Abstract: Effect of lactoferrin and iron have been studied on the multiplication and pseudohyphae production by three pathogenic Candida species viz., C. albicans, C. krusei and C. tropicalis. Results showed that lactoferrin showed significant antifungal effect on the three species tested, while the addition of iron enhance the multiplication of Candida species.
INTRODUCTION
Lactoferrin belongs to the transferrin family of iron binding protein and is a glycoprotein. Its not only found in the human milk but also in most epithelial surface secretions including tears, nasogastric, saliva and bronchial (Brock, 2002). Recently much attention has been given to lactoferrin because of following significant characteristics like, antibacterial, antifungal, anti-viral, antioxidant, immunomodulatory and to acts synergistally with lysozyme to potentiate the activity of proteins. Lactoferrin bind two molecules of iron with very high affinity thus making iron unavailable to pathogen which is an essential element for bacterial and fungal pathogen to survive and multiplication inside the host (Lupetil et al., 2000; Humphrey et al., 2002; Brissot et al., 2000; Di Mario et al., 2003; Weiss, 2002).
During infection, transferrin level increases and iron saturation decreases to allow increased availability of iron binding sites, a phenomenon known as hypoferremic response (Weiss, 2002).
Candida albicans and C. glabrata which causes yeast infections in vagina could be inhibited by activated lactoferrin and to blok yeast adhesion to the vaginal epithelial monolayer (Naidu et al., 2004). Iron chelators like gallic acid and salicylhydroxamate enhance the growth of Candida albicans in vitro (Weiss, 2002; Rehmani et al., 2004; Abe et al., 1990).
Fungicidal effects of lactoferrin have largely been done on Candida albicans because of its high pathogenicity (Viejo Diaz et al., 2004; Yamaguchi and Takakura, 2004; Anil and Samaranayke, 2002). There are very few reports about the antifungal effect of lactoferrin on other Candida species like C. krusei and Candida tropicalis (Nikawa et al., 1993; Xu et al., 1999).
In the presence of iron Candida species showed resistant to antifungal drugs and thus becoming a big challenge to control candidal infections (Bullen et al., 2006; Heyman et al., 2002; Howard, 1999).
Infections caused by Candida albicans remains considered as one of the most pathogenic human and animal Candida species but during recent years other species like C. krusei and C. tropicalis also emerged as a major pathogen of human and animals including all sites (Ruth et al., 2003; Pfaller et al., 2000; Rex et al., 2000).
The aim of present research was to study the effect of lactoferrin, lactoferrin free milk and added lactoferrin with iron on the growth of human pathogenic Candida albicans, C. krusei and C. tropicalis.
MATERIALS AND METHODS
This study was conducted at Department of Biology, College of Science, King Faisal University, Al-Hassa, Saudi Arabia during the period between June 2006-July 2008 and Candida species were collected from different hospitals in Riyadh and Al-Hassa as described latter.
Lactoferrin-free human milk was prepared by removing lactoferrin by treatment with heparin-sepharose. (Davidson et al., 1994). Lactoferrin purchased from Sigma-Aldrich (St. Louis, MO) was then added to the milk to obtain 3x103 μg mL-1 concentration. Sterile distilled water and lactoferrin free milk served as control. Candida albicans. C. krusei and C. tropicalis were isolated from different sources like blood, urine, vagina, sputum and Bronchoalveolar lavage (BAL) were cultured and maintained on Sabouraud dextrose agar. A total number of 100 isolates of each Candida species were collected for experiment.
Stock solution of Candida species were prepared from 24 h old colonies on Sabouraud dextrose agar to get a concentration of 103 cells mL-1 of yeasts into a 100 mL culture tubes. One milliliter from stock solution was added to 9 mL of test medium viz., whole milk, lactoferrin free milk, added lactoferrin and added lactoferrin+iron medium, to get final concentration of yeast cells of 102 cells mL-1. Yeast cells were counted in a hemacytometer at 24 h intervals. The number of CFU were also counted by culturing on SDA medium.
