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

Abstract

INTRODUCTION:

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

METHODS:

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

RESULTS:

Twenty-two C. parapsilosis 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.

CONCLUSIONS:

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.

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 patients11. Suleyman G, Alangaden GJ. Nosocomia fungal infections: epidemiology, infection control, and prevention. Infect Dis Clin North Am. 2016;30(4):1023-52.. Different factors in the nosocomial setting and the hands of healthcare workers may represent important sources for Candida infections22. Sydnor ERM, Perl TM. Hospital epidemiology and infection control in acute-care setting. Clin Microbiol Rev. 2011;24(1):141-73.. Candida parapsilosis complex species (Candida parapsilosis sensu stricto, Candida 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 countries11. Suleyman G, Alangaden GJ. Nosocomia fungal infections: epidemiology, infection control, and prevention. Infect Dis Clin North Am. 2016;30(4):1023-52..

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 C. parapsilosis adherence, tissue penetration, and host invasion33. Junior ADE, Silva AF, Rosa FC, Monteiro SG, Figueiredo PMS, Monteiro CA. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida. Rev Soc Bras Med Trop. 2011;44(3):334-8.. 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 system44. Pulcrano G, Panellis D, Domenico G, Rossadno F, Catarina MR. Ambroxol influences voriconazole resistance of Candida parapsilosis biofilm. FEMS Yeast Res. 2012;12(4):430-8..

Most data regarding the ability to produce hydrolytic enzymes and biofilm formation has been obtained using clinical C. albicans isolates11. Suleyman G, Alangaden GJ. Nosocomia fungal infections: epidemiology, infection control, and prevention. Infect Dis Clin North Am. 2016;30(4):1023-52.,55. Tsui C, Kong EF, Jabra-Rizk MA. Pathogenesis of Candida albicans biofilm. FEMS Pathog Dis. 2016;74(4):1-13.; to date, few studies have investigated these properties in C. parapsilosis complex isolates, particularly those from nosocomial environments. In this context, the present study evaluated the ability of 29 C. parapsilosis 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 C. parapsilosis complex (data not shown).

For this study, yeast genomic deoxyribonucleic acid (DNA) was extracted as previously described66. Valente P, Gouveia FC, Lemos GA, Pimentel D, Elsas JD, Mendonça-Hagler LC, et al. PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures. FEMS Microbiol Lett. 1996;137(2):253-6. and used as template for amplification of a FKS1 gene fragment (1,032bp). Amplicons were digested with EcoRI77. Garcia-Effron G, Canton E, Pemán J, Dilger A, Romá E, Perlin DS. Assessment of two new molecular methods for identification of Candida parapsilosis sensu lato species. J Clin Microbiol. 2011;49(9):3257-61., 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 C. parapsilosis sensu stricto; fragments of 564 and 474bp indicated C. metapsilosis; and fragments of 474bp, 306bp, and 258bp indicated C. orthopsilosis. C. albicans ATCC 1884 was used as the negative control and C. parapsilosis ATCC 22019, C. metapsilosis ATCC 96143, and C. orthopsilosis ATCC 96141 were employed as positive controls.

Phospholipase and aspartyl proteinase production was analyzed and interpreted as previously described88. Price MF. Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia. 1982;20(1):7-14.,99. Aoki S, Ito-Kuma S. Comparative pathogenicity of a wild-type stain and respiratory mutants of Candida albicans in mice. Zentralbl Bakteriol. 1990;273(3):332-43.. 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 values99. Aoki S, Ito-Kuma S. Comparative pathogenicity of a wild-type stain and respiratory mutants of Candida albicans in mice. Zentralbl Bakteriol. 1990;273(3):332-43.. All biofilm assays were conducted 10 times for each isolate1010. Laffey SF, Butler G. Phenotype switching affects biofilm formation by Candida parapsilosis. Microbiology. 2005;151(1):1073-81.. The isolates were classified into four categories: non-producers, weak, moderate, or strong biofilm producers44. Pulcrano G, Panellis D, Domenico G, Rossadno F, Catarina MR. Ambroxol influences voriconazole resistance of Candida parapsilosis biofilm. FEMS Yeast Res. 2012;12(4):430-8.. 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 C. parapsilosis complex (Table 1); 22 (75.9 %) were identified as C. parapsilosis sensu stricto, seven (24.1%) as C. metapsilosis, and none as C. orthopsilosis. The predominance of C. parapsilosis sensu stricto among the species from this complex has previously been observed in clinical and environmental samples1111. Abi-chacra EA, Souza LOP, Cruz LP, Braga-Silva LA, Gonçalves DS, Sodré CL, et al. Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex. FEMS Yeast Res . 2013;13(8):831-48.,1212. Ziccardi M, Souza LOP, Gandra RM, Galdino ACM, Baptista ARS, Nunes APF, et al. Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes. Int J Med Microbiol. 2015;305(8):848-59.. Conversely, C. metapsilosis and C. orthopsilosis 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 regions1111. Abi-chacra EA, Souza LOP, Cruz LP, Braga-Silva LA, Gonçalves DS, Sodré CL, et al. Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex. FEMS Yeast Res . 2013;13(8):831-48.,1212. Ziccardi M, Souza LOP, Gandra RM, Galdino ACM, Baptista ARS, Nunes APF, et al. Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes. Int J Med Microbiol. 2015;305(8):848-59..

