Hyperbaric oxygen treatment impacts oxidative stress markers in patients with necrotizing soft-tissue infection

Abstract

Necrotizing soft-tissue infection (NSTI) is a rare, severe, and fast-progressing bacterial infection associated with a high risk of developing sepsis or septic shock. Increasing evidence indicates that oxidative stress is crucial in the development and progression of sepsis, but its role in NSTI specifically has not been investigated. Some patients with NSTI receive hyperbaric oxygen (HBO₂) treatment as the restoration of oxidative stress balance is considered an important mechanism of action, which HBO₂ facilitates. However, a gap in knowledge exists regarding the effect of HBO₂ treatment on oxidative stress in patients with NSTI. In the present observational study, we aimed to investigate HBO₂ treatment effects on known markers of oxidative stress in patients with NSTI. We measured plasma myeloperoxidase (MPO), superoxide dismutase (SOD), heme oxygenase-1 (HO-1) and nitrite+nitrate in 80 patients with NSTI immediately before and after their first HBO₂ treatment, and on the following day. We found that HBO₂ treatment was associated with a significant increase in MPO and SOD by a median of 3.4 and 8.8 ng/mL, respectively. Moreover, we observed an HBO₂ treatment-associated increase in HO-1 in patients presenting with septic shock (n=39) by a median of 301.3 pg/mL. All markers were significantly higher in patients presenting with septic shock compared to patients without shock, and all markers correlated with disease severity. High baseline SOD was associated with 90-day mortality. In conclusion, HBO₂ treatment was associated with an increase in MPO and SOD in patients with NSTI, and oxidative stress was more pronounced in patients with septic shock.

Keywords

hyperbaric oxygenation sepsis oxidants

Significance of this study

What is already known about this subject?

▸

There is a lack of knowledge of necrotizing soft-tissue infection (NSTI) pathophysiology. Therefore, treatment of NSTI often relies on available evidence from patients without NSTI with sepsis.

▸

Oxidative stress is key in the development and progression of sepsis. However, markers of oxidative stress have never been examined in septic patients with necrotizing soft-tissue infection.

▸

Hyperbaric oxygen (HBO₂) treatment—an adjunct treatment modality for patients with NSTI—is in general considered to induce oxidative stress, but its oxidative effects in NSTI have not been described.

What are the new findings?

▸

This study reports on the oxidative stress markers myeloperoxidase (MPO), superoxide dismutase (SOD), heme oxygenase-1 (HO-1) and nitrite+nitrate in patients with NSTI before and after receiving HBO₂ treatment.

▸

We show that HBO₂ treatment is associated with an increase in plasma MPO and SOD.

▸

High baseline SOD is associated with increased 90-day mortality in NSTI.

How might these results change the focus of research or clinical practice?

▸

These findings increase our understanding of both NSTI pathophysiology and the mechanism of action of HBO₂ treatment.

Introduction

Necrotizing soft-tissue infection (NSTI) is a rare, severe and rapidly progressing bacterial infection.1 The incidence of NSTI is approximately two per 100,000 inhabitants/year and appears to be increasing.2 3 Most patients with NSTI have sepsis at the time of diagnosis, and approximately 30%–50% deteriorate to septic shock.2 4 The severity of the disease is marked by an amputation rate of 22% and a 90-day mortality of 18%4; survivors often have to cope with prolonged rehabilitation stays5 and functional limitations.6

There is a lack of knowledge of NSTI pathophysiology. Therefore, treatment of NSTI often relies on available evidence from patients without NSTI with sepsis. Oxidative stress has been suggested as a major factor in the development and progression of sepsis and septic shock.7 -9 However, it remains unknown if oxidative stress also plays an essential role in NSTI and to what extent interventions during NSTI management induce oxidative stress.

