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Is hyperbaric oxygen or ozone effective

in experimental endocarditis?

Muhammed Turgut Alper Özkan, MD,a,* Ahmet Vural, PhD,b

Ömer Faruk Çiçek, MD,c Ali Ümit Yener, MD,a Sedat Özcan, MD,a
Hüseyin Toman, MD,d Ahmet Ünver, PhD,b and Mustafa Saçar, MDa
a
Department of Cardiovascular Surgery, Çanakkale Onsekiz Mart University, Çanakkale, Turkey
b
Department of Microbiology, Çanakkale Onsekiz Mart University, Çanakkale, Turkey
c
Department of Cardiovascular Surgery, Ankara Yuksek Ihtisas Hospital, Ankara, Turkey
d
Department of Anesthesiology, Çanakkale Onsekiz Mart University, Çanakkale, Turkey

article info abstract

Article history: Background: Infective endocarditis, a disease with high mortality and morbidity, is most
Received 7 August 2015 commonly caused by Staphylococcus aureus; mortality and morbidity further increase in
Received in revised form the presence of methicillin-resistant strains of S. aureus. Linezolid is the first of the oxa-
2 December 2015 zolidinones, a new antibiotic group that has been approved for the treatment of infections
Accepted 8 December 2015 caused by gram-positive cocci. Linezolid reduces the quantity of microorganisms in
Available online 15 December 2015 vegetation to some extent; in addition, the use of hyperbaric oxygen (HBO) and ozone (O3)
therapies is likely to improve targeted antibacterial effect.
Keywords: Materials and methods: Fifty-six adult male Wistar rats weighing 300e350 g were used. The
Cardiovascular surgery subjects were divided into groups as follows: Group 1 (n ¼ 8): control group that was not
Hyperbaric Oxygen inoculated with microorganisms and was untreated; Group 2 (n ¼ 8): control group that was
Ozone inoculated with microorganisms but was untreated; Group 3 (n ¼ 8): linezolid treatment
Linezolide group; Group 4 (n ¼ 8): O3 therapy group; Group 5 (n ¼ 8): HBO therapy group; Group 6 (n ¼ 8):
Endocarditis linezolid þ O3 therapy group; Group 7 (n ¼ 8): linezolid þ HBO therapy group.
Results: In terms of reducing the number of colonies in the aortic valve, linezolid þ HBO
therapy was found to be the most effective treatment. Then, respectively linezolid þ O3,
linezolid, HBO, and O3 were found to be effective.
Conclusions: We found that linezolid significantly reduced the number of bacteria in the
vegetation in the experimental endocarditis model, and HBO therapy increases the effec-
tiveness of linezolid and makes this better than O3.
ª 2016 Elsevier Inc. All rights reserved.

1. Introduction treatment for endocarditis is vancomycin; however, resis-

Infective endocarditis, a disease with high mortality and
morbidity, is most commonly caused by Staphylococcus aureus;
mortality and morbidity further increase in the presence of
methicillin-resistant strains of S aureus (MRSA). The standard
tance rates have been gradually increasing in recent years
[1e5], and the risk of adverse effects of the drug is extremely
high. Linezolid is one of the extremely limited good alterna-
tives to vancomycin. Linezolid is the first of the oxazolidi-
nones, a new antibiotic group that has been approved for the

