Author: Mahmoudabadi, Ali Zarei; Zarrin, Majid; Miry, Sanaz
Date published: October 1, 2010
Candida albicans is the most virulent among the Candida species, and can cause several forms of candidiasis in debilitated human. Several factors were described as virulent for pathogenesis of C. albicans. The secreted aspartyl proteinases, phospholipases, germ tube formation, adherence to host tissues and phenotypic switching have been more discussed in literatures [1-3]. Both hydrolytic enzymes can cause cellular membranes destruction. Four types of phospholipases have been reported in C albicans including phospholipase A, B, C, and D. The extracellular phospholipases of C albicans have a significant role in the pathogenesis of infections and invasion to mucosal epithelia [1,2]. In addition, several studies have shown that clinical isolates of C albicans have higher levels of extracellular phospholipase activity .
Vidotto et al  believe that the correlation between phospholipase activity and high germ tube formation can facilitate the mucosa penetration. Vulvovaginal candidosis is an opportunistic yeast infection that presents as a spectrum of very different clinical variants (e.g. acute candidosis, recurrent and chronic candidosis). Disease is caused by an overgrowth or infection of the vagina by a Candida species. However, C albicans represent more than 80% of isolates from clinical infection . Candi duria is a common nosocomial infection, which involves the urinary tract system as asymptomatic candiduria. The disease is most commonly caused by C albicans .
Pinto et al  compared the level of production of phospholipase in C albicans isolated from the cases of infection and commensals. They observed the higherlevel phospholipase activity in C albicans with infection sources. In a study conducted by Farina et al , 14.5% of vaginal strains of C albicans were without phospholipase activity, whereas 40.6% of isolates have large production of phospholipase. Basu et al.  compared phospholipase activity of C albicans isolated from vagina and urine samples. They found that 66.6% of vaginal isolates have phospholipase activity (Pz value) 0.82-0.86 and 60% urinary isolates have Pz value 0.84-0.89. Although there are several papers in Candida and candidiasis in Iran [4,5,9,10] there is little knowledge about their phospholipase activities.
The present study focused on extracellular phospholipase activities in different isolates of C albicans isolated from vagina and urine samples from Ahvaz, Iran. In addition, phospholipase activities were compared in C albicans isolated from two different sources.
Materials and methods
Isolates and identification
Seventy-two C. albicans strains isolated from patients with vulvovaginal candidiasis and 28 isolates from urine samples from hospitalized patients were studied. The strains were stored in medical mycology laboratory, Ahvaz Jundishapur University of medical sciences. All strains were transferred onto fresh Sabouraud's dextrose agar, SDA (Merck Germany) plates and incubated at 37°C for 24h. Then isolates were re-identified by germ tube test, production chlamydoconidia on Corn meal agar (Difco, USA) and green colour colonies on CHROMagar Candida (CHROMagar Candida, France) .
Each isolate was inoculated onto test tubes contained 10ml SDA and incubated at 37°C for 18h shaking. Each tube centrifuged for 30min and the sediment was washed by PBS for 30min. Supernant was removed and sediment was re-suspended in sterile distilled water. A suspension with turbidity according to the McFarland standard #2 of yeast cells was prepared in distilled water [H].
Phospholipase activity assays
Phospholipase activity assays were performed according to Price et al. [H]. The test medium contained 65g SDA, 58.4g NaCl and 5.5g CaCb. Medium was dissolved in 980ml distilled water and sterilized at 121 0C for 12min. Egg yolk was centrifuged at 5000g for 30 minutes. The supernatant was removed and added to cooled medium (45-500C) (2%), mixed and dispensed in plates. An aliquot (10µ1) of the yeasts suspension was inoculated onto test medium which was then incubated at 37°C for four days. Colony diameter and colony diameter plus precipitation zone were measured for each isolate (Fig. 1). Calculation of the zone of phospholipase activity was performed according to Price et al method [H].
Five classes were described for phospholipase activity including; Pz value = 1 means that the test strain is negative for phospholipase, while a value of Pz O.900.99 = weak phospholipase activity (+), 0.80-0.89 = poor phospholipase activity (++); 0.70-0.79= moderate phospholipase activity (+++) and <0.70 = large phospholipase activity (++++).
Results and discussion
The present study aimed at determining in vitro phospholipase activity in several isolates of C albicans from urogenital samples. As shown in table one, all tested isolates (100 isolates) were found to be positive for the production of extracellular phospholipase. C albicans is the common Candida species that is most frequently associated with the mucosal colonization, oropharyngeal, esophageal, vulvovaginal or urinary system candidiasis among debilitated patients [3,12]. Several studies show that phospholipase [2,8,13], proteinases [3,8], and germ tube production  have been implicated in the invasion and destruction of host tissue as pathogenic factors for C albicans.
Several studies have shown that phospholipase activity is observed only in C albicans strains . Pnito et al.  reported 99.4% of isolates of C albicans with phospholipase activity. However, other researchers described that other Candida species such as C glabrata, C guilliermondii, C tropicalis, C fornata and C inconspicua secreted smaller amounts of phospholipase [7,14,15]. In the present study, 100% isolates of C albicans had detectable phospholipase activity (Table 1). Basu et al.  documented that 48.7% clinical isolates of C albicans and only two isolates from normal healthy people were positive for phospholipase activity. In another study conducted by Samaranayake et al.  80% isolates of C albicans recovered from HIV patients were phospholipase-positive. Reports from different countries show that there are more different phospholipase activities in different regions. Borst and Fluit  believed that virulence factors could be associated with geographical region and infection type.
