ABSTRACT
BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.
Subject(s)
Calorimetry/methods , Escherichia coli/growth & development , Staphylococcus aureus/growth & development , Bacteriological Techniques/methods , Species SpecificityABSTRACT
The microcalorimetry is a method used for recording of the heat produced by a thermodinamic system in a scale of micronanojouls. One of the domains in which this method is used is the one called bacterial microcalorimetry, which studies the heat generated by the bacterial populations. The process of bacterial growth can be monitored in real time by the recording a graph of the generated power over time. The modern isothermal microcalorimeters allow the detection of a signal variation of only one microwatt. The estimated generated power of a bacteria is approximately 1-4pW thus only a small number of bacteria is necessary for the experiments. Recent studies in the field of bacterial microcalorimetry have demonstrated that, in standard conditions, this method can be reproductible and can be used to detect and characterize bacterial growth (through the study of the microcalorimetric growth curve particular to a bacterial species which is called a microcalorimetric fingerprint) and offers the new information in regards to bacterial metabolism. Also, microcalorimetry can offer information about bacterial interaction with different factors in the medium (for example, antibioticsubstances, in which case an antibiogram is obtained in 4-5 hours). In conclusion, we can say that microcalorimetry is a reproducible method, which offers an interesting perspective on bacterial characterization, with great scientific potential, and there are sufficient arguments to continue studies in this field.
