Developing selective media for quantification of multispecies biofilms following antibiotic treatment

The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymicrobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multispecies biofilms is frequently carried out by plating on selective media. However, the influence of the selective agents in these media on quantitative recovery before or after antibiotic treatment is often unknown. In the present study we developed selective media for six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to selective media strongly influence quantitative recovery of (un)treated biofilms. Hence, the developed media were optimized for selectivity and quantitative recovery before or after treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or tobramycin. Finally, in a proof of concept experiment the novel selective media were applied to determine the community composition of multispecies biofilms before and after treatment with tobramycin.

囊性纤维化（cystic fibrosis, CF）患者的肺部长期被多微生物生物膜群落定殖，引发难治性感染。为对抗此类感染，CF患者通常需接受多种抗生素治疗。探究多微生物群落组成在抗生素治疗下的动态变化，对于新型治疗方案的开发至关重要。目前，针对特定多物种生物膜中的单株细菌开展培养依赖型定量分析，通常通过在选择性培养基上涂布培养实现。然而，此类培养基中的筛选试剂对抗生素处理前后的细菌定量回收效果的影响，往往尚不明确。本研究针对六种常从CF患者肺部共分离得到的细菌物种开发了专用选择性培养基，分别为铜绿假单胞菌（Pseudomonas aeruginosa）、金黄色葡萄球菌（Staphylococcus aureus）、咽峡炎链球菌（Streptococcus anginosus）、木糖氧化无色杆菌（Achromobacter xylosoxidans）、粘膜炎罗斯菌（Rothia mucilaginosa）以及溶血孪生球菌（Gemella haemolysans）。研究表明，向选择性培养基中添加特定成分，会显著影响抗生素处理前后生物膜的定量回收效果。因此，本研究针对四大类抗生素——即头孢他啶（ceftazidime）、环丙沙星（ciprofloxacin）、多粘菌素（colistin）以及妥布霉素（tobramycin）——的处理前后，对开发的培养基进行了选择性与定量回收效果的优化。最后，本研究通过概念验证实验，应用新型选择性培养基测定了经妥布霉素处理前后的多物种生物膜群落组成。