Abdelhamid Ahmed G, Yousef Ahmed E
Department of Food Science and Technology, The Ohio State University, Columbus, OH, United States.
Botany and Microbiology Department, Faculty of Science, Benha University, Benha, Egypt.
Front Cell Infect Microbiol. 2024 Feb 16;14:1346813. doi: 10.3389/fcimb.2024.1346813. eCollection 2024.
is a versatile opportunistic pathogen which causes a variety of acute and chronic human infections, some of which are associated with the biofilm phenotype of the pathogen. We hypothesize that defining the intracellular metabolome of biofilm cells, compared to that of planktonic cells, will elucidate the metabolic pathways and biomarkers indicative of biofilm inception. Disc-shaped stainless-steel coupons (12.7 mm diameter) were employed as a surface for static biofilm establishment. Each disc was immersed in a well, of a 24-well microtiter plate, containing a 1-mL Lysogeny broth (LB) suspension of ATCC 9027, a strain known for its biofilm prolificacy. This setup underwent oxygen-depleted incubation at 37°C for 24 hours to yield hypoxic biofilms and the co-existing static planktonic cells. In parallel, another planktonic phenotype of ATCC 9027 was produced in LB under shaking (200 rpm) incubation at 37°C for 24 hours. Planktonic and biofilm cells were harvested, and the intracellular metabolites were subjected to global untargeted metabolomic analysis using LC-MS technology, where small metabolites (below 1.5 kDa) were selected. Data analysis showed the presence of 324 metabolites that differed ( 0.05) in abundance between planktonic and biofilm cells, whereas 70 metabolites did not vary between these phenotypes ( > 0.05). Correlation, principal components, and partial least square discriminant analyses proved that the biofilm metabolome is distinctly clustered away from that of the two planktonic phenotypes. Based on the functional enrichment analysis, arginine and proline metabolism were enriched in planktonic cells, but butanoate metabolism was enriched in biofilm cells. Key differential metabolites within the butanoate pathway included acetoacetate, 2,3-butandiol, diacetyl, and acetoin, which were highly upregulated in the biofilm compared to the planktonic cells. Exogenous supplementation of acetoin (2 mM), a critical metabolite in butanoate metabolism, augmented biofilm mass, increased the structural integrity and thickness of the biofilm, and maintained the intracellular redox potential by balancing NADH/NAD ratio. In conclusion, hypoxic biofilm has a specialized metabolic landscape, and butanoate pathway is a metabolic preference and possibly required for promoting planktonic cells to the biofilm state. The butanoate pathway metabolites, particularly acetoin, could serve as markers for biofilm development.
是一种多能性机会致病菌，可导致多种急性和慢性人类感染，其中一些感染与该病原体的生物膜表型有关。我们假设，与浮游细胞相比，定义生物膜细胞的细胞内代谢组将阐明指示生物膜形成的代谢途径和生物标志物。使用圆盘形不锈钢试片（直径12.7毫米）作为静态生物膜形成的表面。将每个试片浸入24孔微量滴定板的孔中，该孔中含有ATCC 9027的1毫升溶菌肉汤（LB）悬浮液，ATCC 9027是一种以生物膜形成能力强而闻名的菌株。此装置在37°C下进行缺氧培养24小时，以产生缺氧生物膜和共存的静态浮游细胞。同时，在37°C下于摇床（200转/分钟）中培养24小时，在LB中产生ATCC 9027的另一种浮游表型。收获浮游细胞和生物膜细胞，并使用LC-MS技术对细胞内代谢物进行全局非靶向代谢组学分析，其中选择分子量小于1.5 kDa的小分子代谢物。数据分析表明，浮游细胞和生物膜细胞之间有324种代谢物的丰度存在差异（P<0.05），而70种代谢物在这些表型之间没有变化（P>0.05）。相关性分析、主成分分析和偏最小二乘判别分析证明，生物膜代谢组与两种浮游表型的代谢组明显聚类分开。基于功能富集分析，精氨酸和脯氨酸代谢在浮游细胞中富集，但丁酸代谢在生物膜细胞中富集。丁酸途径中的关键差异代谢物包括乙酰乙酸、2,3-丁二醇、双乙酰和乙偶姻，与浮游细胞相比，它们在生物膜中高度上调。外源性添加乙偶姻（2 mM），丁酸代谢中的一种关键代谢物，可增加生物膜质量，提高生物膜的结构完整性和厚度，并通过平衡NADH/NAD比率维持细胞内氧化还原电位。总之，缺氧生物膜具有特殊的代谢格局，丁酸途径是一种代谢偏好，可能是促进浮游细胞转变为生物膜状态所必需的。丁酸途径代谢物，特别是乙偶姻，可作为生物膜发育的标志物。
