In Vitro Evaluation of Ozone Activity on Recent Clinically Isolated Bacterial Strains

This study aims to evaluate the cozone bactericidal activity in different suspension media (saline, broth and whole blood) at different exposure times. Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, ESBLpositive Escherichia coli, MDR Pseudomonas aeruginosa were suspended in different media. We used a bacterial concentration of 0.2 MF for all experiments, as this concentration is consistent with the results of septic shock blood experiments. We performed ozone insufflations in a “sealed environment”. The total number of insufflations for each experiment ranged from one to four. The gas concentration was maintained at 80 mcg/ml. We confirmed the bactericidal activity of ozone on saline for all the bacterial strains. Experiments in broth revealed no changes in the bacterial growth. Ozone is primarily bactericidal against E. coli and bacteriostatic on P. aeruginosa, S. aureus and E. faecalis on whole blood. This study confirms the bactericidal efficacy of topical ozone applications and supports the need for further evaluations of the therapeutic potential of major ozone autohemotherapy. The results in E. coli promote further investigations of ozone activity on other Enterobacteriaceae and its potential use in the treatment of urinary infections. In general, these results suggest that ozone-therapy might be an alternative therapy to overcome antibiotic resistance.


Introduction
The microbicidal activity of ozone has been demonstrated since the late 1800.The first municipal water purification plant dates back to 1906.On June 26, 2001, the US Food and Drug Administration (FDA) formally approved the use of ozone (gaseous phase and ozonized water) as an antimicrobial agent for the treatment, storage and preservation of food products [1].
Ozone is the most powerful oxidizing agent, showing ten times the effectiveness of chlorine, and it's currently used to potabilize water [2][3][4], disinfect swimming pool water [5] and decontaminate bioclean rooms [6].
Using electron microscopy analysis, Thanomsub et al. showed the destruction of the bacterial membrane with consequent cell lysis [19].The treatment of Bacillus subtilis spores with various oxidizing agents (including ozone) damages the inner membrane, making the spores more sensitive to subsequent thermal and osmotic stresses, with the increased rapid penetration of methylamine within the core [20].The literature also confirms the synergic action of ozone with antibiotic therapy in vivo [21][22][23][24][25][26].
The in vitro ozone bactericidal activity is compromised when applied to blood and blood derivatives [27].
The aim of this study is to evaluate the ozone bactericidal activity on different bacterial strains and media under different circumstances.

Microorganisms and Media
These experiments were divided into three phases according to the suspension media used: saline, nutrient broth (BHI) and whole blood.
We focused our study on the four multi-resistant bacterial strains frequently found in nosocomial sepsis [28][29][30][31][32][33].The following bacterial strains were isolated from clinical samples obtained from patients at the Neurosurgery Intensive Care Unit, suspended in glycerol and frozen at −80˚C: The preparation of the bacterial strains was performed using the following steps: 1) Thawing of the bacterial strain for testing; 2) Seeding culture medium Trypticase Soy Agar (TSA); 3) Incubation at 37˚C for 18 -24 hours; 4) Suspension of the bacterial strains in saline and dilution until reaching 0.2 MF (McFarland); 5) Using a drop (0.02 ml) of the bacterial suspension; 6) Inoculating into 2.5 ml of the selected medium.

Ozone Generation
A Medica-srl machine, model E80, was used to generate medical ozone (Ozonline International, Medica S.r.l.Via Sante Vincenzi, 48 -40138 Bologna Italy) from oxygen and electricity.The machine converts medical oxygen into a mixture of O 3 (0.05%) and O 2 (99.95%) through an electrochemical process.The Medica-srl machine is equipped with a photometer, calibrated according to the classic iodometric titration of ozone, and a voltage system which regulates the concentration within a range from 5 to 80 µg/ml.
In all the experiments, we used an ozone concentration of 80 µg/ml, corresponding to the maximum concentration within the therapeutic range recommended in vivo [34][35][36][37][38].
The volume of the O 2 /O 3 gas mixture used for each insufflation was 6 ml, corresponding to 480 µg of ozone.

