The Preliminary Study Of The Effect Of Hypoxia On Biofilm Formation By Cryptococcus Neoformans

Background and PurposeC. neoformans is a heterothallic, basidiomycetous fungus, which is a capsule containing conditional pathogens, widespread in nature. With the increased incidence of HIV infection and organ transplant patients, extensive use of broad spectrum antibiotics and immunosuppressive agents, widely applied of invasive medical device, the opportunistic infections caused bye C. neoformans increased year by year. Inhaled C. neoformans spread through the blood to reach the central nervous system and cause life-threatening meningoencephalitis with the lethality rate of over 90%. The virulence factor of C. neoformans include:capsule, melanin, urease, phospholipase and its ability to grow in the host temperature environment. In recent years,the formation of C. neoformans biofilm has also been identified as an important virulence factor, and gradually attracted people’s attention.A biofilm can be defined as a complex structured community of microorganisms, surrounded by an extracellular matrix of polysaccharides, adhered to each other at a surface or interface, nearly 65% of human infections associated with biofilm. In addition, each year more than 500,000 patients died from biofilm-associated infection. With the wide application of interior medical devices, fungal biofilm-related infections has caused concern among the people. It has been demonstrated that C. neoformans has the ability to colonize surfaces and form biofilms in a wide variety of substances, such as vascular shunts, peritoneal dialysis fistula, hip joints prosthesis and so on.These interior medical devices, can be used as a substrate so that the fungal can adhesion on it and grown into biofilm structure, the cells can from and cause severe fungemia or disseminated infection under certain conditions.These implants related infections are inherently difficult to resolve and may require long-term antifungal therapy and removal of the implant to control the latent or chronic infection. Thus, fungal biofilms become an increasingly important clinical and economic issues, to understand the role of biofilms in C. neoformans infection will promote clinical anti-infective therapy.C. neoformans is an strictly obligate aerobic pathogenic yeast, it’s pathogenic process must face the different oxygen concentration changes in host environment. Domestic and foreign research mostly concentrated in the hypoxic adaptation mechanism of C. neoformans, but the effect and mechanism of hypoxic to its main virulence factor is unclear. We found that the important pathogenic phenotypes of C. neoformans changes under hypoxic conditions in the previous study, as the formation of capsule and melanin decreased, the activity of urease and phospholipids decreased, so C.neoformans in the host enters a latent or chronic state, leading to the delayed healing. This study further explored the impact of hypoxia on C. neoformans biofilm, an important virulence factor, in order to investigate the influence of hypoxia in the mechanism of C. neoformans latent or chronic infection.MethodsIn this study, low oxygen content (1%) culture conditions was build by hypoxic incubator, and the atmospheric conditions are normal oxygen content (21%) culture conditions, study with the standard strain H99 strain of C. neoformans (serotype A, mating type a), established the biofilm model in vitro using DMEM medium cultured in 24-well 96-well plates. Research the difference of biofilm cultured in 1% an 21% oxygen concentration at the 2h,4h,8h,12h,24h,48h and 72 h time points by optical microscope, scanning laser confocal microscope and MTT method to observe the biofilm thickness, morphology, growth kinetics and other indicators.ResultsSuccessful construction of C. neoformans biolflim vitro model in both hypoxia and normoxia condition was done. The progress to form the biofilm in the two condition is similarily, they experienced the adhesion, aggregation, micro-colony formation and biofilm maturation process, and ultimately formated the mature biofilms about 16μm thick. The cell density and growth activity of the biofilm increased with incubation time, and gradually stabilized after a certain maturity. At each time point, the biofilm cell density and growth activity under 1% oxygen concentration is relatively large, compared with 21% oxygen concentration.ConclusionC. neoformans can form biofilm in hypoxia. The ability of C. neoformans to form biofilm in different oxygen concentration, hypoxic conditions may promote the formation of C. neoformans biofilm.