As shown in table 1, 100%) of urine and 94.43%) of vaginalis isolates were produced the higher amount of phospholipase. In our study phospholipase activity with higher Pz values seems to be more commonly present in vaginal isolates (84.7%o isolates with Pz value <0.70) compared with 75%o in urine isolates. In the present study, we tested C albicans isolated from vaginal candidiasis with acute, chronic or relapse disease. Probably variable levels of phospholipase activity in our study are related to source of C albicans. Oksuz et al.  detected phospholipase activity in 53.08%o of tested C albicans that originated from healthy adults. In addition this activity was found more frequently in oral yeasts followed by fecal yeasts. Borst and Fluit  also found difference phospholipase activity between samples originated from urine, blood and wound.
There is no previous study of phospholipase activity of Iranian isolates of Candida species. In the present study, 100% clinical isolates of C albicans from vaginitis and urine samples demonstrated phospholipase activity.
This study is based on an MD thesis (Sanaz Miry) which was supported by a grant (No. U-88187) from Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
1) da Costa KRC, Ferre ira JC, Komesu MC, Candido RC. Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity. Mycopathologia. 2009; 167: 73-9.
2) Samaranayake YH, Dassanayake RS, Jayatilake JAMS, et al. Phospholipase B enzyme expression is not associated with other virulence attributes in Candida albicans isolates from patients with human immunodeficiency virus infection. J Med Microbiol. 2005; 54: 583-93.
3) Vidotto V, Yumi Koga-Ito C, Milano R, Fianchino B, Pontón J. Correlation between germ tube production, phospholipase activity and serotype distribution in Candida albicans. Rev Iberoam Mi col. 1999; 16: 208-10.
4) Shahbazian N, Najafian M, Raj ab Zadeh AR. Comparison of efficacy of clotrimazole with fluconazol in vulvovaginal candidiasis. Sci Med J Ahwaz Uni Med Sci. 2006; 49: 519-22.
5) Zarei Mahmoudabadi A, Keradmand AR, Enayatollahi N. Frequency of candiduria in inpatients and outpatients in department of urology, Golestan hospital, Ahvaz, Iran. IJKD. 2009; 3: 114-5.
6) Pinto E, Ribeiro IC, Ferreira NJ, Fortes CE, Fonseca PA, Figueiral MH. Correlation between enzyme production, germ tube formation and susceptibility to fluconazole in Candida species isolated from patients with denture-related stomatitis and control individuals. J Oral Pathol Med. 2008; 37: 587-92.
7) Farina C, Saleri N, Lombart JP, et al. Epidemiological phenotypic characteristics of vaginal yeasts at the Comoros. Mycoses. 2009;52:458-61.
8) Basu S, Gugnani HC, Joshi S, Gupta N. Distribution of Candida species in different clinical sources in Delhi, India, and proteinase and phospholipase activity of Candida albicans isolates. Rev Iberoam Micol. 2003: 20: 137-40.
9) Jamilian M, Mashadi E, Sarmadi F, et al. Frequency of vulvovaginal candidiasis species in nonpregnant 15-50 years old women in spring 2005 in Arak. J Arak Uni Med Sci. 2007; 2: 7-14.
10) Jafari Nodoushan AA, Dehghani M, Mirbagheri SM. In vitro antifungal effect of aqueous Garlic (Allium sativum) extract and its combination with fluconazole against five common clinical Candida isolated from candidiasis lesions. J Kerman Uni Med Sci. 2007; 3: 153-62.
11) Price MF, Wilkinson ID, Gentry LO. Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia. 1982; 20: 7-14.
12)A1-Abeid HM, Abu-Elteen KH, Elkarmi AZ, Hamad MA. Isolation and characterization of Candida spp. in Jordanian cancer patients: prevalence, pathogenic determinants, and antifungal sensitivity. Jpn J Infect Dis. 2004; 57: 27984.
13) Ma L, Xie LX, Dong XG, Shi WY. Virulence of extracellular phospholipase B of Candida albicans in rabbit experimental keratomycosis. Zhonghua Yan Ke Za Zhi. 2008;44:237-43.
14) Mohan das V, Ballai M. Proteinase and phospholipase activity as virulence factors in Candida species isolated from blood. Rev Iberoam Micol. 2008; 25: 208-10.
15) Oksuz S, Sahin I, Yildirim M, et al. Phospholipase and proteinase activities in different Candida species isolated from anatomically distinct sites of healthy adults. Jpn J Infect Dis. 2007; 60: 280-3.
16) Borst A, Fluit AC. High levels of hydrolytic enzymes secreted by Candida albicans isolates involved in respiratory Infections. J MedMicrobiol. 2003: 52: 971-4.
Ali Zarei Mahmoudabadi*1,2, Majid Zarrin2, Sanaz Miry2
1 Infectious and Tropical Diseases Research Centre, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2 Department of Medical My copar asitology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
How to cite this article:
Zarei Mahmoudabadi A, Zarrin M, Miiy S. Phospholipase activity of Candida albicans isolated from vagina and urine samples. Jundishapur J Microbiol. 2010; 3(4): 169-73.
Received: May 2010 Accepted: June 2010
* Address for correspondence:
Ali Zarei Mahmoudabadi, Infectious Diseases and Tropical Research Centre and Department of Medical My copar asitology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Tel: +98611 3330074; Fax: +98611 3332036; Email: zarei40(a)yhotmail.com