| With the widely application of the biomedical material in clinical, biomaterial centered infection has become a very common nosocomial infection. Especially for the mixed infection between the long-term endotracheal intubation patients implanted with biological material with the microbe, characterized of refractory, repeatability and resistance, which will result in clinical infection and difficult healing. Previous study has reported that Staphylococcus epidermidis-Candida albicans mixed species are the common clinical conditional pathogens infected the implant biological material. However, there is little known about the research of mixed infection of implanted biological material. In this present study, the in vitro model of the endotracheal tube surface mixed-biofilm will be formed to investigate the mixed-biofilm infection due to the implanted biological material. The difference between the endotracheal tube surface mixed microbial biofilms and single microbial biofilms will be investigated, and the results will offer a new idea for the prevention of mixed infection by implanted biological material. The study will explore the function of Farnesol and Tyrosol to the endotracheal tube surface Staphylococcus epidermidis-Candida albicans mixed-species biofilm, and the findings will offer a beneficial insight into the clinical treatment to biological materials related to mixed microbial infection.Part I Study on the formation of endotracheal tube surface bacteria and fungi mixed-species biofilm and related gene expression[Objective]To copy the in vitro model of the endotracheal tube surface mixed-species biofilm, and to investigate the difference of growth characters, in vitro growth dynamics, ultrastructure and gene expression between Staphylococcus epidermidis-Candida albicans mixed-species biofilm and single microbial biofilm.[Methods]The polyvinyl chloride (PVC) endotracheal tube was chosen as the experimental material. The experiments were divided into five groups:single culture of Staphylococcus epidermidis ATCC35984 (biofilm formation-positive S. epidermidis, Group SE+), Staphylococcus epidermidis ATCC12228 (biofilm formation-negative S. epidermidis, Group SE-), Candida albicans ATCC10231 (Group CA), co-culture of Staphylococcus epidermidis ATCC35984 and Candida albicans ATCC10231 (Group MIX+), co-culture of Staphylococcus epidermidis ATCC 12228 and Candida albicans ATCC 10231 (Group MIX") to build in vitro biofilm model. To copy the single and mixed microbes in the endotracheal tube surface in vitro biofilm model, the ability of biofilm formation for each group was detected by using crystal violet semi-quantitative adherence assay at 2,4,6,8,12,24,48 and 72 h post incubation. XTT assay was performed to determine the growth kinetics in the same time. Confocal Laser Scanning Microscope was employed to observe the in vitro growing status of biofilm. Scanning electron microscopy was used to observe the ultrastructure of the biofilms 24 and 72 h post incubation. The gene expressions of icaA, aap, fbe, als3 and hwpl were analyzed by real time PCR.[Results]①. Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 h post incubation in Group SE+ and Group MIX+; the biofilm in Group MIX1+ was thicker than that in Group SE+ after co-cultured for 72 h. There was an equilibrium growth of biofilm reaching at the 24 h post incubation and the increase thickness was continuing as the time went on. There was a significant difference between two groups at any other time (P<0.05) except at 72 h post incubation. In Group CA, the biofilm started to grow at 12 h post incubation, but the biofilm was significantly thinner than that in the Group MIX+ in all the time points (P<0.05). In Group MIX- and Group SE-, there was no biofilms to be formed.②. XTT assay showed that the OD value in Group MIX+ was lower than that in Group CA at 2 and 4 h post incubation, but there is no significant difference (P>0.05); the OD value of Group MIX+ was significantly higher than that of Group CA after 6 h post incubation (P<0.05). The growth speed in Group MIX+ was lower than that in the Group SE+ before 48 h post incubation, and it was faster at 48,72 h post incubation; there was no significant difference in the growth speed between Group MIX+ and Group SE+ in any other time points (P>0.05) except 12 h post incubation. From 2 to 72 h post incubation, the OD value in Group MIX- was higher than that in Group SE- at 2 and 48 h post incubation, but there is no significant difference (P>0.05) except at 6 and 8 h post incubation. The OD value in Group MIX- was lower than that in Group CA at 2 and 4 h post incubation significant difference was shown(P<0.05) except at 2,4 and 72 h post incubation.