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Journal of Thermal Analysis and Calorimetry

, Volume 133, Issue 1, pp 773–777 | Cite as

Study of bacterial sensitivity in zinc sulfate solutions by microcalorimetry

  • Ricardo Aveledo
  • Alberto Aveledo
  • Cristina Vázquez
  • Natividad Lago
  • Marta M. Mato
  • José L. Legido
Article

Abstract

Zinc sulfate is an inorganic compound and dietary supplement. It has shown antimicrobial effects on certain pathogens and may contribute to the treatment or prevention of infections. In this study, the bacterial growth of Pseudomonas aeruginosa, which is a pathogen involved in several life-threatening infections to the human body, is assessed by microcalorimetry using different concentrations of zinc sulfate. Bacteria growth monitoring has been demonstrated using microcalorimetric techniques. Dissolutions of zinc sulfate were prepared with concentrations from 0 to 250 mM. A suspension of 106 CFU mL−1 of Pseudomona aeruginosa and as a culture medium, a liquid soya-casein-digested liquid were used. The measurements were carried out in a Calvet microcalorimeter at constant temperature of 309.65 K. The reference cell was filled with 6 mL of culture medium, 1 mL of the metallic dissolution, and 1 mL of mineral-medicinal water. In the experimental cell, the latter was replaced by the bacterial suspension. The data were collected by a data acquisition and processing system, at intervals of 22.2 s for 48 h. Representing the difference in heat output generated between the experimental and control cells versus time, the bacterial growth curves were obtained and the thermograms were compared using different concentrations of the metallic dissolution. This study highlights the role of zinc sulfate in suppressing bacteria growth at certain concentrations measured by microcalorimetric techniques. Such thermodynamic technique evidences the potential use of metallic dissolution in the medical industry, among others, in order to take advantage of its bactericide property.

Keywords

Microcalorimetry Metabolism Bacteria Pseudomonas aeruginosa Zinc sulfate 

Introduction

Microcalorimetry deals with the measure of the thermal properties of reactions or physical changes, in which the heat exchange is very weak. It is a highly sensitive experimental technique that allows to determine the energy released by any process or transformation [1].

This technique has potential to be used in biological experiments, since heat flow is strongly related to the kinetics and thermodynamics of biological processes. Heat variations resulting from chemical reactions, which take place during metabolism, can be used to monitor bacterial growth, and monitor the influence of external agents [2, 3, 4].

All living beings produce heat during metabolism. The rate of heat production is an adequate measure of the metabolic activity of the organisms and their constituent parts, cells, and subcellular levels [5]. The heat generated by a single cell is in the range of 1–80 pW. Human connective tissue cells have metabolic rates of approximately 25–80 pW per cell. In contrast, microorganisms produce small amounts of heat, in the order of 1–3 pW per cell [6]. Despite the low levels of heat produced by bacteria, their exponential growth in the culture medium allows their detection using microcalorimetry within a few hours [7], even from samples with a low concentration, e.g., 10 CFU mL−1 [5, 8].

Microcalorimetry has been used as an analytical tool in microbiology for a long time. However, in the last few years, microcalorimetric investigations of microbial processes are becoming increasingly popular [9].

Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative, aerobic strict opportunistic bacterium. It causes several types of infections in the human body, including infections in different systems such as respiratory, urinary, digestive, musculoskeletal, among others. Sometimes, these infections are serious and life-threatening. [10, 11]. In the field of orthopedic surgery, the formation of the biofilm around metallic implants has been a field of great interest in research due to the important consequences to the health and life of patients.

Zinc sulfate

Zinc sulfate is an inorganic compound and dietary supplement. As a supplement, it is used to treat zinc deficiency and to prevent conditions in those at high risk [12]. Metals such as copper, silver, arsenic, or zinc have been used in various ways for their antimicrobial properties [13]. Zinc sulfate has shown antimicrobial effects on certain pathogens and may contribute to the treatment or prevention of infections [14].