HBO₂ treatment is used as an adjunctive intervention to standard care in NSTI, due in part to its antimicrobial mechanisms.10 11 HBO₂ treatment regulates oxidative stress by the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS),12 which are believed to destroy the invading bacteria.13 Interestingly, markers of oxidative stress are increased in sepsis non-survivors.14 It has been suggested that ROS plays a crucial part in the innate immune response and that some of their by-products have protective effects in the acute inflammatory response.15 16

Myeloperoxidase (MPO), an oxidative stress marker and key component of the innate immune response and neutrophil-mediated bacterial killing,17 18 is associated with increased mortality in patients with sepsis.19 It is of note that MPO seems to be important in the HBO₂-mediated regulation of oxidative stress, which is required for inhibition of β2-integrin in the endothelial adherence by neutrophils.20 21 Moreover, HBO₂-mediated alterations of the two antioxidants, heme oxygenase-1 (HO-1) and superoxide dismutase (SOD), have been demonstrated in experimental models of sepsis associated acute lung injury, in sepsis, and as part of a protective mechanism against oxidative DNA damage.22 -27 Multiple HBO₂ treatments have been shown to elevate RNS by enhancing production of nitrite+nitrate in selected cohorts,28 but the systemic effect on RNS remains unresolved.29 30 Of notable interest is a SOD-mediated regulatory function of NO expression31 and an inhibitory function of HO-1 on the NO synthase expression,32 indicating closely interconnected regulatory mechanisms between RNS and ROS.

Oxidative stress in patients with NSTI has not been investigated extensively.33 It is therefore unclear if high levels of markers of oxidative stress are associated with increased mortality in NSTI, and if HBO₂ treatment could impact these key factors of the innate immune response. Clinical treatment protocols of HBO₂ involve short bursts of HBO₂, and the resultant innate antioxidant defences have been demonstrated to be sufficient for making biochemical ROS stress a reversible process after HBO₂ treatment.24 34 -38 This antioxidant defence does not lead to human innate immunocompromise when HBO₂ treatment is used in bacterial infection control and sepsis.21 39 Therefore, intermittent hyperoxia mediated by HBO₂ treatment and the resulting elevation in ROS production is considered reversible and harmless to the patient while having the effect of stimulating endogenous antioxidant systems and providing antimicrobial activity. However, the human data on the pathophysiological effects of HBO₂ treatment in patients with NSTI are scarce, and a better understanding of the effects in this group of patients is needed.

We aimed to investigate HBO₂-induced modifications of the oxidative stress markers MPO, SOD, HO-1 and nitrite+nitrate in patients with NSTI, and to associate baseline levels with severity of disease and outcome. We hypothesized that HBO₂ treatment may affect markers of oxidative stress as well as antioxidant scavengers, and that high baseline levels of MPO and SOD are associated with increased mortality in NSTI.

This article was prepared following the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.40

Study design and subjects

This was an observational cohort study of adult patients with NSTI admitted to Copenhagen University Hospital (Rigshospitalet) between February 2013 and March 2017. The cohort consisted of the Danish subgroup of patients that participated in the international multicenter INFECT study (ClinicalTrials.gov, NCT01790698) and included adult patients (≥18 years) with a diagnosis of NSTI, based on surgical findings of necrosis engaging any layer of the soft-tissue compartment; those patients where surgical findings did not reveal signs of NSTI were excluded.4 The present study included 80 randomly selected subjects from this cohort using a computer-generated allocation table from which some data have been presented elsewhere.41

Patient management and data collection

All patients were treated according to our standardized treatment protocol, as previously described in detail elsewhere.4 The treatment protocol aimed to perform a minimum of three sessions of HBO₂ treatment of 90 min duration at 284 kPa (18 m seawater equivalent), with a compression/decompression rate of 5–15 min. It was preferable that two sessions were performed within the first 24 hours from admission. No exact criteria for the termination of HBO₂ treatment was stipulated, but in general, HBO₂ therapy was provided for at least three sessions or could be continued until infection control. All HBO₂ sessions were performed in an intensive care unit (ICU)-capable multichamber (Drass Galeazzi S.p.A., Type HPO4000, HPE50.2.A).

Data on patient characteristics, biochemistry, microbiological findings, disease severity scores (Simplified Acute Physiology Score II (SAPS II) and Sequential Organ Failure Assessment (SOFA) score), amputation, renal-replacement therapy (RRT) and vital status at day 90 were obtained from an electronic database.42 Dedicated personnel sampled blood at scheduled time points through an arterial line. Blood samples were collected in EDTA tubes, placed on ice and centrifuged at 3500 rpm (2400 G) for 10 min. Thereafter, plasma was pipetted into cryotubes and stored at −80°C until analysis.

The present study analyzed plasma samples collected immediately before the first session of HBO₂ treatment (baseline), immediately post the first session of HBO₂ treatment and on the following day (between 08:00 and 12:00).

