders showing different clinical features and inheritance, and displaying distinct phenotypes in vitro.

Candiduria is a common finding in hospitalized patients with indwelling urine-draining devices. Animal models for candiduria are not well-developed and, despite its prevalence and associated mortality, candiduria is understudied. The presence of Candida in urine does not imply disease because it is also a commensal. Biofilm formation on catheters and the host-pathogen interaction are likely to be important factors that contribute to the pathogenesis. The objective of this study was to establish a candiduria model in mice with indwelling catheters. Our data demonstrate that biofilm formation on indwelling catheters and persistent candiduria can be established in mice. The study supports the concept that biofilm formation contributes to persistence. It also outlines differences between catheter-related candiduria in mice and humans. Specifically, mice exhibit higher levels of leukocyturia. In addition, mean daily fungal burden in urine in the murine model is 10-to 100-fold lower than that in humans. These important findings must be taken into consideration when using this model to study host-pathogen interaction in the setting of candiduria.