③. Confocal laser scanning microscopy showed that there were almost living microorganism in biofilm of Group MIX+ by using the LIVE/DEAD bacterium staining methods 24 h post incubation. There were predominant living bacterium with some dead bacterium 48 h post incubation, while there is an increase of dead bacterium in the biofilm 72 h post incubation. There were a few of dead bacterium 72 h post incubation whereas almost living bacterium at 24 and 48 h post incubation in biofilm of Group MIX-. In Group MIX+ good growth state and a few pseudohyphae were observed in the mixed-species biofilm 24 h post incubation by using fluorescence in situhybridzation, and there were much more pseudohyphae 72 h post incubation. In Group MIX- Staphylococcus epidermidis were growing randomly 24 h post incubation, while Candida albicans clusters were observed in some places. There is an increase growth of Staphylococcus epidermidis 72 h post incubation whereas the Candida albicans generated the pseudohyphae in some places.④. Compared three-dimensional structures formed from Group MIX+ with Group SE+ and Group CA at 24 h post incubation, a mixed growth of Staphylococcus epidermidis and Candida albicans was observed with formation of the spore and pseudohyphae of Candida albicans, which suggested that the primary three-dimensional structure was formed. Spores and pseudohyphae of C. albicans were observed in Group MIX- and Staphylococcus epidermidis was growing randomly tiled, three-dimensional structures were not formed in Group MIX-⑤. The real-time PCR results showed the expressions of fbe, icaA and aap genes in Group MIX+ were up-regulated 1.93,1.52 and 1.46 fold respectively 72 h post incubation compared with the Group SE+, als3 and hwpl genes in Group MIX+ were up-regulated 4.22 and 2.56 fold compared with the Group CA. Aap and fbe genes in Group MIX- were down-regulated 0.25 and 0.47 fold compared to Group SE-, while als3 gene was up-regulated 2.21 fold and hwpl gene was down-regulated 0.57 fold in Group MIX- compared with Group CA. [Conclusions]1. Compact and highly organized multicellular group structure, or matured endotracheal tube surface mixed-species biofilm could be formed by the mixed growth of biofilm formation-positive S. epidermidis, and C. albicans at 24 h post incubation.2. Mixed-species biofilm was formed in Group MIX+, and was thicker and more complex than that of single species biofilm of C. albicans, which might be one of the reasons for difficulties for healing the clinical biological materials mixed microbial infection. Genes of icaA、aap、fbe in Group MIX+ were up-regulated compared with Group SE+, while genes of als3 and hwpl were also up-regulated compared with Group CA during the process of mixed-species biofilm formation, which might be related to the complexity of the mixed-species biofilm.3. There was no thicker mixed-species biofilm formed in the co-culture of biofilm formation-negative S. epidermidis and C. albicans (Group MIX-) compared with single species biofilm. Down-regulated gene expression of aap, fbe and hwpl suggested that the biofilm formation-negative S. epidermidis can not induce the formation of mixed-species biofilm via the co-culture with C. albicans.Part Ⅱ The mechanism and effect of fungal quorum-sensing molecules on the formation of Staphylococcus epidermidis-Candida albicans mixed-species biofilms[Objective]To explore the mechanism and effect of fungal quorum-sensing molecules, including Farnesol and Tyrosol on the formation of the endotracheal tube surface Staphylococcus epidermidis-Candida albicans mixed species biofilm.[Methods]The polyvinyl chloride (PVC) endotracheal tube was chosen as the experimental material. Staphylococcus epidermidis ATCC35984 (biofilm formation-positive S. epidermidis) and Candida albicans ATCC10231 were co-cultured and divided into three groups. Species of biofilms were treated with quorum-sensing molecule Farnesol (Group DF), with quorum-sensing molecule Tyrosol (Group DT) and with double-distillation h2O (Group DD). Biofilm mass was semi-quantified by crystal violet semi-quantitative adherence assay at 2,4,6,8,12,24,48,72 and 96 h post incubation. XTT assay was performed to determine the growth kinetics at the same time. Confocal laser scanning microscopy was used to observe biofilm growing status. Scanning electron microscopy was employed to observe the ultrastructure of the biofilms at 24 and 72 h post incubation. The gene expressions of icaA, aap, fbe, als3, hwp1 and efg1 were analyzed by real time PCR.