Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection
Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection
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DOI：
10.1007/s00253-012-4179-9
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发表时间：
2013-01-01
影响因子：
5
通讯作者：
Bryers, James D.
中科院分区：
文献类型：
--
作者：
Ma, Hongyan;Bryers, James D.
大部分人类微生物感染都是由生物膜引起的。根除生物膜感染的尝试依赖于消毒剂和抗生素。不幸的是，生物膜细菌对抗生素应激源的反应明显低于浮游细菌。亚致死剂量的抗生素实际上可以增强生物膜的形成。在这里，我们开发了一种非侵入性显微图像分析来量化正在发育的生物膜内的质粒缀合。通过不依赖于培养的流式细胞术分析和选择性平板计数方法来分析证实的破坏性样品，以培养转接合子。在没有任何抗生素选择压力的情况下，底物负载的增加改变了生物膜的 3-D 结构，并随后影响了质粒缀合的频率（至少减少两倍）。更重要的是，暴露于亚致死剂量卡那霉素的生物膜中的供体群体表现出含有卡那霉素抗性基因的质粒的转移效率提高了十倍。然而，当用不同的抗生素亚胺培南胁迫时，含有kan(R+)基因的质粒的转移并没有增强。这些初步结果表明，生物膜细菌“感知”它们所耐药的抗生素，从而增强了耐药性的传播。共焦扫描显微镜与我们的非侵入性图像分析相结合，能够估计整个生物膜景观的平均值或局部单个生物膜簇的质粒接合转移效率。
Biofilms cause much of all human microbial infections. Attempts to eradicate biofilm-based infections rely on disinfectants and antibiotics. Unfortunately, biofilm bacteria are significantly less responsive to antibiotic stressors than their planktonic counterparts. Sublethal doses of antibiotics can actually enhance biofilm formation. Here, we have developed a non-invasive microscopic image analyses to quantify plasmid conjugation within a developing biofilm. Corroborating destructive samples were analyzed by a cultivation-independent flow cytometry analysis and a selective plate count method to cultivate transconjugants. Increases in substrate loading altered biofilm 3-D architecture and subsequently affected the frequency of plasmid conjugation (decreases at least two times) in the absence of any antibiotic selective pressure. More importantly, donor populations in biofilms exposed to a sublethal dose of kanamycin exhibited enhanced transfer efficiency of plasmids containing the kanamycin resistance gene, up to tenfold. However, when stressed with a different antibiotic, imipenem, transfer of plasmids containing the kan(R+) gene was not enhanced. These preliminary results suggest biofilm bacteria "sense" antibiotics to which they are resistant, which enhances the spread of that resistance. Confocal scanning microscopy coupled with our non-invasive image analysis was able to estimate plasmid conjugative transfer efficiency either averaged over the entire biofilm landscape or locally with individual biofilm clusters.