* Corresponding author. Department of Cardiovascular Surgery, Faculty of Medicine, 18 Mart University, Çanakkale, Turkey. Tel.: þ90 505
610 4606; fax: þ90 286 218 3738.
E-mail address: mtaozkan@yahoo.com (M.T.A. Özkan).
0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jss.2015.12.006
 j o u r n a l o f s u r g i c a l r e s e a r c h 2 0 2 ( 2 0 1 6 ) 6 6 e7 0
treatment of infections caused by gram-positive cocci. In our
earlier study on an experimental endocarditis model, linezolid
significantly reduced the number of bacteria in vegetations,
but, it did not provide complete sterilization [6]. This suggests
the need for adjuvant therapy. Linezolid reduces the number
of colonies of microorganisms in vegetations to some extent
[7], but the additional use of hyperbaric oxygen (HBO) and
ozone (O3) therapies is likely to improve the targeted anti-
bacterial effect.
HBO therapy can be simply described as breathing 100%
oxygen at pressures greater than 1 atm in a totally closed
chamber [8]. It is synergistic and additive to the effects of
some antimicrobial agents [9]. The efficacy of HBO therapy for
experimental mediastinitis and endocarditis has been re-
ported [10,11].
O3 is an unstable molecule consisting of three oxygen (O)
atoms [12]; it is also a strongly oxidizing gas with antibacterial
properties, which provide additional support for different
antibiotics [13,14]. In the health sector, O3 therapy is based on
the application of an O2/O3 mixture in an amount that does
not exceed the antioxidant capacity of biological systems.
This study aimed to investigate whether HBO or O3 therapy
increases the efficacy of linezolid in the treatment of experi-
mental endocarditis. This study is expected to contribute to
the treatment of endocarditis.
2. Materials and methods
The methods used for animal experiments were in accor-
dance with the international guiding principles for biomedical
research involving animals recommended by the World
Health Organization. Permission was granted by Canakkale
Onsekiz Mart University Animal Experiments Local Ethics
Committee (protocol number: 2013/05-05, date of approval:
May 30, 2013). This study was conducted in Canakkale Onsekiz
Mart University Experimental Research Center.
In the study, 56 adult male Wistar rats weighing 300e350 g
were used. We determined the minimum number of rats so as
to accurately reflect the study results and provided normal
food and water to the rats ad libitum.
The rats were anesthetized by the intramuscular admin-
istration of a 2:1 mixture of 0.75 mg/kg of ketamine hydro-
chloride (100 mg/mL) and xylazine hydrochloride (20 mg/mL).
A polyethylene catheter was passed from the right carotid
artery to the left ventricle to injure the aortic valve. Insertion
of the catheter caused aortic lesions and induced bacteremia,
which led to the development of aortic valve vegetations
[15,16]. After 18e24 h, all animals, except those in the non-
contaminated control group, were intravenously adminis-
tered 106 colony-forming units (CFU) of MRSA in 1 mL of 0.9%
NaCl. The catheters were left in place during the entire study.
The rats in the untreated groups (contaminated and non-
contaminated groups) were killed 48 h after bacterial inocu-
lation to determine the microbial load. Treatment was started
48 h after inoculation and lasted for 5 d. The rats in the
treatment groups were killed 12 h after the last dose.
The subjects were divided into groups as follows:
Group 1 (n ¼ 8): control group that was not inoculated with
microorganisms and was untreated.
67
Group 2 (n ¼ 8): control group that was inoculated with
microorganisms but was untreated.
Group 3 (n ¼ 8): linezolid treatment group.
Group 4 (n ¼ 8): O3 therapy group.
Group 5 (n ¼ 8): HBO therapy group.
Group 6 (n ¼ 8): linezolid þ O3 therapy group.
Group 7 (n ¼ 8): linezolid þ HBO therapy group.
The treatment protocols are summarized in Table 1.
2.1. Assessment of infection
After the extracted vegetations were weighed and titrated in
sterile tubes, serial dilutions were performed by homogenizi-
ng the vegetations in 1 mL of sterile phosphate saline buffer.
These solutions were plated on blood agar plates with 7%
sheep blood. All plates were incubated for 48 h at 37 C.
Isolated colonies were evaluated for MRSA. The number of
bacteria was determined and calculated for each plate in
terms of CFU/g tissue.
3. Statistics
Quantitative variables were presented as arithmetic
mean  standard deviation of log10 CFU/g of vegetation.
Differences among the groups were investigated using the
Student t or ManneWhitney U test for continuous variables.
KruskaleWallis testing was used for comparison between the
groups. A P value < 0.05 was considered statistically signifi-
cant. All statistical studies were carried out with the Statistical
Package for the Social Sciences (SPSS) program (version 15.0,
SPSS, Chicago, IL).
4. Results
No sign of infection was observed on the valves of the rats in
the non-contaminated group (Group 1); there was also no
bacterial growth in cultures. Purulence was observed on the
valve leaflets of the rats in the contaminated group. The dis-
tribution of CFU values according to the groups is summarized
Table 1 e Treatment protocols.
Agent Route of administration
Linezolid Intravenously, 75 mg/kg/8 h on the first day; then
75 mg/kg/12 h. Treatment was continued for 5 d.
Ozone O3/O2 gas mixture was given intraperitoneally
0.7 mg/kg. O3 production was performed with an
ozone generator, which is in commercial use. O3/O2
(97% O2, 3% O2) flow speed was kept constant at 3 L/
min, at a concentration of 60 mg/mL. Tygon polymer
tubes and silicone-coated polypropylene disposable
syringes were used for continuous administration of
O3 at the desired concentration. Treatment was
continued for 5 d.
Hyperbaric Hyperbaric oxygen therapy at 2.8 atmospheric
oxygen pressure was given to this group of rats for 90 min
every day. Treatment was continued for 5 d.
68 j o u r n a l o f s u r g i c a l r e s e a r c h 2 0 2 ( 2 0 1 6 ) 6 6 e7 0