In the last few years, microcalorimetry has been used to monitor bacterial growth under different conditions. Several studies focused on the study of a single microorganism [5, 8, 15, 16, 17, 18, 19]. In addition, it has been employed to investigate the relationship between two microorganisms [20, 21, 22, 23, 24, 25]. Esarte et al. [26] analyzed the microcalorimetric behavior of P. aeruginosa and its susceptibility against two antibiotics, ceftazidime and piperacillin–tazobactam. Vazquez et al. [27] evaluated the effects of ultrasound waves on the viability of several bacteria that usually occur in mineral waters such as spa thermal waters.

Despite the above references, no previous thermodynamic studies based on bacterial behavior in different concentrations of zinc have been reported in the literature. In this study, the bacterial growth of P. aeruginosa is assessed by microcalorimetry using different concentrations of zinc sulfate.

Experimental

Sample preparation

Bacteria samples were obtained from the American Type Culture Collection: P. aeruginosa (ATCC 27853). This bacterium was inoculated on blood agar plates and incubated at 309.65 K in an incubator for 24 h. The blood agar plates with multiple bacterial colonies were then used to prepare a bacterial suspension, whose concentration was adjusted to the corresponding 0.5 on the McFarland scale. This suspension was diluted with 0.9% sterile saline to obtain a final concentration of 106 CFU mL−1.

On the other hand, the solutions of zinc sulfate were prepared at different concentrations expressed in millimolar (0–250 mM). As a culture medium, a liquid soya-casein-digested liquid was used.

The reference cell was filled with 6 mL of culture medium, 1 mL of the metallic dissolution, and 1 mL of mineral-medicinal water. In the experimental cell, the latter was replaced by the bacterial suspension. Both cells were cleaned and sterilized by autoclaving before using.

Experimental equipment

The measures were carried out in a Calvet microcalorimeter designed by Professor Paz Andrade [28]. It is equipped with a device allowing operation in the absence of vapor phase and having two Teflon® screw capped stainless steel cells of approximately 10 cm3 (experimental and reference) [24]. Both cells were introduced, from the upper part of the microcalorimeter in the internal thermopile chamber through two cylindrical holes aligned in parallel, which extended from the upper part of the microcalorimeter to the internal thermopile chamber. The large distance that separates the cells from the entrance ensures the minimization of heat flow to the exterior [21]. A Philips PM2535 multimeter and a data acquisition system were connected to the microcalorimeter. A Setaram EJP30 stabilized current source was used to perform an electrical calibration. The precision in calorimetric signal achieved was ± 1 µV [28]. Further details about the experimental method have been published [28].

A constant temperature of 309.65 K was maintained in the outer chamber of the calorimeter. The data were collected by a data acquisition and processing system, at intervals of 22.2 s for 48 h. The calorimetric curve is defined by a series of electromotive force points recorded by the data acquisition system and corresponds to the energy exchange that occurs during the culture period.

In order to measure the modification of the medium pH by the residues that the bacterial metabolism produces, samples were subject to pH control both before and after each experiment using a basic 20 + pH meter.

Results and discussion

The heat voltage difference versus time was recorded, and therefore, the bacterial growth curves were obtained at different concentrations of the metallic dissolutions. The results obtained are shown as heat flow versus time curves in Figs. 16. The differences in shape of curves allow us to visualize the trend of bacterial growth when increasing the concentration of zinc sulfate.
Fig. 1

Calorimetric heat flow versus time for P. aeruginosa

Fig. 2

Calorimetric heat flow versus time for P. aeruginosa + 10 mM zinc sulfate

Fig. 3

Calorimetric heat flow versus time for P. aeruginosa + 25 mM zinc sulfate

Fig. 4

Calorimetric heat flow versus time for P. aeruginosa + 100 mM zinc sulfate

Fig. 5

Calorimetric heat flow versus time for P. aeruginosa + 150 mM zinc sulfate

Fig. 6

Calorimetric heat flow versus time for P. aeruginosa + 250 mM zinc sulfate

The shape of the heat flow curve of P. aeruginosa (Fig. 1) is characterized by the presence of just one phase. It shows an initial ascending part with two main leaps, and then a descending curve showing an exponential shape, which is prolonged over time. When the experiment is performed with the different concentrations of zinc sulfate (Figs. 25), the maximum voltage peak signal gradually decreases, reaching almost complete signal suppression using 250 mM of zinc sulfate (Fig. 6). At higher concentrations of zinc sulfate, the growth is completely inhibited.