Elisa

Plasma MPO, SOD and HO-1 were measured using ELISAs (MPO, 501410, Cayman Chemical, Michigan, USA; SOD, ab119520, Abcam, Amsterdam, Netherlands. HO-1: ab207621, Abcam, Amsterdam, Netherlands) according to manufacturers’ instructions. All samples from one patient were measured in duplicates on the same plate. The analysts were blinded from patient and outcome. Samples were prepared in 96-well microtiter plates and absorbances were recorded in a plate reader at 450 nm (Multiskan FC, Thermo Scientific, Copenhagen, DK).

Chemiluminescent assay nitrite and nitrate concentrations were measured from the thawed plasma samples. The frozen samples were thawed and placed in 96-well microtiter plates and analyzed using a modified Griess nitrite/nitrate colorimetric assay (780001, Cayman Chemicals, Michigan, USA) according to manufacturer's instructions and as previously described .43 In short, nitrite concentration was estimated by incubation with the Griess reagent, converting nitrite to a purple azo-compound detected by absorbance at 540 nm in a plate reader (Multiskan FC, Thermo Scientific, Copenhagen, DK). Nitrate was first converted to nitrite by incubation with nitrate reductase and subsequent addition of the Griess reagent to detect total nitrate plus nitrite. The background absorbance was estimated as the absorbance before staining with the Griess reagent and was subtracted from the absorbance of samples stained with the Griess reagent. The nitrate concentration was calculated as the difference between the total nitrite and nitrate concentration.

Outcome measures

The primary outcome was change in MPO before and after HBO₂ treatment. Secondary analyses included changes in SOD, HO-1 and nitrite+nitrate before and after HBO₂ treatment, associations of baseline levels of MPO, SOD, HO-1, nitrite/nitrate with microbiological findings, SOFA score, SAPS II, blood lactate, presence of septic shock at admission, RRT, amputation and 90-day mortality.

Sample size

To our knowledge, plasma levels of MPO have never been examined in patients with NSTI. We based our sample size calculation on a plasma MPO of 60 ng/mL, as found previously in 124 septic intensive care patients that were believed to be comparable to our cohort.19 An HBO₂ treatment mediated modification of MPO activity of approximately 40% has been observed in patients with chronic wounds.20 Using G*Power V.3.1.9.2 (Kiel, Germany) for sample size calculation in a paired design (power 0.90 at a 5% significance level), we found that a minimum of 41 patients should be included. In order to use all wells in the ELISA plate, thereby streamlining the laboratory analyses and to comply with the above-stated uncertainties, we chose to include 80 patients. This number of subjects was defined in a parallel experiment.41

Statistical analysis

Categorical data were presented as absolute numbers with percentage (%). Continuous data were presented as medians with IQR. As data were not normally distributed, we used Wilcoxon rank-sum test for non-paired continuous data and Wilcoxon signed-rank test for paired data (before/after HBO₂ treatment). Correlations were examined using Spearman's rank correlation test. Logistic regression analysis using the Youden Index optimal cut-off point44 was performed to assess the association between baseline MPO, SOD, HO-1, nitrite+nitrate levels and 90-day mortality, and the results were addressed with OR and 95% CI. Receiver operating characteristic (ROC) curves were analyzed, and diagnostic accuracy in predicting 90-day mortality were evaluated using area under the curve (AUC), sensitivity, specificity, positive-predictive value and negative-predictive value (NPV). Patient lost to follow-up were excluded from regression analysis.

P values were reported as exact unless <0.001. P values below 0.05 were considered statistically significant. Statistical analyses were performed using RStudio V.1.0.153 (RStudio). Figures were prepared in GraphPad Prism V.8.0.2. (GraphPad, La Jolla, California, USA).

Results

A total of 260 patients with NSTI were included at the Rigshospitalet, Copenhagen University Hospital, Denmark, between February 2013 and March 2017. Of these, 224 (86%) received HBO₂ treatment as part of their standardized NSTI treatment protocol, and from this group, we randomly selected 80 patients for inclusion in the present study. Patient characteristics are presented in table 1. Baseline blood samples (before the first HBO₂ treatment) were collected after a median of 4 hours and 42 min (3 hours 20 min–8 hours 15 min) from admission. One patient was lost to follow-up at day 90, resulting in an overall follow-up rate of 98.8%. Additional description of the included cohort has been published previously.41