Suspension Medium: Saline
The four bacterial strains were suspended in saline and diluted until reaching 0.2 MF in a final volume of 2.5 ml for each suspension.The obtained bacterial suspensions were sown onto TSA culture medium (Bio-Mérieux Italia) as (T 0 ).We standardized the inoculum using a calibrated loop of 10 µl and seeding in four quadrants.We placed a 21-gauge needle (Troge/Hamburg), premounted with a three-way cock (Axel S.r.l.connector), on the tubes containing the bacterial suspensions (BD 7-ml Vacutainer Red tube, Belliver Industrial Estate, Plymouth), thereby avoiding any leakage of the gas during the insufflation ("insufflation in closed tube").
Because of excessive pressure, the insufflation could open the tubes.To avoid this problem, we aspirated the air from the tubes using a 60-ml syringe (Luer Lock Omnifix/B.Braun) for a total of 360 ml, creating a vacuum.
We proceeded with the insufflation of 6 ml of O 2 /O 3 mixture.
This procedure was applied in all experiments.The tubes were subsequently shaken in a monodirectional oscillator for 40 minutes.
The experiment was terminated with a second seeding (T 1 ).

Suspension Medium: Brain Heart Infusion (BHI)
The bacterial strains were suspended in saline, diluted until 0.2 MF and suspended in 2.5 ml of BHI (Bio-Mérieux Italia).
We proceeded with the insufflation of 6 ml of O 2 /O 3 mixture.
The tubes were subsequently shaken in a monodirectional oscillator for 40 minutes.
The experiment was terminated with a second seeding (T 1 ).

Suspension Medium: Whole Blood
We performed the same procedure on medium containing the fresh whole blood of healthy donors.The blood was collected using a 21-gauge butterfly needle (Pic Indolor, Mirage, Artsana S.p.a.Grandate, CO, Italy) and BD Vacutainer Light Blue tubes containing citrate (Belliver Industrial Estate, Plymouth).
We performed seven different experiments: 1) We inoculated the bacterial suspensions in 2.5 ml of whole blood; the obtained bacterial suspensions were subsequently sown onto TSA culture media (Bio-Mérieux Italia) as (T 0 ).
2) We inoculated the bacterial suspensions in 2.5 ml of whole blood; the obtained bacterial suspensions were subsequently sown onto TSA culture media (Bio-Mérieux Italia) as (T 0 ).
3) We inoculated the bacterial suspensions in 2.5 ml of whole blood, and the obtained bacterial suspensions were subsequently sown onto TSA culture media (Bio-Mérieux Italia) as (T 0 ).

5)
We inoculated the bacterial suspensions in 2.5 ml of whole blood, and the obtained bacterial suspensions were subsequently sown onto TSA culture media (Bio-Mérieux Italia) as (T 0 ).
The experiment continued according to the following steps: Before and after each experiment on whole blood, we performed a complete blood count.
All experiments, regardless of the suspension medium, were consistent with a control bacterial growth curve obtained without ozone.

Statistical Analysis
The results were expressed as the means ± SD of three independent measurements for each experiment.The statistical evaluations were performed using the statistical software SPSS ver.10.Significance was defined as a P value < 0.05.

Suspension Medium: Saline
After contact with the O 2 /O 3 mixture, we observed a total reduction of the bacterial load and the absence of growth for all four bacterial strains under examination (Figure 1).

Suspension Medium: Whole Blood
We performed seven experiments, adding ozone to the bacterial suspensions to verify the total dosage and intervals between insufflations.
E. coli: reduction of bacterial growth at T 1 (5') and total absence of colonies at T 2 and T 3 .Bactericidal activity (Figure 2).
E. coli: reduction of the bacterial growth at T 1 (5') and total absence of colonies at T 2 .Bactericidal activity (Figure 3).
Experiment III -Ozone: 480 µg × 3; total time of mechanical agitation: 45'.Staph.aureus and Ent.faecalis remained in the two quadrants in each seeding, while Ps.aeruginosa showed a growth reduction at T 1 (5') and also remained in one quadrant at T 2 (45').Bacteriostatic activity.
Ent. faecalis: although a higher susceptibility to ozone than the first two bacteria was observed, these bacteria showed an evident resumption of growth at T 4 (65').
The results for Staph.aureus and Ps.aeruginosa were similar: after an evident reduction at T 2 , the bacterial growth resumed.
Ent. faecalis: bacterial growth was observed in three quadrants at T 0 and in two quadrants at T 1 (5'); the growth returned in three quadrants at T 2 (20') and remained constant until the last seeding (T 4 : 80').Bacteriostatic activity.
E. coli: the evident reduction of bacterial growth at T 1 (5') and total absence of colonies in subsequent seedings.Bactericidal activity (Figure 8).
The blood counts showed no significant changes.The curves of the control bacterial growth showed a progressive increase in the bacterial growth.