in Table 2. Comparison between groups showed significantly

Table 3 e Comparisons between groups.
different number of bacteria (P < 0.05; Table 3). The pairwise
comparisons are summarized in Table 4. Groups CFU (log 10)
The number of colonies in Groups 3, 5, 6, and 7 was n Mean  SD
significantly lower than that in the control group (P ¼ 0.023,
Contaminated group 8 9.6  0.9
0.002, 0.001, and 0.001, respectively). There was no statistically
Linezolid group 8 7.4  0.9
significant difference between the O3 group and the control Ozone group 8 9.1  0.7
group (P > 0.05). The number of colonies in Groups 4 and 5 was Hyperbaric oxygen group 8 8.5  0.6
not significantly different (P > 0.05). Compared with the linezolid þ ozone group 8 5.9  1.1
number of colonies in Group 4, the number of colonies in linezolid þ HBO group 8 4.8  0.5
Groups 3, 6, and 7 were significantly lower (P ¼ 0.003, 0.001, pa 0.001

and 0.001, respectively). CFU ¼ colony-forming units; HBO ¼ hyperbaric oxygen; SD ¼

The number of colonies in Group 5 was lower than that in standard deviation.
a
the control group but was significantly higher than that in KruskaleWallis test.
Groups 3, 6, and 7 (P ¼ 0.024, 0.002, and 0.001, respectively).
The number of colonies in Groups 6 and 7 was significantly
lower than that in Group 3 (P ¼ 0.015 and 0.001, respectively).
The number of colonies in Group 7 was significantly lower Currently, drug resistance is managed by the use of
than that in Group 6 (P ¼ 0.015). The number of colonies of different antibiotics or combinations of antibiotics, which has
linezolid þ HBO group was significantly lower than all other led to an increase in renal or hepatic side effects [20]. The use
groups (P ¼ 0.001). of adjuvant therapy instead of additional antibiotics may
reduce the incidence of such adverse effects. Normally, the
addition of adjuvant therapy enhances the efficacy and re-
duces the needed dose of an antibiotic, leading to reduced
5. Discussion potential for renal and hepatic side effects, as well as a lower
cost of treatment. However, it remains necessary to thor-
In this study, a combination of linezolid and HBO was deter-
oughly investigate the efficacy and side effects of adjuvant
mined as the most effective treatment for experimentally
therapy options.
induced endocarditis. It was seen that O3 treatment or HBO
HBO therapy centers are increasingly widespread. At these
therapy alone was not sufficient.
centers, HBO therapy is applied for conditions such as stasis
Although there are insufficient data on the use of line-
ulcer, osteomyelitis, crush syndrome, carbon monoxide
zolid for the treatment of endocarditis, the current results
poisoning, decompression, gas embolism, cyanide poisoning,
are contradictory. Some studies have reported that linezolid
and acute cerebral edema [21]. The number of O3 therapy
was extremely successful in the treatment of endocarditis,
centers is increasing but not as much as the number of HBO
but, many have reported partial success or failure because of
therapy centers. These centers perform O3 therapy for wound
the bacteriostatic effect [17e19]. However, when vancomycin
healing and acceleration of metabolism as well as for anti-
is contraindicated due to resistance or side effects, linezolid
oxidant, antiaging, and antiallergic effects [22]. The increase
is currently one of the two drugs that can be used in our
in the number of these centers facilitates the evaluation of
country. In this study, we aimed to improve the efficacy of
HBO and O3 as adjuvant therapies for infections.
linezolid using adjuvant therapies such as HBO and O3.
Oxygen may enhance the bactericidal or bacteriostatic ef-
Although there are studies that investigated HBO and O3
fects of antibiotics. HBO therapy can play an important role in
adjuvant therapies for infections, there are only a few
the treatment of many patients with bacterial infections.
studies that compared both. There has been no article that
There are many studies that have reported the wound healing
investigated the effect of HBO and O3 therapies for infective
and antimicrobial effects of HBO [23e26]. However, appro-
endocarditis.
priate selection of the pressure level used for HBO therapy is
also important. In one study, lower pressure (2.5 atm) was
used as a regeneration stimulus, whereas higher pressure
(2.8 atm) was used for antibacterial support [27]. In another
Table 2 e CFU values of groups. study, HBO was applied at a pressure of 2.8 atm for necrotizing
Group CFU (log10) soft-tissue infections [28]. In our study, HBO therapy was
applied at 2.8 atm to aim for antibacterial effects.
n Min Max Median Mean
Yu et al. [29] found that HBO alone improved the treatment
Group 1, (non-contaminated) 8 d d d d of sternal infections and osteomyelitis after cardiac surgery.
Group 2, (contaminated) 8 8.1 10.9 9.6 9.6 Inanmaz et al. [30] demonstrated that HBO prophylaxis
Group 3, (linezolid) 8 6.1 8.4 7.1 7.4
reduced the incidence of infection after neuromuscular sur-
Group 4, (ozone) 8 7.9 10.1 9.1 9.1
Group 5, (HBO) 8 7.7 9.4 8.5 8.5
gery. In our study, HBO therapy reduced the signs of infection
Group 6, (linezolid þ ozone) 8 4.6 7.9 5.6 5.9 and bacterial growth, but the effect was not adequate. In our
Group 7, (linezolid þ HBO) 8 4.1 5.4 4.9 4.8 study, linezolid þ HBO therapy was found to be more effective
than linezolid therapy alone. In an experimental mediastinitis
CFU ¼ colony-forming units; HBO ¼ hyperbaric oxygen.
model, adjuvant HBO therapy increased the effectiveness of
 j o u r n a l o f s u r g i c a l r e s e a r c h 2 0 2 ( 2 0 1 6 ) 6 6 e7 0 69