Table 1

Patients’ characteristics

Characteristics	NSTI, n=80
Age (years)	61 (52—68)
Sex, male	52 (65)
BMI (kg/m²)	26 (24—31)
Comorbidities
Cardiovascular disease	35 (44)
Chronic kidney disease	5 (6)
COPD	6 (8)
Diabetes	21 (26)
Immune deficiency	2 (3)
Liver cirrhosis	1 (1)
Malignancy	7 (9)
Peripheral vascular disease	11 (14)
Rheumatoid disease	4 (5)
Polyicrobial versus monomicrobial
Monomicrobial infections	30 (37)
Polymicrobial infections	42 (53)
Negative findings	8 (10)
Biochemistry
Leukocyte count (10⁹/L)	15 (10–22)
C reactive protein (mg/L)	221 (148–324)
Creatinine (µmol/L)	109 (78–181)
Lactate (mmol/L)	2.3 (1.3–3.3)
Other
SOFA score*	8 (6–10)
SAPS II^†	44 (35–51)
LRINEC^‡	8 (7–10)
Septic shock on admission^§	39 (49)
Amputation within 7 days	13 (16)
RRT within 7 days	12 (15)
Mortality, day 90	12 (15)

Continuous data are presented as medians (IQR) and categorical data as absolute numbers (percentage, %).

*SOFA score, day 1; data were missing for three (4%) patients.

†Data were missing for three (4%) patients.

‡Data were missing for eight (10%) patients.

§Septic shock defined as lactate>2 mmol/L and use of vasopressor or inotrope.

BMI, Body Mass Index; COPD, chronic obstructive pulmonary disease; LRINEC, Laboratory Risk Indicator for Necrotizing Fasciitis Score; NSTI, necrotizing soft-tissue infection; RRT, renal-replacement therapy; SAPS II, Simplified Acute Physiology Score II; SOFA, Sequential Organ Failure Assessment.

Baseline MPO, SOD, HO-1 and nitrite +nitrate was 160.5 ng/mL (101.4—230.4), 118.1 ng/mL (74.5—270.2), 7575 pg/mL (5,113—13,416) and 3.6 µM (1.6—6.9) respectively. Correlations between baseline MPO, SOD, HO-1 and nitrite +nitrate are presented in online supplemental materials (online supplemental table 2).

Table 2

Effects of HbO₂ treatment on ROS and RNS

Plasma ROS/RNS	Median difference
	Immediately before to after HBO₂			After HBO₂ to follow-up sample
	All	Septic shock	Non-shock sepsis	All	Septic shock	Non-shock sepsis
MPO (ng/mL)	3.4* ↑	4.6	3.4* ↑	−11.0* ↓	−14.2* ↓	1.6
SOD (ng/mL)	8.8* ↑	−1.9	14.4	−19.1** ↓	−24.4* ↓	−10.1* ↓
HO-1 (pg/mL)	17.9	301.3* ↑	−172.3	−11.6	−513.5	65.6
Nitrite+nitrate (µM)	0.05	0.16	0.01	−0.19	0.01	−0.20

Data are compared immediately before and after the first HbO₂ treatment session (left), as well as after the first HbO₂ treatment session and following day (right). Differences are presented as median differences across the overall cohort (n=80), patients admitted in septic shock (n=39) and patients without septic shock (n=41). Superscripts indicate level of statistical significance performed by Wilcoxon signed-rank test. A positive value indicates significant upregulation (↑); a negative value indicates significant downregulation (↓).

**P<0.05, **P<0.01.

HBO₂, hyperbaric oxygen; HO-1, heme oxygenase-1; MPO, myeloperoxidase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase.

Effects of HBOT on ROS and RNS

Paired analysis of MPO found a significant increase from baseline to post-HBO₂ treatment by a median of 3.4 ng/mL, and a subsequently decreased of −11.0 ng/mL on the following day (table 2). No significant difference was observed from baseline HBO₂ treatment to the following day (data not shown, p=0.30).

In addition, SOD increased significantly from baseline to post-HBO₂ treatment with a subsequent decrease on the following day (table 2). SOD was significantly lower on the following day, compared with baseline by a median of −14.5 ng/mL (p=0.02).

No immediate effect was observed on HO-1 in the overall cohort, but in the subgroup of patients presenting with septic shock (n=39), HO-1 was found to increase significantly from baseline to post-HBO₂ treatment (table 2). No difference was observed from baseline to the following day (data not shown, p=0.27). No differences were observed in nitrite+nitrate levels (table 2).

