Home » Volumes » Volume 50 July/August 2017 » In vitro evaluation of hydrolytic enzyme activity and biofilm formation of Candida parapsilosis species complex from a nosocomial environment

In vitro evaluation of hydrolytic enzyme activity and biofilm formation of Candida parapsilosis species complex from a nosocomial environment

Shaiana Paula-Mattiello1 Sílvia Dias de Oliveira1 Renata Medina-Silva1

1Faculdade de Biociências, Laboratório de Imunologia e Microbiologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brasil.

DOI: 10.1590/0037-8682-0032-2017

The nosocomial environment may act as a reservoir for C. parapsilosis complex isolates with phenotypic features that could possibly lead to nosocomial infections and health complications in hospital patients.



Candida parapsilosis complex species, frequently found in hospital environments, have gained importance as etiological agents of candidemia.


Candida parapsilosis complex isolates from a nosocomial environment were identified and their hydrolitic enzyme activity and ability to form biofilm were characterized.


Twenty-two Cparapsilosis sensu stricto isolates produced proteinase and three produced phospholipase. Most Candida metapsilosis isolates produced proteinase and one also produced phospholipase. All 29 isolates formed biofilms.


The nosocomial environment may act as a reservoir for Cparapsilosis complex isolates with phenotypic features that could possibly lead to nosocomial infections and health complications in hospital patients.

Keywords: Candida parapsilosis complex; Biofilm; Hydrolytic enzymes

Candida species are major agents involved in nosocomial fungal infections worldwide, especially infections of endogenous origin and those related to immunocompromised patients1. Different factors in the nosocomial setting and the hands of healthcare workers may represent important sources for Candida infections2Candidaparapsilosis complex species (Candida parapsilosis sensu strictoCandida orthopsilosis, and Candida metapsilosis) are often isolated from nosocomial environments; since the 1990s, they have gained importance as etiological agents of candidemia in hospitals in different countries1.

The pathogenicity of Candida species may be at least partly due to their ability to produce hydrolytic enzymes, such as phospholipases (PLs) and aspartyl proteinases (SAPs), which are considered important factors for Cparapsilosis adherence, tissue penetration, and host invasion3. Moreover, the ability to form biofilms can induce a significant reduction in yeast antifungal susceptibility and an increase in their capacity to evade the immune system4.

Most data regarding the ability to produce hydrolytic enzymes and biofilm formation has been obtained using clinical Calbicans isolates1,5; to date, few studies have investigated these properties in Cparapsilosis complex isolates, particularly those from nosocomial environments. In this context, the present study evaluated the ability of 29 Cparapsilosis complex isolates present in hospital settings to produce hydrolytic enzymes and form biofilms. These isolates were collected between December 2009 and February 2010, strictly from the nosocomial environment and medical devices in a general patient care unit at a tertiary care teaching hospital in Porto Alegre (RS-Brazil). Following the collections, the isolated yeasts were biochemically identified as belonging to the Cparapsilosis complex (data not shown).

For this study, yeast genomic deoxyribonucleic acid (DNA) was extracted as previously described6 and used as template for amplification of a FKS1 gene fragment (1,032bp). Amplicons were digested with EcoRI7, separated by electrophoresis on a 2% agarose gel stained with ethidium bromide (0.5µg/mL), and analyzed using a Gel Doc L-Pix Image System (Loccus Biotecnologia, SP, Brazil). Fragments of 1,032bp indicated Cparapsilosis sensustricto; fragments of 564 and 474bp indicated Cmetapsilosis; and fragments of 474bp, 306bp, and 258bp indicated CorthopsilosisCalbicans ATCC 1884 was used as the negative control and Cparapsilosis ATCC 22019, Cmetapsilosis ATCC 96143, and Corthopsilosis ATCC 96141 were employed as positive controls.

