The use of poloxamer hydrogels for the assessment of biofilm susceptibility towards biocide treatments
The use of poloxamer hydrogels for the assessment of biofilm susceptibility towards biocide treatments
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DOI：
10.1111/j.1365-2672.1998.tb05262.x
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发表时间：
1998-12-01
影响因子：
4
通讯作者：
Wood, P
中科院分区：
文献类型：
--
作者：
Gilbert, P;Jones, MV;Wood, P
泊洛沙姆F127是一种无毒的聚氧乙烯和聚氧丙烯的二嵌段共聚物。水溶液（30%w/v）显示热可逆凝胶化，在15 ℃的温度下为液体。将冷冻的泊洛沙姆（30%在胰蛋白胨大豆肉汤中）与铜绿假单胞菌（10（4）cfu ml（-1））的接种物混合，并将100 μ l液滴置于单独的玻璃盖片上。将其置于含有润湿的棉絮的密封培养皿中，并在35 ℃下孵育。通过将盖玻片转移到< 15 ℃的稀释液中，可以对泊洛沙姆凝胶进行活菌计数。凝胶和液体分批培养中的生长曲线彼此难以区分，固定期细胞密度在16 h时均约为5 x 10（10）cfu/ml。细胞被膜制备物的SDS-PAGE显示泊洛沙姆生长的细胞表现出生物膜而不是增殖表型。对不同浓度的氯己定，碘和过氧化氢的敏感性进行了评估，为10分钟，在35 ℃的肉汤生长的细胞悬浮液和孵育的泊洛沙姆凝胶（1和16小时）。将凝胶浸入其玻璃载体上的杀生物剂中，然后转移到10 ℃的中和剂中，在5分钟内完全溶解。进行进一步的系列稀释和平板计数。虽然对所有杀生物剂的敏感性适度降低与接种物与凝胶的结合（1小时孵育）相关，但在长时间孵育和适应生物膜表型（16小时孵育）后注意到实质性变化。凝胶群体模拟在生物膜中观察到的局部高细胞密度，并且还将经受与天然生物膜中发现的相同的营养和化学梯度。热可逆凝胶化使得测试接种物能够完全回收而没有进一步的创伤。因此，他们提供了一个有效的模型，用于评估生物膜对杀生物剂的敏感性，并将适合筛选方案。
Poloxamer F127 is a non-toxic, di-block copolymer of polyoxyethylene and polyoxypropylene. Aqueous solutions (30% w/v) show thermoreversible gelation, being liquid at temperatures 15 degrees C. Chilled poloxamer (30% in tryptone soya broth) was mixed with an inoculum of Pseudomonas aeruginosa (10(4) cfu ml(-1)) and placed as 100 mu l drops onto separate glass cover-slips. These were placed into sealed Petri dishes containing moistened cotton wool and incubated at 35 degrees C. Viable counts could be performed on the poloxamer gels bg transfer of the coverslips to diluents at < 15 degrees C. Growth curves in the gels and in liquid batch cultures were indistinguishable from one another with stationary phase cell densities, being approximately 5 x 10(10) cfu ml(-1) in each at 16 h. SDS-PAGE of cell envelope preparations showed the poloxamer-grown cells to exhibit a biofilm rather than planktonic phenotype. Susceptibility towards various concentrations of chlorhexidine, iodine and hydrogen peroxide was assessed for 10 min at 35 OC for suspensions of broth-grown cells and for incubated poloxamer-gels (1 and 16 h). The gels were immersed in biocide, on their glass supports, before transfer to neutralizer at 10C where dissolution was complete within 5 min. Further serial dilutions and plate counts were made. While modest decreases in susceptibility towards all biocides were associated with incorporation of the inoculum with the gel(1 h incubation), substantial changes were noted after prolonged incubation and adaptation to a biofilm phenotype (16 h incubation). The gel populations mimic the localized high cell densities observed in biofilms and will also be subject to the same nutrient and chemical gradients as found within natural biofilms. Thermoreversible gelation enables complete recovery of the test inoculum without further trauma. They therefore provide an effective model for assessing biofilm susceptibility towards biocides and would be suitable for screening programmes.