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MICROBIOLOGY REPORT  
Year : 2018  |  Volume : 10  |  Issue : 3  |  Page : 163-165
Evaluation of exoenzyme activities, biofilm formation, and co-hemolytic effect in clinical isolates of Candida parapsilosis species complex


1 Department of Parasitology and Mycology, Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

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Date of Web Publication8-Aug-2018
 

   Abstract 


Candida parapsilosis species complex is considered as important emerging pathogens and little is known about their pathogenicity factors and co-hemolytic activity with different bacteria species. The aim of this study was to determine in vitro exoenzyme activities, biofilm formation, and co-hemolytic effect of different bacteria species on clinical C. parapsilosis complex isolates. In total, 67 C. parapsilosis complex isolates consist of C. parapsilosis sensu stricto 63/67 and Candida orthopsilosis 4/67 were used in this study. To determine the hemolytic activity of these species, Sabouraud dextrose sheep blood agar was used. Evaluation of the CAMP-like phenomenon carried out in the presence of Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and Streptococcus agalactiae. Tube test method with ethylenediaminetetraacetic acid-rabbit plasma was used to determine coagulase activity, and biofilm formation was assessed by the tube method in assist of Sabouraud glucose broth (8%) medium. Fisher's exact tests were used for data statistical analysis. Sixty-six of 67 (98.5%) and 3/67 (4.5%) of the species showed hemolysin and coagulase activity, respectively. Fifty-five of 67 (82.1%) of species had ability for biofilm formation, and none of the samples exhibited co-hemolytic effect in the presence of four mentioned bacteria. No significant difference was found between the level of enzyme production and biofilm formation among the isolates.

Keywords: Biofilm, CAMP-like, Candida orthopsilosis, Candida parapsilosis, coagulase

How to cite this article:
Pakshir K, Ravandeh M, Khodadadi H, Motamedifar M, Zomorodian K, Alipour S. Evaluation of exoenzyme activities, biofilm formation, and co-hemolytic effect in clinical isolates of Candida parapsilosis species complex. J Global Infect Dis 2018;10:163-5

How to cite this URL:
Pakshir K, Ravandeh M, Khodadadi H, Motamedifar M, Zomorodian K, Alipour S. Evaluation of exoenzyme activities, biofilm formation, and co-hemolytic effect in clinical isolates of Candida parapsilosis species complex. J Global Infect Dis [serial online] 2018 [cited 2019 Aug 21];10:163-5. Available from: http://www.jgid.org/text.asp?2018/10/3/163/238710





   Introduction Top


Candida species have been recognized as normal flora of human skin. Although Candida albicans is the most frequently isolated fungal species from human infections, Candida parapsilosis has become the second or third most common cause of fungal infections in human.[1] Nowadays, these species have been reclassified as a C. parapsilosis species complex comprising three distinct species: C. parapsilosis, Candida orthopsilosis, and Candida metapsilosis, using different molecular methods.[2] Several factors, such as adherence, persistence, germ tube formation, phenotypic switching, interference with host defense systems, synergism with bacteria, and the production of hydrolases, have been proposed to be Candida spp. virulence factors.[3] The ability of C. albicans to form biofilms and adhere to host tissues is important pathogenesis factor. Biofilm formation can act as a reservoir of agents, allow coinfection with other pathogens, promote the persistence of infection, and increase mortality.[4] The role of exoenzymes as a virulence factor of C. albicans has been intensively investigated, but its contribution to the virulence of C. parapsilosis species complex remains uncertain.[5],[6] The co-hemolytic effect which was named the “CAMP reaction,” first described by Christie, Atkins, and Munch-Peterson in 1944. The cooperative (CAMP-like) lytic processes are the result of the interaction of at least two membrane-active agents of bacteria, with biological membranes. Streptococcus agalactiae (B group streptococci) produces a thermostable, extracellular, diffusible protein that acts synergistically with the Staphylococcus aureus b-lysine to produce a zone of enhanced lysis in ovine and bovine erythrocyte cultures.[7]

According to our knowledge, there are no data about co-hemolytic effect of bacteria species which are commonly part of normal skin flora or any association in mucocutaneous area with infected lesions caused by Candida infections. The aim of this study was to evaluate hemolysin and coagulase activities, biofilm formation, and determination of co-hemolytic effect (CAMP-like factor) of C. parapsilosis species complex with standard strain of S. aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and S. agalactiae.


