Dynamic Monitoring And Initial Screening Of The Related Genes Of The Persisters Emerging In The Candida Albicans Biofilm

ObjectiveCandida albicans (albicans Saccharomyces) is an opportunistic pathogen whose biofilm shows high antibiotic resistance. Biofilm is the most common mode of microorganism gowth in nature and its mechanism of resistance is very complicated. The possible mechanisms include:physical barrier function of the extracellular matrix in the biofilm hinder the drug’s penetration, the retardation of biofilm strains growth led to declined sensitivity to drugs, resistant mutant strains, but these can not fully explain biofilm resistance.K. lewis discovered a small number of strains which were not simply antibiotic-resistant mutants in the Pseudomonas aeruginosa biofim in2001, they were called "peresisters" due to its resistance to high drug concentration and its multidrug tolerance. In2006, Lafleur study group discovered that C. albicans biofilms also produce multidrug tolerance peresisters. However, unlike bacterial persisters, C. albicans peresisters were only observed in the biofilm, but not in planktonic stationary population. Much studies suggest that persisters may be largely responsible for the multidurg tolerance of the fungal biofilm and are closely connect with the chronic inflammation in clinical. In the study, making the dynamic monitoring of the persisters emerging in Candida albicans biofilm first, analysising its impact factors and characteristics, determining two states which have large changes on persisters numbers; Then screening genes differentially expressed between the two states by SSH, lays foundation for further use of researching the related genes of the persisters and opening its generation mechanism and related pathways.Materials and methods(1) Dynamic monitoring of the persisters emerging in Candida albicans biofilm and its analysisBuild Candida albicans biofilm model in vitro by individual yeast cells and hyphal compartments respectively, count up the number of progenitive fungal cells and persisters at different phases of the biofilm formation; In addition, observing the biofilm morphological change by confocal scanning laser microscopy.(2) Construction the cDNA subtractive library between the two states of Candida albicans, screening a portion of genes related to the persister’s emerging.Screene of the genes differentially expressed between the two states of Candida albicans by SSH (bidirectional hybridization), identify recombinant clonal expansion plasmids by PCR, select positive clones randomly and are sequenced for homology in the GenBank database.Results(1) The biofilms which formed by individual yeast cells and hyphal compartments at different phases has no significant difference both on the number of progenitive fungal cells and persisters. The number of progenitive fungal cells shows "S" growth curve until12hours; The persisters emergs within0.5hour and it’s number reach a steady state after2hours, meanwhile the biofilm is in the period of microcolony formation under the microscopy.(2) The subtractive library was successfully constructed. The EST fragments were identified and the average insert size were measured around300bp-600bp, the recombination rate is more than95%; The capacity of the forward and the reverse subtractive library is2800and2600respectively;200clones were seleccted respectively for sequenced, a total of388valid ESTs and238unigenes were obtioned, including53contigs and185singlets after clustering mosaic. Conclusion(1) The persisters emerging in candida albicans biofilm are closely connected with the attachment to a surface(within2hours of the biofilm formation)and has little association with the maturity of the biofilm and its dimorphism.(2) SSH is a useful technique with high sensitivity for the detection of differential genes expressed in differential status. Lays foundation for researching the mechanism of occurrence and development of the persisters emerging in the Candida albicans biofilm.