Phospholipase and aspartyl proteinase production was analyzed and interpreted as previously described8,9. The average Pz values – the ratio between the colony diameter (dc) and dc plus precipitation zone (dcp) – were calculated and the isolates were grouped into five categories according to their Pz values9. All biofilm assays were conducted 10 times for each isolate10. The isolates were classified into four categories: non-producers, weak, moderate, or strong biofilm producers4. Statistical comparisons regarding biofilm formation were performed employing the Student’s t test with p-value ≤ 0.05, using the software Statistical Package for the Social Sciences (SPSS)version 22.0 (IBM, USA), available at PUCRS.

All isolates were confirmed by FKS1 gene amplification as belonging to the Cparapsilosis complex (Table 1); 22 (75.9 %) were identified as Cparapsilosis sensu stricto, seven (24.1%) as Cmetapsilosis, and none as Corthopsilosis. The predominance of Cparapsilosis sensu stricto among the species from this complex has previously been observed in clinical and environmental samples11,12. Conversely, Cmetapsilosis and Corthopsilosis are not usually found or occur at very low frequencies in nosocomial environments; previous reports have suggested they are associated mainly with clinical samples and that their relative frequency varies among hospitals and geographic regions11,12.

The ability of the isolates to produce proteinase and PL is detailed in Table 1. Our results indicate that both Cparapsilosis sensu stricto and Cmetapsilosis produce mainly proteinase; most (93.1%) isolates were classified as very strong producers. The isolates also exhibited PL production; however, at a lower frequency (13.7%). Specifically, all 22 Cparapsilosis sensu stricto isolates demonstrated proteinase production, but only three showed PL activity. These data are similar to studies indicating that this species is a frequent strong proteinase producer, but often a non-PL producer3,11. Of the seven Cmetapsilosis isolates, five produced proteinase, and interestingly, one was found to produce both enzymes. Phospholipase production is not usual for this species; the absence of PL activity in C. metapsilosis isolates has been previously described13. Although the role of PL in Candida virulence remains unclear, some authors recognize the secretion of proteinases as an important virulence factor, which is immunogenic during infection and able to promote the degradation of host defense proteins3.

TABLE 1 Species identification, original collection site, and proteinase and phospholipase activities of nosocomial Candidaparapsilosis complex isolates. 

Isolates Origin Proteinase activity (Pz) Rating Phospholipase activity (Pz) Rating
CP.01 Chair 1 0.40 + + + + 1.0
CP.02 Chair 2 0.31 + + + + 1.0
CP.03 Bed 4 0.46 + + + + 0.55 + + + +
CM.04 Medication trolley 2 0.54 + + + + 1.0
CP.05 Sphygmomanometer 2 0.47 + + + + 1.0
CP.06 Sphygmomanometer 3 0.36 + + + + 1.0
CP.07 Glove 1 0.35 + + + + 0.52 + + + +
CP.08 Room table 1 0.55 + + + + 1.0
CM.09 Sphygmomanometer 4 1.0 1.0
CP.10 Room table 2 0.28 + + + + 0.60 + + + +
CP.11 Medication table 1 0.39 + + + + 1.0
CP.12 Sphygmomanometer 1 0.43 + + + + 1.0
CP.13 Glove 2 0.42 + + + + 1.0
CP.14 Wheelchair 1 0.38 + + + + 1.0
CP.15 Bed 5 0.52 + + + + 1.0
CP.16 Sphygmomanometer 4 0.47 + + + + 1.0
CP.17 Feed table 1 0.40 + + + + 1.0
CP.18 Medication trolley 1 0.37 + + + + 1.0
CM.19 Sphygmomanometer 3 0.57 + + + + 1.0
CP.20 Feed table 2 0.40 + + + + 1.0
CP.21 Medication trolley 2 0.40 + + + + 1.0
CP.22 Medication trolley 3 0.47 + + + + 1.0
CM.23 Medication trolley 4 0.55 + + + + 1.0
CP.24 Medication trolley 5 0.36 + + + + 1.0
CM.25 Sphygmomanometer 5 0.38 + + + + 1.0
CP.26 Feed table 3 0.37 + + + + 1.0
CM.27 Bed 1 1.0 1.0
CM.28 Feed table 1 0.60 + + + + 0.42 + + + +
CP.29 Feed table 4 0.36 + + + + 1.0
Cparapsilosis ATCC 22019 1.0 1.0
Calbicans ATCC 18804 0.95 + 0.87 ++