The ability of the isolates to produce proteinase and PL is detailed in Table 1. Our results indicate that both C. parapsilosis sensu stricto and C. metapsilosis 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 C. parapsilosis 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 producer33. Junior ADE, Silva AF, Rosa FC, Monteiro SG, Figueiredo PMS, Monteiro CA. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida. Rev Soc Bras Med Trop. 2011;44(3):334-8.,1111. Abi-chacra EA, Souza LOP, Cruz LP, Braga-Silva LA, Gonçalves DS, Sodré CL, et al. Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex. FEMS Yeast Res . 2013;13(8):831-48.. Of the seven C. metapsilosis 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 described1313. Silva BV, Silva LB, Oliveira DB, Silva PR, Ferreira-Paim K, Andrade-Silva LE, et al. Species distribution, virulence factors, and antifungal susceptibility among Candida parapsilosis complex isolates recovered from clinical specimens. Mycopathologia. 2015;180(5):333-43.. 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 proteins33. Junior ADE, Silva AF, Rosa FC, Monteiro SG, Figueiredo PMS, Monteiro CA. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida. Rev Soc Bras Med Trop. 2011;44(3):334-8..

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

The development of the biofilm has been described as an important virulence factor for Candida1414. Araujo D, Henriques M, Silva S. Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 2017;25(1):62-75.. In this context, the development of biofilm by C. parapsilosis complex has gained considerable attention because isolates from these species have been observed in an extensive variety of biotic and abiotic surfaces1414. Araujo D, Henriques M, Silva S. Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 2017;25(1):62-75.. 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 C. parapsilosis sensu stricto. Furthermore, C. parapsilosis ATCC 22019 and C. albicans 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 stricto; CM: Candida metapsilosis; ATCC 22019: Candida parapsilosis; ATCC 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 C. parapsilosis complex isolates from hospital environment origins because they demonstrate the maintenance of important virulence factors when these yeast species leave host tissues1515. Ferreira AM, Barcelos LS, Rigotti MA, Andrade D, Andreotti JT, Almeida MG. Areas of hospital environment: A possible underestimated microbes reservoir? - Integrative Review. J Nurs. 2013;7(5):4171-82., which may represent a potential risk for reinfection of hospitalized patients. Thus, our results contribute to the characterization of the clinical risk posed by C. parapsilosis sensu stricto and C. metapsilosis 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 C. parapsilosis complex occurring in the nosocomial environment.

Acknowledgements

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

REFERENCES

  • 1
    Suleyman G, Alangaden GJ. Nosocomia fungal infections: epidemiology, infection control, and prevention. Infect Dis Clin North Am. 2016;30(4):1023-52.
  • 2
    Sydnor ERM, Perl TM. Hospital epidemiology and infection control in acute-care setting. Clin Microbiol Rev. 2011;24(1):141-73.
  • 3
    Junior ADE, Silva AF, Rosa FC, Monteiro SG, Figueiredo PMS, Monteiro CA. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida Rev Soc Bras Med Trop. 2011;44(3):334-8.
  • 4
    Pulcrano G, Panellis D, Domenico G, Rossadno F, Catarina MR. Ambroxol influences voriconazole resistance of Candida parapsilosis biofilm. FEMS Yeast Res. 2012;12(4):430-8.
  • 5
    Tsui C, Kong EF, Jabra-Rizk MA. Pathogenesis of Candida albicans biofilm. FEMS Pathog Dis. 2016;74(4):1-13.
  • 6
    Valente P, Gouveia FC, Lemos GA, Pimentel D, Elsas JD, Mendonça-Hagler LC, et al. PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures. FEMS Microbiol Lett. 1996;137(2):253-6.
  • 7
    Garcia-Effron G, Canton E, Pemán J, Dilger A, Romá E, Perlin DS. Assessment of two new molecular methods for identification of Candida parapsilosis sensu lato species. J Clin Microbiol. 2011;49(9):3257-61.
  • 8
    Price MF. Plate method for detection of phospholipase activity in Candida albicans Sabouraudia. 1982;20(1):7-14.
  • 9
    Aoki S, Ito-Kuma S. Comparative pathogenicity of a wild-type stain and respiratory mutants of Candida albicans in mice. Zentralbl Bakteriol. 1990;273(3):332-43.
  • 10
    Laffey SF, Butler G. Phenotype switching affects biofilm formation by Candida parapsilosis Microbiology. 2005;151(1):1073-81.
  • 11
    Abi-chacra EA, Souza LOP, Cruz LP, Braga-Silva LA, Gonçalves DS, Sodré CL, et al. Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex. FEMS Yeast Res . 2013;13(8):831-48.
  • 12
    Ziccardi M, Souza LOP, Gandra RM, Galdino ACM, Baptista ARS, Nunes APF, et al. Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes. Int J Med Microbiol. 2015;305(8):848-59.
  • 13
    Silva BV, Silva LB, Oliveira DB, Silva PR, Ferreira-Paim K, Andrade-Silva LE, et al. Species distribution, virulence factors, and antifungal susceptibility among Candida parapsilosis complex isolates recovered from clinical specimens. Mycopathologia. 2015;180(5):333-43.
  • 14
    Araujo D, Henriques M, Silva S. Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 2017;25(1):62-75.
  • 15
    Ferreira AM, Barcelos LS, Rigotti MA, Andrade D, Andreotti JT, Almeida MG. Areas of hospital environment: A possible underestimated microbes reservoir? - Integrative Review. J Nurs. 2013;7(5):4171-82.
  • 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).

Publication Dates

  • Publication in this collection
    Jul-Aug 2017

History

  • Received
    05 Apr 2017
  • Accepted
    20 June 2017
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