Conceptual Model of Biofilm Antibiotic Tolerance That Integrates Phenomena of Diffusion, Metabolism, Gene Expression, and Physiology
Conceptual Model of Biofilm Antibiotic Tolerance That Integrates Phenomena of Diffusion, Metabolism, Gene Expression, and Physiology
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DOI：
10.1128/jb.00307-19
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发表时间：
2019-11-01
影响因子：
3.2
通讯作者：
Wallqvist, Anders
中科院分区：
文献类型：
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作者：
Stewart, Philip S.;White, Ben;Wallqvist, Anders
转录学、代谢学、生理学和计算建模方法被结合在一起，以深入了解体外生物膜系统中抗生素耐受的机制。铜绿假单胞菌生物膜是在滴流反应器中培养的，其基质是模拟慢性伤口渗出物的。4d后，生物膜厚度为114亩m，菌落数为9.45log(10)cfu cm(-2)。相对于指数期浮游细胞，这些生物膜表现出对环丙沙星后续处理的耐受性。通过元素平衡估算出生物膜的比生长速率约为0.37h(-1)，反应扩散模型为0.32h(-1)，即浮游细胞最大比生长速率的三分之一。基因表达的全局分析表明，与对数生长期浮游细胞相比，生物膜中核糖体基因和其他合成代谢功能基因的转录水平较低，并揭示了生物膜细胞中多种应激反应的诱导，包括与生长停滞、锌限制、低氧和酰基高丝氨酸内酯群体感应相关的应激反应。吩嗪生物合成和反硝化的代谢途径在生物膜中转录激活。定制的反应-扩散模型预测，当这些生物膜厚度超过约40微米时，将形成陡峭的氧浓度梯度。在生物膜中培养时，缺乏PSL多糖合成、严格反应、静止相反应和膜应激反应的突变菌株表现出对环丙沙星的敏感性增加。这些结果支持导致生物膜耐药的一系列现象，包括氧气限制、电子受体饥饿和生长停滞、诱导相关的应激反应以及分化为受保护的细胞状态。生物膜中的细菌受到保护，免受抗生素的杀伤，这种降低的敏感性导致感染的持久性，如囊性纤维化肺和慢性伤口中的感染。提出了一个广义的生物膜耐药概念模型，包括以下机制步骤：(I)在代谢底物和产物中建立浓度梯度；(Ii)在局部化学微环境中对这些变化作出积极的生物反应；(Iii)生物膜细胞进入一系列状态，包括交替代谢、应激反应、生长缓慢、生长停止或休眠(所有这些状态都在抗生素治疗之前)；(Iv)对抗生素暴露的适应性反应；以及(V)在通过这些变化获得的某些生理状态中，微生物细胞对抗生素挑战的敏感性降低。
Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip flow reactors on a medium composed to mimic the exudate from a chronic wound. After 4 days, the biofilm was 114 mu m thick with 9.45 log(10) CFU cm(-2). These biofilms exhibited tolerance, relative to exponential-phase planktonic cells, to subsequent treatment with ciprofloxacin. The specific growth rate of the biofilm was estimated via elemental balances to be approximately 0.37 h(-1) and with a reactiondiffusion model to be 0.32 h(-1) , or one-third of the maximum specific growth rate for planktonic cells. Global analysis of gene expression indicated lower transcription of ribosomal genes and genes for other anabolic functions in biofilms than in exponential-phase planktonic cells and revealed the induction of multiple stress responses in biofilm cells, including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing. Metabolic pathways for phenazine biosynthesis and denitrification were transcriptionally activated in biofilms. A customized reaction-diffusion model predicted that steep oxygen concentration gradients will form when these biofilms are thicker than about 40 mu m. Mutant strains that were deficient in Psl polysaccharide synthesis, the stringent response, the stationary-phase response, and the membrane stress response exhibited increased ciprofloxacin susceptibility when cultured in biofilms. These results support a sequence of phenomena leading to biofilm antibiotic tolerance, involving oxygen limitation, electron acceptor starvation and growth arrest, induction of associated stress responses, and differentiation into protected cell states.IMPORTANCE Bacteria in biofilms are protected from killing by antibiotics, and this reduced susceptibility contributes to the persistence of infections such as those in the cystic fibrosis lung and chronic wounds. A generalized conceptual model of biofilm antimicrobial tolerance with the following mechanistic steps is proposed: (i) establishment of concentration gradients in metabolic substrates and products; (ii) active biological responses to these changes in the local chemical microenvironment; (iii) entry of biofilm cells into a spectrum of states involving alternative metabolisms, stress responses, slow growth, cessation of growth, or dormancy (all prior to antibiotic treatment); (iv) adaptive responses to antibiotic exposure; and (v) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.