CP: Candida parapsilosis sensu strictoCM: Candida metapsilosisC : Candida; Pz = 1.0 (-): no activity; Pz0.90-0.99 (+): weak activity; Pz 0.80-0.89 (+ +): mild activity; Pz 0.70-0.79 (+ + +): strong activity; Pz < 0.69 (+ + + +): very strong activity.

The development of the biofilm has been described as an important virulence factor for Candida14. In this context, the development of biofilm by Cparapsilosis complex has gained considerable attention because isolates from these species have been observed in an extensive variety of biotic and abiotic surfaces14. In our study, all 29 isolates formed biofilms; 12 were classified as weak and 15 as moderate biofilm producers (Figure 1). Only two isolates were strong biofilm producers and both were identified as Cparapsilosis sensu stricto. Furthermore, Cparapsilosis ATCC 22019 and Calbicans ATCC 18804 were classified as moderate and weak biofilm producers, respectively. No significant difference in biofilm production was detected between Candida species (p-value=0.71). However, over half of the isolates (51.7%) were characterized as strong or moderate producers. This indicates a significant frequency of potentially pathogenic Candida species that can adhere efficiently to surfaces and tissues, representing an important risk when occurring in nosocomial environments.

FIGURE 1 Biofilm production by Candida parapsilosis complex isolates. The biofilm formation of all 29 isolates on polystyrene plates was quantified by measuring absorbance (A570nm). CP: Candida parapsilosis sensu strictoCM: Candida metapsilosisATCC 22019: Candida parapsilosisATCC 18804: Candida albicans. The error bars represent the variation of 10 replicates. The dashed line indicates the cut-off for biofilm classification (A570nm = 0.048). 

The data presented in this study revealed that all Candida isolates investigated were able to form biofilms and most also exhibited the ability to efficiently produce proteinase. These findings emphasize the potential pathogenicity of nosocomial Cparapsilosis complex isolates from hospital environment origins because they demonstrate the maintenance of important virulence factors when these yeast species leave host tissues15, which may represent a potential risk for reinfection of hospitalized patients. Thus, our results contribute to the characterization of the clinical risk posed by Cparapsilosis sensu stricto and Cmetapsilosis isolates occurring in nosocomial environments and provide pertinent information regarding the behavior of pathogenic microorganisms in hospital settings. These findings may be used in the design and application of daily disinfection and antisepsis practices for healthcare workers.

Our findings improve information concerning phenotypic properties of clinically important yeast species that efficiently contaminate surfaces and objects in hospital settings. Thus, they are relevant for the understanding of both sporadic cases and outbreaks of invasive fungal infections in hospitals, especially in immunocompromised patients. Further analyses, aiming measure the levels of hydrolytic enzymes expression, as well as to evaluate other virulence factors – like the mechanisms of antimicrobial resistance of the isolates in the biofilm condition – would importantly contribute to enhance the characterization of yeast species of the Cparapsilosis complex occurring in the nosocomial environment.


We wish to thank Professor Brasílio Ricardo Cirillo da Silva for support with statistical analyses.


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Financial support: This work was financially supported by the Brazilian Funding Agencies: Fundação de Amparo à Pesquisa no Estado do Rio Grande do Sul (FAPERGS) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Received: April 05, 2017; Accepted: June 20, 2017

Corresponding author: Msc. Shaiana Paula-Mattiello. e-mail:shaiana_m@hotmail.com

Conflict of interest: The authors declare that have no conflicts of interest.