# Microcalorimetry--Versatile Method of Describing Bacterial Growth
## 3 Results  
The strains collected (from the 3 implants, marked as P1, P2, P3) were identified by MALDI-TOF as P1--_Staphylococcus epidermidis_, P2--_Staphylococcus aureus_, P3--_Staphylococcus aureus_ and using classic microbiology on blood agar and Sabouraud agar medium (Figures 1 and 2). The results showed that all the isolated strains were resistant to some beta-lactam antibiotics (penicillin and ceftroline). The _S. epidermidis_ strain, besides the resistance to beta-lactams, was also resistant to tetracycline and the tested macrolides (azithromycin and erythromycincin) (Table 1, Figure 3). The analyzed strains showed different virulence factors. The obtained results are presented in Figure 4.  
Figure 3: Antibiogram of analyzed strains P1—_S. epidermidis_ (first row of images), P2—_S. aureus_ (second row of images), P3—_S. aureus_ (the third row of images). Following the classic microbiology tests described above, we can see that, although the two bacterial, strains P2--_Staphylococcus aureus_ and P3--_Staphylococcus aureus_, are different strains collected from different patients, they seem very similar and we cannot make a very clear difference between the two. This aspect is very beneficial for the current study, which aims to compare the classical method of bacterial diagnosis with microcalorimetry. As we will describe in the following experiments, in relation to the bacterial investigation in terms of the thermal growth curves generated by microcalorimetry, we can see changes in kinetics that help us differentiate the two. Of course, through further studies in which we can change different parameters, these differences may become even more obvious. The aim of this study and of our team in general is to find ways that we can characterize the pathogens with increasing sensitivity and specificity and then apply this method clinically, which, in our opinion, has an increased ability to bring innovation in medicine and in understanding the development of pathogens. Figure 4: Aspects of expression of virulence factors on culture media: top-left—medium with casein for highlighting caseinase; right-middle—medium with lecithin to highlight lecithinase; bottom-left—medium for highlighting lipases; right—medium with seculin for highlighting the ability of the tested strains to hydrolyze seculin. We conducted experiments with 2 _S. aureus_ strains and one _S. epidermidis_ strain obtained from patients intraoperatively after the sonication of the infected implants. All the experiments were conducted with samples of 0,6 mL to ensure reproducibility and at 37 \({}^{\circ}\)C, the normal temperature of the body at which the bacteria develop. All the experiments used the same bacterial concentration. The media used for the bacteria was Mueller-Hinton (MH). All the experiments displayed complex thermal signals. For a better description of the growth curves of the bacteria, we proposed and analyzed the thermogram using key points on a set of quantitative parameters. These points are: thermal signal detection, establishment of the exponential growth, the peak, and the return to baseline. The associated quantities to these points (times, i.e., the corresponding positions or intervals on the time scale and heat flow values) can be used to characterize the raw bacterial growth thermograms as well as to differentiate the microorganisms. We analyzed the thermogram of all three Staphylococcus strains and, as can be seen from the figures below, we observed similarities for all the experiments (Figures 5-8); the time needed for the bacteria to grow as well as the heat flow generated by their growth was, for both experiments, in the same parameters. Figure 5: Graphical representation of the growth curve generated by the heat released by the active metabolism of _S. aureus—P2_. Experiments performed at 37 \({}^{\circ}\)C and with a microcalorimetric cell load of 600 \(\mu\)L.  
Figure 6: Graphical representation of the growth curve generated by the heat released by the active metabolism of _S. Aureus_—_P3_.