Association between ROS and RNS levels and NSTI severity and outcome

Patients with septic shock at admission (n=39) had significantly higher baseline MPO, SOD, HO-1 and nitrite+nitrate compared with patients without shock (MPO: 197.3 vs 140.1 ng/mL, SOD: 207.2 vs 89.3 ng/mL, HO-1: 10 402 vs 6692 pg/mL, nitrite+nitrate: 4.6 vs 2.7 µM; figure 1). This was in line with the correlation analysis that demonstrated significant correlations between MPO, SOD, HO-1, nitrite+nitrate and SOFA score (table 3). Additionally, SOD, HO-1 and nitrite+nitrate was found to correlate with SAPS II, whereas MPO, SOD and HO-1 correlated with baseline blood lactate levels (table 3).

Figure 1

Baseline MPO, SOD, HO-1 and nitrite +nitrate levels according to; septic shock vs non-shock, amputation vs no amputation and RRT vs no RRT. Medians with interquartile ranges are illustrated. HO-1, heme oxygenase-1; MPO, myeloperoxidase; RRT, renal-replacement therapy; SOD, superoxide dismutase.

Table 3

Spearman rank correlation between severity of disease and baseline ROS and RNS levels

	SAPS II		SOFA		Lactate
	Rho	P value	Rho	P value	Rho	P value
MPO	0.16	0.15	0.40	<0.001	0.33	0.003
SOD	0.55	<0.001	0.70	<0.001	0.59	<0.001
HO-1	0.35	0.003	0.68	<0.001	0.39	<0.001
Nitrite+nitrate	0.27	0.02	0.25	0.03	0.15	0.19

HO-1, heme oxygenase-1; MPO, myeloperoxidase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPS II, Simplified Acute Physiology Score II; SOD, superoxide dismutase; SOFA, Sequential Organ Failure Assessment.

Patients undergoingng an amputation had higher levels of SOD (284.5 vs 110.6 ng/mL) and HO-1 (14 376 vs 6829 µM) at admission compared with non-amputee patients (figure 1). Patients who received RRT within the first 7 days from admission had higher baseline MPO (286.9 vs 172.0 ng/mL), SOD (472.8 vs 108.7 ng/mL) and HO-1 (19 471 vs 7575 pg/mL) compared with those not who did not receive RRT (figure 1).

In univariate and age-sex adjusted logistic analysis, high baseline SOD was found to be an independent predictor of 90-day mortality (table 4). However, in age–sex-SOFA score adjusted analysis no markers were found to be associated to 90-day mortality. Diagnostic accuracy of the studied markers in predicting mortality is shown in table 5. ROC curves of 90-day mortality according to baseline MPO, SOD, HO-1 and nitrite+nitrate are displayed in online supplemental figure 1.

Table 4

Univariate and multivariate logistic regression analyses of 90-day mortality based on low versus high baseline ROS and RNS levels according to the optimal cut-off values

	Unadjusted			Adjusted analysis: age and sex			Adjusted analysis: age, sex and SOFA Score
	OR	95% CI	P	OR	95% CI	P value	OR	95% CI	P value
MPO
Low <320.4	1 Ref.			1 Ref.			1 Ref.
High ≥320.4	1.87	0.37 to 7.62	0.41	1.51	0.28 to 6.55	0.59	0.38	0.04 to 2.26	0.31
SOD
Low <114.9		1 Ref.			1 Ref.			1 Ref.
High ≥114.9	12.1	2.16 to 226.92	0.02	9.91	1.67 to 190.41	0.04	4.24	0.52 to 92.97	0.23
HO-1
Low <7891		1 Ref.			1 Ref.			1 Ref.
High ≥7891	4.88	1.11 to 33.95	0.06	3.66	0.75 to 26.74	0.14	1.14	0.13 to 11.22	0.90
Nitrite+nitrate
Low <18.62		1 Ref.			1 Ref.			1 Ref.
High ≥18.62	1.48	0.20 to 7.02	0.65	1.73	0.23 to 8.99	0.54	0.94	0.11 to 5.41	0.95

HO-1, heme oxygenase-1; MPO, myeloperoxidase; Ref., reference; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; SOFA, Sequential Organ Failure Assessment.