Discussion
The study was divided into three phases, according to the suspension medium used for the bacterial cultures (saline, BHI and whole blood), to understand the interaction between the ozone, bacterium and the suspension medium.Indeed, the milieu in which microbes are present determines the effectiveness and outcome of the ozone treatment [39].
As shown in previous studies [7,11,16], we confirmed the ozone bactericidal activity on all bacterial strains when suspended in saline.Interestingly enough, ozone loses bactericidal activity with BHI suspension medium.
The results are interesting and discordant when the suspension medium is whole blood.Burgassi et al. observed that fresh plasma (as a biological substance with antioxidant systems), compromises ozone bactericidal activity.
Moreover, these authors suggested the incompatibility of using whole blood because it coagulates in the presence of a bacterial suspension [27].
To overcome this technical problem, we used tubes containing citrate, the same anticoagulant present in the bags for major ozone autohemotherapy (AHT-O 3 ).Compared to the results reported in the literature, our study on whole blood shows that the single insufflation with an O 2 /O 3 mixture is sufficient to achieve a bacteri-cidal effect on E. coli.We also found that a second insufflation was essential for improving the results; specifically, the most encouraging results were obtained in ex-  periment VI, where we performed a second insufflation at 45 minutes after the first.We observed that the bacterial growth typically resumed after approximately 40 minutes from the first exposure to the ozone oxidative insult, suggesting that the best results were obtained in experiment VI.
Although additional ozone insufflations prolonged bactericidal activity (Staph.aureus, Ent.faecalis, Ps. aeruginosa), the effects of these treatments were not significantly different from those obtained in the experiments with only two insufflations.
Based on the differential results obtained on whole blood between E. coli and Ps.aeruginosa, and the failure of the experiments on broth culture, suggested that the mechanism of ozone, contrary to the literature [19,20], could also have a metabolic basis.On the other hand, changes in the size and morphology of the colonies, observed in all the bacterial strains after exposure to ozone, confirm the oxidative mechanism.
The bactericidal activity on E. coli, contrasts with the increased effectiveness of ozone against Gram-positive bacteria [19].The primary target of ozone on E. coli is the sulfhydryl group in the bacterial membrane [40].Thus, we confirmed the correlation between ozone bactericidal activity and membrane permeability, which is specific for each microorganism [39].
The results of our study on whole blood represents an important confirmation of the ozone bactericidal activity in the topical treatment of wounds, and the daily persistence of topical ozone preparations kills even the most resistant bacteria [25,41].
The in vitro results were not consistent with the observations of AHT-O 3 in vivo, except for the portion of blood directly exposed to ozone.In fact, these results are consistent with those of Burgassi et al. [27], showing that the AHT enriched with ozone, even at highest ozone concentration (80 µg/ml), is not able to oxidize and destroy circulating bacteria in the blood.
However, the evaluation of the ozone bactericidal activity on whole blood performed in the present study in-
To clarify the mechanism of ozone, it would be useful to examine its affects on other Enterobacteriaceae (suspension medium: whole blood), such as Serratia and Klebsiella, and Aspergillus.Indeed, Aspergillus produces large colonies whose variations in size or color might be an important index for metabolic alterations.
In conclusion, we confirmed the bactericidal efficacy of topical ozone preparations, according to the evidence of antibody-catalyzed ozone formation in bacterial killing [57], and suggest the need for further evaluations of the therapeutic potential of AHT-O 3 .

Conclusion
Further in vitro investigation on whole blood might reveal the synergic action between traditional antibiotics and ozone treatment, and might provide the basis for developing successive studies in vivo in patients affected through multidrug resistant nosocomial infections.