[Results]①. Results of crystal violet semi-quantitative adherence assay showed that there was a difference of ability to biofilm formation among Group DF, Group DT and Group DD through the whole culture cycle (from 2 to 96 h post incubation). Additionally, there was a difference of ability to biofilm formation between Group DD and Group DF/DT. The biofilms in Group DF is thiner than that in Group DD from 2 to 96 h except at 4 and 72 h post incubation, and there was no significant difference between two groups at the other time (P>0.05) except at 12 and 24 h post incubation (P<0.05). From 2 to 96 h post incubation, the biofilms in Group DT is thicker than that in Group DD, and there was no significant difference between two groups at the other time(P>0.05) except at 2,4 and 6 h (P<0.05) post incubation.②. Growth kinetics of the biofilms was detected by using XTT assay, and the results showed there was no difference of among the Group DF, Group DT and Group DD through the whole culture cycle (from 2 to 96 h post incubation). The growth kinetics in Group DF was smaller than that in the Group DD except at 72 h post incubation through the whole culture cycle. There was a significant difference (P< 0.05) between Group DD and Group DF at 4 and 48 h post incubation. From 2 to 96 h, the growth kinetics in Group DT was greater than that in the Group DD at 4,12 and 72 h post incubation, and there was a significant difference between Group DD and Group DT at 12 h post incubation (P<0.05).③. Microbe growth status in three group biofilm were observed by using confocal laser scanning microscopy and LIVE/DEAD bacterium staining methods, the results showed that living microbes were predominate at 24 h post incubation, while dead microbes were increasing at 48 h post incubation and then shifting into the dead microbes were predominate at 72 h post incubation.④. The scanning electron microscopy results showed that spores and pseudohyphae were growing together in S. epidermidis-C. albicans co-cultured Group DD at 24 h post incubation which suggested that mixed-species biofilm with 3-D structure was formed. More thicker and longer pseudohyphae gowth in Group DD at 72 h post incubation. There was no pseudohyphae growing at 24 h post incubation, and a little of spores and pseudohyphae were observed in S. epidermidis-C. albicans co-cultured Group DF at 72 h post incubation. S. epidermidis embed in array of mesh stereochemical structure formed in Group DT by the spores and pseudohyphae 24 h post incubation. S. epidermidis was growing adhere on the pseudohyphae and forming a complicated three-dimensional structure biofilm. The biofilms were more complicated and more mature at 72 h post incubation in group DT.⑤. The results of real-time PCR showed that the gene expressions of icaA, aap and fbe in Group DF were down-regulated 0.83 fold,0.59 fold and 0.75 fold respectively at 6 hours post incubation compared with the Group DD. Aap gene was down-regulated 0.67 fold and icaA, fbe genes were up-regulated 2.36 fold and 1.99 fold at 24 hours post incubation compared with Group DD. Als3,hwp1 and efg1 genes in Group DF were down-regulated 0.41 fold,0.43 fold and 0.27 fold at 6 hours post incubation compare to Group DD. IcaA, aap and fbe genes in Group DT were up-regulated 1.61 fold,1.05 fold and 1.86 fold respectively at 6 hours post incubation compared with the Group DD, and that up-regulated 25.59 fold,17.65 fold and 21.46 fold respectively at 24 hours post incubation. Als3, hwp1 and efg1 genes were down-regulated in Group DT 0.27 fold,0.26 fold and 0.30 fold at 6 hours post incubation compare to Group DD, and that decreased 0.79 fold,0.59 fold and 0.65 fold at 24 hours post incubation.[Conclusions]1. Quorum-sensing molecule Farnesol can inhibit the formation of Staphylococcus epidermidis-Candida albicans mixed-species biofilm, which suggest that the Farnesol might become the target on the treatment of infected clinical mixed microbial biofilm. Farnesol can decrease the ability to biofilm formation, growth kinetics and inhibit the structure formation of biofilm via the effect of quorum-sensing system on the biofilm formation. Inhibition of Farnesol for the biofilms might be related to the early down-regulation of icaA, aap and fbe, and continuous down-regulation of als3, hwpland efg1.2. Quorum-sensing molecule Tyrosol can promote the formation of Staphylococcus epidermidis-Candida albicans mixed-species biofilm. Tyrosol can increase the ability to biofilm formation and growth kinetics of biofilm. It could be a novel idea for the management of mixed-microbial infection when the methods were found to inhibit or decrease the production of Tyrosol. Function of Tyrosol to the biofilms might be related to the combined function of up-regulation of icaA, aap and fbe, and down-regulation of als3, hwp1 and efg1. |
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