Five sets of each yeast in triplicate were prepared for each type of medium and incubated at 37 °C for 24, 48 and 72 h. After each 24 h intervals cells were counted in the hemacytometer. The correlation between cell count and CFU was very high, results here reported are only for cell counts.
Added lactoferrin medium was prepared by adding 3000 μg of commercial lactoferrin per milliliter while 3x103 μg mL-1 of lactoferrin and 300 μg of ferrous sulphate were added to lactoferrin free milk to prepare lactoferrin + iron medium.
Candida species were isolated on Sabouraud dextrose agar and chromoagar medium (CHROMAGAR, Paris, France) and identified by Api Aux identification system (Biomerieux, France).
Candida species were collected from Riyadh Medical city, Central Hospital and various private hospitals in Riyadh and Al-Hassa region. These Candida species were isolated from Blood, Urine, Vagina, Sputum and Branchoalveolar Lavage (BAL). This project is not funded by any organization or a part of a research project.
RESULTS AND DISCUSSION
Almost similar type of increase in cell counts were shown by the candida Albicans, C. krusei and C. tropicalis (Table 1) after 24, 48 and 72 h of incubation. An insignificant decrease in cell counts was observed after 24, 48 and 72 h incubation period in lactoferrin free milk as compared to whole milk. The number of cell counts were significantly decreases in the case of added lactoferrin (3x103 μg mL-1) but trends reversed in the medium with added iron and lactoferrin. Even the cell counts were more than the cell counts in the whole milk. There were gradual increase observed in all the cases after each 24 h of incubation period. In the medium containing added lactoferrin a significant reduction of cell count was seen even after 24 h of incubation for example. Only 650 cells were seen after 24 h of incubation in added lactoferrin as compared to 3350 cell in lactoferrin free milk and 3500 cells counts in the whole milk in the case of Candida albicans. A similar trends were also observed in other two species. In the case of lactoferrin + iron medium the cell counts were highest and even more than the cell counts observed in the whole milk medium. The results here clearly shows that lactoferrin does effect the growth of Candida species.
This is in coincide with earlier reports that lactoferrin has antifungal properties and affect the prevalence of pathogen in the host by binding iron molecules and making them unavailable to pathogen (Brissot and Guyadar, 2000; Di Mario et al., 2003). When iron added to the medium, even in the presence of lactoferrin increases the number of cell counts significantly. This is due to the availability of free iron in the medium which is an essential element for the multiplication of pathogen (Rehmani et al., 2004; Abe et al., 1990).
Results showed when lactoferrin added to the milk, no pseudohyphae were produced
by any of the Candida species even after 72 h. of incubation but when
iron added with lactoferrin, Candida species produced pseudohyphae although
after 72 h of incubation as compared to whole milk where these Candida species
produced pseudohyphae after 48 h of incubation (Table 2).
Production of pseudohyphae of Candida species helps in faster spreading
of Candidal infection in vivo, but lactoferrin not only checks
the multiplication of yeast cells but also stopped the production of pseudohyphae
by Candida species (Lupetil et al., 2000;
Humprey et al., 2002; Brissot and Guyader, 2000).
| Table 1: | Effect of whole milk, lactoferrin free milk, added lactoferrin and added lactoferrin with iron on the cell concentrations of Candida species (initial conc. 1000 cells mL-1) |
| ± SD from the mean | |
| Table 2: | Production of pseudohyphae in the whole milk, lactoferrin free milk, added lactoferrin added lactoferrin with iron by Candida species |
| +: Present, -: Absent | |
| Table 3: | Source of isolation of Candida species |
A final total number of 100 isolated of each Candida species were collected from different types of specimen (Table 3). There were no significant differences were found as for as reaction to lactoferrin is concerned. All these isolates behaved similarly (Brock, 2002; Naidu et al., 2004; Nikawa et al., 1993; Xu et al., 1999).
In conclusion lactoferrin could be used safely as an antibiotics to checks bacterial and fungal infection (Lupetil et al., 2000; Humphrey et al., 2002; Di Mario et al., 2003).
ACKNOWLEDGMENTS
Author is very thankful to Riyadh Medical City, Central Hospital Riyadh and Private Hospitals in Riyadh and Al-Hassa region for providing the Candida species.