Using the curves displayed by the microcalorimeter, the amount of heat released (Q) during the culture time can be determined using the following equation:
$$Q = K \times A$$
where A (μV h) is the area, calculated by the trapezoidal method, and K represents a constant whose value is 23.8 μV−1 h−1, which was calculated from the electric calibration performed by the Joule effect on the equipment.
The maximum voltage peak (Vmax), the area under the curve (AUC48), and the amount of heat exchanged (Q48) during the 48 h of the experiment of P. aeruginosa with the different solutions of zinc sulfate are shown in Table 1.
Table 1

Maximum voltage peak, Vmax/μV, area under the curve, AUC48/μV h, and heat generated during 48 h, Q48/J, of Pseudomona aeruginosa with different concentrations of zinc sulfate

Zinc sulfate concentration/mM

Vmax/μV

AUC48/μV h

Q48/J

0

119

8348.77

198.699

10

93

8942.77

212.836

25

91

9397.45

223.658

100

84

8501.95

202.345

150

58

4507.50

107.278

250

2

233.85

5.565

As illustrated in Table 1, when comparing the maximum voltage peak signal of the bacteria with the different concentrations of zinc sulfate, an inverse proportionality was appreciated; therefore, the heat flow decreased when increasing the concentration of this metallic dissolution. The highest voltage peak was 119 μV using without the exposure of metallic dissolution (0 mM of zinc sulfate), then it decreased gradually showing a signal of 58 μV using a concentration of 150 mM, reaching almost complete signal suppression using 250 mM of zinc sulfate.

With respect to the total heat exchanged, unlike the Vmax values, the lowest four concentrations of zinc sulfate (mean = 209.384 J) did not reveal inverse proportionally (zinc sulfate concentration VS heat generated). This phenomenon could be explained by the depletion of bacterial nutrients in the culture medium caused by an initial elevated voltage. However, using concentrations > 100 mM, the total heat generated dropped significantly.

The residues produced by the bacteria metabolism usually are acid, resulting in a decreased pH in the culture medium. However, as shown in Table 2, this trend only happened with the sample with pure bacteria and in the sample with the addition of 250 mm of zinc sulfate. On the rest of the experiences, the pH of the experimental cells after 48 h in the calorimeter (final pH) was alkalized. When reviewing the literature, studies have revealed the property of the P. aeruginosa to increase the pH when the biofilm is produced [29].
Table 2

pH values before and after each experiment of Pseudomona aeruginosa with different concentrations of zinc sulfate

Zinc sulfate concentration/mM

Initial pH

T/°C

Final pH

T/°C

0

7.10

26.7

6.94

30.3

10

6.80

26.2

8.06

31.5

25

6.63

27.8

7.36

31.5

100

5.70

26.7

7.34

28.4

150

5.53

27.1

6.62

30.2

250

4.91

24.3

4.81

27.5

Conclusions

Bacteria metabolism was inhibited as the concentration of zinc sulfate increased. Therefore, this study highlights the negative effect that zinc sulfate has in bacteria growth at certain concentrations measured by microcalorimetry. Such thermodynamic technique evidences the potential use of zinc sulfate in the medical industry, among others, in order to take advantage of its bactericide property.

Notes

Acknowledgements

The authors are grateful to Miguel Ramos for the support in the preparations of the metallic dissolutions. They thank Maria Perfecta Salgado Gonzalez for her collaboration with the technical measures. Financial support was provided by the projects EM 2012/141, CN 2012/285, and “Agrupación Estratégica de Biomedicina (INBIOMED)” by “Xunta de Galicia” and the project FIS 2011-23322 funded by Ministry of Science and Innovation of Spain. All these projects are co-funded with FEDER funds.

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Copyright information

© Akadémiai Kiadó, Budapest, Hungary 2018

Authors and Affiliations

  1. 1.Department of Applied PhysicsUniversity of VigoVigoSpain
  2. 2.Pharmacy ServiceAlvaro Cunqueiro HospitalVigoSpain

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