Table 4 e Pairwise comparisons of the groups.

Contaminated Linezolid Ozone group Hyperbaric Linezolid þ ozone Linezolid þ HBO
group group oxygen group group group

Contaminated group Pa ¼ 0.002 P ¼ 0.268 P ¼ 0.023 P¼ 0.001 P ¼ 0.001

Linezolid group P ¼ 0.002 P ¼ 0.003 P ¼ 0.024 P¼ 0.015 P ¼ 0.001
Ozone group P ¼ 0.268 P ¼ 0.003 P ¼ 0.091 P¼ 0.001 P ¼ 0.001
Hyperbaric oxygen group P ¼ 0.023 P ¼ 0.024 P ¼ 0.091 P¼ 0.002 P ¼ 0.001
Linezolid þ ozone group P ¼ 0.001 P ¼ 0.015 P ¼ 0.001 P ¼ 0.002 P ¼ 0.015
Linezolid þ HBO group P ¼ 0.001 P ¼ 0.001 P ¼ 0.001 P ¼ 0.001 P ¼ 0.015

CFU ¼ colony-forming units; HBO ¼ hyperbaric oxygen.

a
Mann-Whitney U test.

linezolid and teicoplanin [31]. Furthermore, the adjuvant use
of HBO therapy for diabetic foot ulcers was found to promote
and improve linezolid absorption in the ischemic area [32].
O3 is therapeutically used in medicine because it effectively
inactivates bacteria, spores, and viruses [33e35]. O3 alone is
used as a surface disinfectant and as an antibacterial agent in
dentistry [36e38]. However, few studies have investigated the
use of O3 alone for systemic infections [39]. O3 as an adjuvant
therapy increases antibacterial activity in urinary tract in-
fections and vulvovaginitis [40]. In another study, O3 increased
the efficacy of antibiotic therapy for mediastinitis caused by
MRSA [41]. Studies on experimental infection models have
shown that O3 was comparably effective to HBO therapy in
reducing oxidative and inflammatory cytokines, in addition to
enhancing the efficacy of antibiotic therapy [42]. In our study,
the treatment efficacy of linezolid þ O3 was significantly better
than linezolid alone or O3 alone. Interestingly, the efficacy of
linezolid þ O3 was similar to the efficacy of linezolid þ HBO.
The antimicrobial mechanism of O3 and HBO may be
associated with the stimulation of inflammatory cytokines,
activation of reactive oxygen radicals, and oxidation of
phospholipids and lipoproteins, which eventually breaks the
membrane integrity of pathogenic bacteria.
HBO and O3 applications are increasing every day, and
indication fields are expanding. Easy access of HBO and O3
facilitates the studies to be done on them, and these studies
are expected to make adjuvant treatment options find a place
in clinical practice.
6. Conclusion
In our study, HBO þ antibiotic therapy was found to be more
effective than O3 þ linezolid and isolated linezolid therapy.
Accordingly, HBO therapy increases the effectiveness of line-
zolid and makes this better than O3. However, to reach a
definitive judgment, more studies are needed to compare O3
and HBO.
Acknowledgment
This study was supported by Canakkale Onsekiz Mart
University Scientific Research Projects Commission (no: TSA-
2013-168).
Authors’ contributions: M.T.A.Ö. and M.S. contributed to
study conception and design. H.T. and A.V. contributed to
acquisition of data. Ö.F.Ç. and A.Ü. contributed to analysis and
interpretation of data. M.T.A.Ö. and A.Ü.Y.contributed to
drafting of the article. M.S. and A.Ü. contributed to critical
revision.
Disclosure
The authors declared no conflicts of interest with respect to
the authorship and/or publication of this article.
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