The potential of therapeutic hyperthermia to eradicate Staphylococcus aureus bacteria
Description
We hypothesize that hyperthermia might be a new therapeutic option for infectious diseases as a sole agent or in combination with different antimicrobials. Our hypothesis is that hyperthermia alone (45 °C) may inhibit growing of the Staphylococcus aureus strains and hyperthermia in combination with antimicrobial drugs may result in synergistic effects and reduce resistance to our study antibiotics. In our study, we used two different temperature levels; normal body temperature and hyperthermia temperature level (37 °C and 45 °C) for bacterial incubation and antimicrobial susceptibility testing (Kirby-Bauer disc diffusion method) and we assessed growth rates on two different temperatures using a turbidimetric method (McFarland optic reader) and determined inhibition zone diameters on agar plates zones for several antibiotics. Inhibition zone diameters were interpreted according to EUCAST criteria. 1.In order to determine the activity of hyperthermia (alone) on the growth rate of the bacterial strains, bacterial suspensions of the two strains were prepared in Mueller-Hinton broth (MHB) tubes. Initially, the bacterial density was adjusted to a 0.2 McFarland standard using a McFarland densitometer (DensiCHEK Plus, bioMérieux, France). Prepared suspensions were incubated at both 37 °C and 45 °C for 20 hours. 2. Thermo-antibiograms of both strains were performed to investigate the presence of heat-antibiotic synergy (Gazel and Yılmaz, 2018). Erythromycin, vancomycin, oxacillin, clindamycin, cefoxitin, ciprofloxacin, gentamicin, penicillin, amikacin and tetracycline standard antibiotic discs (Bioanalyse, Turkey) were used in the study. Thermo-antibiogram was applied using Kirby-Bauer disk diffusion method (Gazel and Yılmaz, 2018). Bacterial suspensions of 0.5 McFarland standard were prepared for both strains. Samples taken from the prepared suspensions were inoculated on Mueller-Hinton agar (MHA) media Findings 1. Significant inhibition on bacterial growth was observed (S. aureus ATCC 29213 strain) by the effect of hyperthermia alone at 45 °C when compared with 37 °C. Raw data is given in the Excel file. 2. We observed larger inhibition zones indicating increased susceptibility for most of the antibiotics at 45 °C when compared with 37 °C (Kirby-Bauer disc diffusion method; Images of the agar plates showing antibacterial inhibition zones against antibiotics are upload as figure files)
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1st step: In order to determine the activity of hyperthermia (alone) on the growth rate of the bacterial strains, bacterial suspensions of the two strains were prepared in Mueller-Hinton broth (MHB) tubes. Initially, the bacterial density was adjusted to a 0.2 McFarland standard using a McFarland densitometer (DensiCHEK Plus, bioMérieux, France). Prepared suspensions were incubated at both 37 °C and 45 °C for 20 hours. The turbidity/density values of the tubes were measured every 20 minutes with the densitometer throughout the incubation period. The mean of the measurements taken for each hour was recorded and the growth rate curve was graphed (Table 1, Figure 1). Growth curves were created using Microsoft Office Excel. Changes in the growth rate of bacterial strains were evaluated statistically using the Wilcoxon signed-rank test. A value of p < .05 was accepted as statistical significance. 2nd step: Thermo-antibiograms of both strains were performed to investigate the presence of heat-antibiotic synergy (Gazel and Yılmaz, 2018). Erythromycin, vancomycin, oxacillin, clindamycin, cefoxitin, ciprofloxacin, gentamicin, penicillin, amikacin and tetracycline standard antibiotic discs (Bioanalyse, Turkey) were used in the study. Thermo-antibiogram was applied using Kirby-Bauer disk diffusion method (Gazel and Yılmaz, 2018). Bacterial suspensions of 0.5 McFarland standard were prepared for both strains. Samples taken from the prepared suspensions were inoculated on Mueller-Hinton agar (MHA) media. Within 15 minutes after inoculation, antibiotic discs of erythromycin, vancomycin, oxacillin, clindamycin, ciprofloxacin, gentamicin and penicillin were placed on the surface of the MHA and the media were incubated at 37 °C and 45 °C for 18 hours. After incubation, the inhibition zones formed around the antibiotic discs and the diameters of the inhibition zones were measured and recorded. The interpretation of inhibition zone diameters (susceptible or resistant) was made in compliance with EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines (EUCAST, 2022).