Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes

Abstract

Candida parapsilosis (sensu lato), which represents a fungal complex composed of three genetically related species – Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis, has emerged as an important yeast causing fungemia worldwide. The goal of the present work was to assess the prevalence, antifungal susceptibility and production of virulence traits in 53 clinical isolates previously identified as C. parapsilosis (sensu lato) obtained from hospitals located in the Southeast of Brazil. Species forming this fungal complex are physiologically/morphologically indistinguishable; however, polymerase chain reaction followed by restriction fragment length polymorphism of FKS1 gene has solved the identification inaccuracy, revealing that 43 (81.1%) isolates were identified as C. parapsilosis sensu stricto and 10 (18.9%) as C. orthopsilosis. No C. metapsilosis was found. The geographic distribution of these Candida species was uniform among the studied Brazilian States (São Paulo, Rio de Janeiro and Espírito Santo). All C. orthopsilosis and almost all C. parapsilosis sensu stricto (95.3%) isolates were susceptible to amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. Nevertheless, one C. parapsilosis sensu stricto isolate was resistant to fluconazole and another one was resistant to caspofungin. C. parapsilosis sensu stricto isolates exhibited higher MIC mean values to amphotericin B, fluconazole and caspofungin than those of C. orthopsilosis, while C. orthopsilosis isolates displayed higher MIC mean to itraconazole compared to C. parapsilosis sensu stricto. Identical MIC mean values to voriconazole were measured for these Candida species. All the isolates of both species were able to form biofilm on polystyrene surface. Impressively, biofilm-growing cells of C. parapsilosis sensu stricto and C. orthopsilosis exhibited a considerable resistance to all antifungal agents tested. Pseudohyphae were observed in 67.4% and 80% of C. parapsilosis sensu stricto and C. orthopsilosis isolates, respectively. The secretion of phytase (93% versus 100%), aspartic protease (88.4% versus 90%), esterase (20.9% versus 50%) and hemolytic factors (25.6% versus 40%) was detected in C. parapsilosis sensu stricto and C. orthopsilosis isolates, respectively; however, no phospholipase activity was identified. An interesting fact was observed concerning the caseinolytic activity, for which all the producers (53.5%) belonged to C. parapsilosis sensu stricto. Collectively, our results add new data on the epidemiology, antifungal susceptibility and production of potential virulence attributes in clinical isolates of C. parapsilosis complex.

Introduction

Candida parapsilosis (sensu lato) is the second most frequently opportunistic yeast isolated from bloodstream infections in different clinical settings around the world, especially in Latin America and Asia (Pfaller et al., 2008, Diekema et al., 2009, Nucci et al., 2010, Lockhart et al., 2012). In a relevant way, C. parapsilosis (sensu lato) is responsible for 20–30% of all fungal infections, particularly those related to the usage of catheter and other medical devices. For all these reasons, C. parapsilosis (sensu lato) is considered an important public health concern (Lockhart et al., 2008, Diekema et al., 2009, Gonçalves et al., 2010, Nucci et al., 2010, Garcia-Effron et al., 2012). Taking into account the current taxonomy, C. parapsilosis (sensu lato) was reclassified as a fungal complex formed by three genotypically distinguishable species, C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, which reflects the great heterogeneity of isolated strains at both biochemical and genetic levels (Tavanti et al., 2005). Within the complex, C. parapsilosis sensu stricto represents the most predominant species, while C. orthopsilosis and C. metapsilosis are recovered at a much lower incidence (Lockhart et al., 2008, Silva et al., 2009, Bertini et al., 2013). Although the number of epidemiologic investigations is increasing, the current literature still contains little information about the distribution of these opportunistic pathogenic yeasts in clinical samples. In the same way, there is a paucity of data on the effect of different classes of antifungal agents against these three species of Candida.

Opportunistic fungal pathogens, such as Candida spp., have developed sophisticated means to establish the infectious process. In this sense, the expression of a set of virulence attributes, including surface adhesins, phenotypic switching, morphogenesis, biofilm formation and production of hydrolytic enzymes (e.g., lipases, proteases and phosphatases), decisively contributes to the pathogenesis of candidiasis, allowing the fungal cells to escape and/or overcome the host defenses (Schaller et al., 2005, Cuéllar-Cruz et al., 2012, Singh and Mukhopadhyay, 2012, Deorukhkar et al., 2014, Singaravelu et al., 2014). However, these determinants of pathogenicity remain largely unexplored for the species of the C. parapsilosis complex. Aggravating this scenario, many contradictory results have been generated regarding the production of potential virulence traits in species belonging to the C. parapsilosis complex. The ability to form biofilm on polystyrene surface is a good example of this conflicting subject (Song et al., 2005, Tavanti et al., 2007, Lattif et al., 2010, Toro et al., 2011, Abi-chacra et al., 2013).

In light of the knowledge, with no doubt, there is a scarcity of information concerning the epidemiology and antifungal susceptibility of C. parapsilosis complex worldwide, especially in our country (Brazil), as well as the capability of the clinical isolates in producing virulence attributes associated to the infectious process. These reasons led us to perform a study focused on the most developed areas of Brazil, located in the Southeast region. In this context, 53 clinical isolates were collected from hospitals located at three Brazilian States (São Paulo, Rio de Janeiro and Espírito Santo), which were initially identified as C. parapsilosis (sensu lato), in order to evaluate the prevalence of species forming the C. parapsilosis complex as well as their antifungal susceptibility patterns and the production of well-known phenotypic characteristics associated with fungal pathogenesis, including filamentation capability, secretion of hydrolytic enzymes (e.g., proteases, lipases and phytases) and hemolysins as well as biofilm formation capacity. In addition, the effects of antifungal drugs on biofilm-forming cells were also assessed. To finalize, a survey of the epidemiology of C. parapsilosis complex was summarized in this paper in order to present a global perspective of these clinically relevant Candida species during the last 20 years.