Stewart Philip S, Franklin Michael J, Williamson Kerry S, Folsom James P, Boegli Laura, James Garth A
Department of Chemical and Biological Engineering and Center for Biofilm Engineering, Montana State University, Bozeman, Montana, USA
Department of Microbiology and Center for Biofilm Engineering, Montana State University, Bozeman, Montana, USA.
Antimicrob Agents Chemother. 2015 Jul;59(7):3838-47. doi: 10.1128/AAC.00433-15. Epub 2015 Apr 13.
Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.
生物膜相关细菌对抗生素治疗的耐受性增强可能是多种因素共同作用的结果，包括细菌适应生物膜生长时细胞生理学的变化以及生物膜细菌固有的生理异质性。在本研究中，采用转录组学方法来鉴定铜绿假单胞菌生物膜生长过程中差异表达的基因。这些基因被测试是否与对应应激反应及其他假定抗生素保护机制的独立编制基因列表存在统计学上的显著重叠。所测试的基因组包括那些与生物膜对妥布霉素或环丙沙星的反应、药物外排泵、酰基高丝氨酸内酯群体感应、渗透压休克、热休克、缺氧应激和稳定期生长相关的基因。与Anr介导的缺氧应激、RpoS调控的稳定期生长和渗透压应激相关的调控子在生物膜诱导的基因集中显著富集。缺失rpoS、relA和spoT或anr的突变菌株在生物膜中培养，并接受环丙沙星和妥布霉素的挑战。当用环丙沙星挑战时，与野生型菌株0.9个对数级的活细胞减少相比，突变菌株生物膜的活细胞减少了2.4至2.9个对数级。有趣的是，与野生型生物膜相比，没有一个突变体在妥布霉素敏感性上表现出统计学上的显著变化。这些结果与一个模型一致，即在该模型中，由重叠的饥饿或应激反应控制的多个基因有助于保护铜绿假单胞菌生物膜免受环丙沙星的影响。一种独特且尚未被发现的机制保护生物膜细菌免受妥布霉素的影响。
