BMC Microbiol. 2013 Jul 24;13:171. doi: 10.1186/1471-2180-13-171.
Microcalorimetric bacterial growth studies have illustrated that thermograms differ significantly with both culture media and strain. The present contribution examines the possibility of discriminating between certain bacterial strains by microcalorimetry and the qualitative and quantitative contribution of the sample volume to the observed thermograms. Growth patterns of samples of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were analyzed. Certain features of the thermograms that may serve to distinguish between these bacterial strains were identified.
The thermograms of the two bacterial strains with sample volumes ranging from 0.3 to 0.7 ml and same initial bacterial concentration were analyzed. Both strains exhibit a roughly 2-peak shape that differs by peak amplitude and position along the time scale. Seven parameters corresponding to the thermogram key points related to time and heat flow values were proposed and statistically analyzed. The most relevant parameters appear to be the time to reach a heat flow of 0.05 mW (1.67 ± 0.46 h in E. coli vs. 2.99 ± 0.53 h in S. aureus, p < 0.0001), the time to reach the first peak (3.84 ± 0.5 h vs. 5.17 ± 0.49 h, p < 0.0001) and the first peak value (0.19 ± 0.02 mW vs. 0.086 ± 0.012 mW, p < 0.0001). The statistical analysis on 4 parameters of volume-normalized heat flow thermograms showed that the time to reach a volume-normalized heat flow of 0.1 mW/ml (1.75 ± 0.37 h in E. coli vs. 2.87 ± 0.65 h in S. aureus, p < 0.005), the time to reach the first volume-normalized peak (3.78 ± 0.47 h vs. 5.12 ± 0.52 h, p < 0.0001) and the first volume-normalized peak value (0.35 ± 0.05 mW/ml vs. 0.181 ± 0.040 mW/ml, p < 0.0001) seem to be the most relevant. Peakfit® decomposition and analysis of the observed thermograms complements the statistical analysis via quantitative arguments, indicating that: (1) the first peak pertains to a faster, "dissolved oxygen" bacterial growth (where the dissolved oxygen in the initial suspension acts as a limiting factor); (2) the second peak indicates a slower "diffused oxygen" growth that involves transport of oxygen contained in the unfilled part of the microcalorimetric cell; (3) a strictly fermentative growth component may slightly contribute to the observed complex thermal signal.
The investigated strains of Staphylococcus aureus and Escherichia coli display, under similar experimental conditions, distinct thermal growth patterns. The two strains can be easily differentiated using a selection of the proposed parameters. The presented Peakfit analysis of the complex thermal signal provides the necessary means for establishing the optimal growth conditions of various bacterial strains. These conditions are needed for the standardization of the isothermal microcalorimetry method in view of its further use in qualitative and quantitative estimation of bacterial growth.
微量热细菌生长研究表明，热谱图在培养基和菌株方面存在显著差异。本研究旨在通过微量热法来鉴别某些细菌菌株，并探讨样品体积对观察到的热谱图的定性和定量影响。对金黄色葡萄球菌（ATCC 25923）和大肠杆菌（ATCC 25922）的样本生长模式进行了分析。确定了可能用于区分这些细菌菌株的热谱图特征。
对样本体积在 0.3 至 0.7ml 之间且初始细菌浓度相同的两种细菌菌株的热谱图进行了分析。两种菌株均呈现出大致 2 峰形状，其峰幅度和沿时间轴的位置不同。提出并统计分析了与热谱图关键点相关的 7 个参数，这些参数与时间和热流值有关。最相关的参数似乎是达到热流 0.05mW 的时间（大肠杆菌为 1.67±0.46h，金黄色葡萄球菌为 2.99±0.53h，p<0.0001）、达到第一个峰的时间（3.84±0.5h 与 5.17±0.49h，p<0.0001）和第一个峰的值（0.19±0.02mW 与 0.086±0.012mW，p<0.0001）。对体积归一化热流热谱图的 4 个参数进行的统计分析表明，达到体积归一化热流 0.1mW/ml 的时间（大肠杆菌为 1.75±0.37h，金黄色葡萄球菌为 2.87±0.65h，p<0.005）、达到第一个体积归一化峰的时间（3.78±0.47h 与 5.12±0.52h，p<0.0001）和第一个体积归一化峰的值（0.35±0.05mW/ml 与 0.181±0.040mW/ml，p<0.0001）似乎是最相关的。Peakfit®分解和观察到的热谱图分析通过定量论据补充了统计分析，表明：（1）第一个峰与更快的“溶解氧”细菌生长有关（初始悬浮液中的溶解氧作为限制因素）；（2）第二个峰表明较慢的“扩散氧”生长，涉及填充微热量计细胞未填充部分的氧气的传输；（3）严格的发酵生长成分可能会对观察到的复杂热信号略有贡献。
在相似的实验条件下，金黄色葡萄球菌和大肠杆菌的研究菌株表现出不同的热生长模式。使用所选参数可以轻松区分两种菌株。对复杂热信号的 Peakfit 分析为确定各种细菌菌株的最佳生长条件提供了必要的手段。鉴于等温微量热法在定性和定量估计细菌生长方面的进一步应用，需要这些条件来标准化该方法。