   Microbiology Report Top


A subset of 67 clinical isolates including 63 C. parapsilosis sensu stricto and four C. orthopsilosis were included in this study. Hemolytic was evaluated with a sheep blood (7% v/v and 3% w/v glucose) plate assay[8] The presence of a distinctive translucent halo around the inoculum site indicated positive hemolytic activity. Coagulase activity was evaluated with standard tube test method using ethylenediaminetetraacetic acid-rabbit plasma.[9] The presence of a clot formation in tube tests expresses as positive coagulase test. Biofilm formation was evaluated by the tube method, visual detection (Sabouraud dextrose broth 8%).[10] The tubes were examined for the presence of an adherent visible film at the bottom of the tubes and the results were expressed as negative–, weak+, moderate++, and strong+++. Standard strains of bacteria including S. aureus (ATCC25923), S. saprophyticus (PTCC1440), S. epidermidis (PTCC1435), and S. agalactiae (PTCC1768) were used for the evaluation of co-hemolytic effects. After 2 days of incubation for assessing the hemolytic activity, a loop was used to streak each bacterium in straight lines across the plate at a distance of 10 mm from the edge of the border of yeast colony. A distinct arrowhead of hemolysis at the intersection of the tester strain and the Candida colony streaks considered as a positive result for CAMP-like reactions.[7] Chi-square and Fisher's exact tests were used for data statistical analysis. Out of 67clinical isolates, 66/67 (98.5%) presented hemolysin activity. Beta-hemolysin activity was detected in 52/67 (78.78%) isolates. Coagulase activity was detected in 3/4 (4.5%) isolates which all belongs to C. parapsilosis sensu stricto [Table 1]. There was no co-hemolytic effect between positive hemolytic isolates in association with bacteria species [Table 1] and [Figure 1]. A total of 55 (82.1%) of the 67 Candida strains were positive for biofilm production [Figure 2].
Table 1: Distribution profile of enzymatic activity, biofilm production and CAMP-like effect on clinical species of C. parapsilosis complex

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Figure 1: Hemolysin and co-hemolytic (CAMP-like) effect. (A), C. parapsilosis complex with Staphylococcus aureus (CAMP negative). (B -), C. albicans as control with Staphylococcus aureus (CAMP positive , a zone of half-moon shape hemolysis)

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Figure 2: Biofilm formation: (a) Negative (control), (b) Positive (weak+), (c) Positive (mediate++), and (d) positive (strong+++)

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   Conclusion Top


In general, our study reveals that secretion of hemolysin and biofilm-forming ability are two major factors that play role in pathogenesis of C. parapsilosis species complex and none of the bacteria species did used in this study had any role in cooperative (CAMP-like) hemolytic activities with these strains.

Acknowledgments

This study was extracted from an MS thesis by Mostafa Ravandeh and was funded by Deputy of Research and Technology of Shiraz University of Medical Sciences, Shiraz, Iran (Grant No. 11975).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Tavanti A, Davidson AD, Gow NA, Maiden MC, Odds FC. Candida orthopsilosis and Candida metapsilosis spp. Nov. To replace Candida parapsilosis groups II and III. J Clin Microbiol 2005;43:284-92.  Back to cited text no. 1
    
2.
Almirante B, Rodríguez D, Cuenca-Estrella M, Almela M, Sanchez F, Ayats J, et al. Epidemiology, risk factors, and prognosis of Candida parapsilosis bloodstream infections: Case-control population-based surveillance study of patients in Barcelona, Spain, from 2002 to 2003. J Clin Microbiol 2006;44:1681-5.  Back to cited text no. 2
    
3.
Yigit N, Aktas E, Dagistan S, Ayyildiz A. Investigating biofilm production, coagulase and hemolytic activity in Candida species isolated from denture stomatitis patients. Eurasian J Med 2011;43:27-32.  Back to cited text no. 3
    
4.
Mukhia RK, Urhekar AD. Biofilm production by various Candida species isolated from various clinical specimens. Int J Sci Res 2016;5:2388-92.  Back to cited text no. 4
    
5.
Treviño-Rangel Rde J, González JG, González GM. Aspartyl proteinase, phospholipase, esterase and hemolysin activities of clinical isolates of the Candida parapsilosis species complex. Med Mycol 2013;51:331-5.  Back to cited text no. 5
    
6.
Pakshir K, Zomorodian K, Karamitalab M, Jafari M, Taraz H, Ebrahimi H, et al. Phospholipase, esterase and hemolytic activities of Candida spp. Isolated from onychomycosis and oral lichen planus lesions. J Mycol Med 2013;23:113-8.  Back to cited text no. 6
    
7.
Döğen A, Gümral R, Īlkit M. Haemolytic and co-haemolytic (CAMP-like) activity in dermatophytes. Mycoses 2015;58:40-7.  Back to cited text no. 7
    
8.
Luo G, Samaranayake LP, Yau JY. Candida species exhibit differential in vitro hemolytic activities. J Clin Microbiol 2001;39:2971-4.  Back to cited text no. 8
    
9.
Rodrigues AG, Pina-Vaz C, Costa-de-Oliveira S, Tavares C. Expression of plasma coagulase among pathogenic Candida species. J Clin Microbiol 2003;41:5792-3.  Back to cited text no. 9
    
10.
Seker S, Ozenc E.In vitro biofilm activity of Candida species isolated from anatolian boffaloes with mastitis in Western Turkey. Veterinarski Arh 2011;81:723-30.  Back to cited text no. 10
    

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Correspondence Address:
Dr. Keyvan Pakshir
Department of Parasitology and Mycology, Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jgid.jgid_93_17

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