| Objectives:Candida albicans is the main opportunistic pathogen which is also the common member of oral microflora. In immunocompromised patients, C. albicans can cause life-threatening candidaemia and disseminated infections, and Candida spp. are now the fourth most common hospital-acquired infection. This fungus has the unique capacity to colonize the surfaces of mucosal tissues and prostheses, and the biofilms it formed are highly tolerant to antifungal agents, especially compared with planktonic ones. Lewis K. reported, C. albicans forms a small subpopulation (～1%) of persister cells that are completely tolerant to the currently used systemic antifungals. These live cells present in a biofilm killed by exposure to a high level of amphotericin B (AMB), which vital staining shows, and these cells can be sorted out from the bulk. Persisters exhibit multidrug tolerance, which is a significant feature of a biofilm infection.Persister cells are clinically relevant, and antimicrobial therapy selects for high-persister (hip) strains in vivo.131isolates of C. albicans from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day had been got, and their persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of AMB and plating for survivors. The persister levels of the isolates varied from0.2to9%, and all of the hip isolates had an AMB MIC that was the same as that for the wild type, indicating that these strains were drugtolerant rather than drug-resistant mutants. Biofilms of the majority of hip strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. Persisters may play a major role in the recalcitrance of chronic infections, and their drug tolerance may be a crucial component contributes to antimicrobial drug failure and relapsing infections. In order to test this possibility, we used a live-animal infection model. Caenorhabditis elegans, which is soil nematode, has been used widely during the past decade for the study of various pathogens. A large number of human pathogens, including Candida species can infect and kill this worm, which makes it an useful and simple model host. C. albicans colonizes the intestine of nematodes, and it forms biofilm and kills the worms through the formation of a complicated meshwork of filaments that penetrate through their cuticle, when they are transferred into a liquid medium environment. Given the above, C. elegans infection model has been used to evaluate the antifungal effectiveness of chemical compounds. In our work, we infected C. elegans with C. albicans isolates of different persister levels, treated them with AMB and compared the survival rate to illuminate the role that persisters play in the recalcitrance of chronic infections and the effect of persisters on antifungal therapy.MethodChoice of C. albicans and nematode strains:6hip strains and6low-persister (lop) strains were selected for this study. These hip strains form8～9%persister cells tolerant to AMB, and the level for lop strains is less than0.3%. Lab strain3153a was included, which forms about1% persister cells. The C. elegans strain glp-4;sek-1was used.Antifungal agent:amphotericin BSynchronization of C. elegans:Worms need to be incubated on NGM agar plates with E. coli, as the food provided to worms, for about6d at15℃to become gravid and give the maximum number of eggs. Eggs were isolated by washing the gravid worms off the plates using the M9buffer, vortexing the suspension plus bleach solution at high speed for4min. Released egg were centrifugated and washed three times in M9buffer, hatched overnight, and the L1-stage worms were plated onto lawns of E. coli on NGM agar media. Worms were synchronized and grown to sterile to L4-stage at25℃for3d. Establishment of C. elegans infection model:The synchronized L4-stage worms were washed and concentrated using M9buffer, put on BHI lawn of each C. albicans strain, and non-infected worms were used as the blank. All the plates were incubated at25℃for2h. Worms on every plate were centrifugated and washed three times, concentrated in M9buffer.15～20worms infected with each C. albicans specie in20μl M9buffer were dispensed per well of the96-well plate. The final volume was150μl per well by adding40%BHI in M9with AMB and the respective concentrations of AMB were0.5,1.0,2.0,4.0,8.0and16.0μg/ml for each strain group. Wells filled without any AMB were used as negative controls intra-group. Plates were incubated at25℃for5days, and the survival rate of worms in each well was counted by observing with microscope.ResultsThere were not notable differences between survival rates of infected worms without AMB in each group, which indicated that the virulence of these stains was similar without drug treatment. Compared with negative controls, survival rate of drug worms in each group increased. With the increase of drug concentration, the mortality rate of non-infected worms was U-trend, suggesting that the cytotoxicity of AMB was high. The survival rate of the infected worms increased with treatment concentration increasing, with the peak at the concentration of2～4μg/ml, which maybe the most appropriate drug concentration in treating the worms. In the same concentration, the increase for hip group was significantly lower than that for lop group, although that for worms infected with different strains in the same group are not identical. Increase and drug dose were nonlinearly related for the two group. With increasing drug concentration, survival rates of worms increased, as well as the deviation between hip group and lop group. Both of them increased to the maximum when concentration of AMB was2～4μg/ml. The results indicated that the effects of AMB for worms infected with lop C. albicans strains were much better than hip ones. Treatment with antifungal drugs could not get satisfactory efficacy for infection with hip strains.Conclusion C. elegans provides an alternative model for the study of persisters and antifungal pharmacodynamics. Persisters play a major role in the recalcitrance of chronic infections, and the drug tolerance of persisters may be a critical component responsible for antifungal drug failure and relapsing infections. Persister cells may serve as an important indicator of therapeutic failure for fungicidal agents and prognosis of chronicinfectious diseases. C. elegans infection models can be used to create high-throughput screen to identify chemical compounds with antifungal activity against persisters. |
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