Table 5

Accuracy of high baseline biomarker (defined by being above the optimal cut-off point) level in predicting 90-day mortality

	MPO	SOD	HO-1	Nitrite+nitrate
Sensitivity	0.25 (0.05–0.57)	0.92 (0.62–1.00)	0.80 (0.44–0.97)	0.17 (0.02–0.48)
Specificity	0.85 (0.74–0.92)	0.52 (0.40–0.65)	0.55 (0.42–0.68)	0.88 (0.78–0.95)
PPV	0.23 (0.05–0.54)	0.26 (0.14–0.42)	0.22 (0.10–0.39)	0.20 (0.03–0.56)
NPV	0.85 (0.74–0.92)	0.97 (0.85–1.00)	0.94 (0.81–0.99)	0.86 (0.75–0.93)
AUC-ROC	0.48 (0.31–0.66)	0.69 (0.51–0.87)	0.69 (0.49–0.88)	0.61 (0.43–0.79)

Data are presented as fractions (95% CI).

AUC-ROC, area under the receiver operating characteristic curve; HO-1, heme oxygenase-1; MPO, myeloperoxidase; NPV, negative predictive value; PPV, positive predictive value; SOD, superoxide dismutase.

Microbiological findings and levels of ROS and RNS

Forty-two (n=42) patients had polymicrobial infection, 30 monomicrobial infection and eight patients had no microbial findings in culture from either blood or soft tissue. No differences were found at baseline between polymicrobial and monomicrobial infections (MPO: 159.6 vs 174.9 ng/mL, p=0.87; SOD: 112.8 vs 157.5 ng/mL, p=0.48; HO-1: 7733 vs 9585 pg/mL, p=0.35; nitrite +nitrate: 2.95 vs 3.82 µM, p=0.36). Patients with group A Streptococcus NSTI (n=19) had higher levels of baseline MPO compared with non-group A Streptococcus NSTI (223.4 vs 160.5 ng/mL, p=0.01). No differences were found in SOD (p=0.21), HO-1(p=0.32) and nitrite +nitrate levels (p=0.06) in patients infected with group A Streptococcus compared with to other microbes (data not shown).

Discussion

This observational study of 80 patients with NSTI is the first to demonstrate that HBO₂ treatment is associated with an overall increase in MPO and SOD, and an increase in HO-1 in a subgroup of patients with NSTI with septic shock. Moreover, we observed markers of oxidative stress to be associated with disease severity, and a high baseline SOD to be associated with 90-day mortality.

MPO and SOD are essential in oxygen-dependent bacterial killing by human neutrophils.45 46 Studies indicate that oxidative stress is crucial for the release of MPO from neutrophils, monocytes and macrophages,47 48 and that superoxide may serve as a substrate that MPO use to elicit killing of the ingested bacteria in the neutrophil phagosomes.49 50 SOD possesses fundamental antioxidant effects51 and its activity has been found to be decreased in patients with sepsis admitted to intensive care.52 In the present study, we observed that HBO₂ treatment was associated with an increase in levels of MPO and SOD, suggesting that HBO₂ treatment may promote immunomodulatory effects by inducing oxidative stress and increase antioxidant capacity in patients with NSTI, as demonstrated by the initial upregulation of MPO and SOD.

HO-1 plays a fundamental role in control of bacterial infections.53 Experimental sepsis models have indicated that deficiency of HO-1 increases mortality risk.54 55 Interestingly, HBO₂ treatment has been shown to increase HO-1 in sepsis models.22 23 We were unable to detect any overall fluctuations in plasma HO-1 in relation to the first HBO₂ treatment session. However, we did observe significant differences in HO-1 responses between patients in septic shock and those not in septic shock, suggesting that the effect of HBO₂ treatment may differ across patients with various degrees of host response to infection. In this context, clinical studies indicate that the treatment effect of HBO₂ may be more pronounced in the most severely affected patients with NSTI.56 57 We also observed that patients in septic shock had the highest level of MPO and SOD at baseline, as well as the greatest decrease after HBO₂ to the following day but without any significant increase immediately before or after HBO₂, which is in contrast to the patients without shock admitted with a lower level of oxidative stress, but who were able to increase in MPO and SOD from before to after HBO₂.

It should be noted that in patients with septic shock, HBO₂ treatment caused super induction of HO-1; this enzyme is known to stimulate endogenous anti-inflammatory cytokines and antioxidant defense mechanisms,58 to be associated with improved survival in both preclinical polymicrobial models and human sepsis,59 and to be favorably stimulated by HBO₂ treatment causing higher sepsis survival.23 These results may indicate a two-sided effect of HBO₂ with the ability to increase oxidative stress and thereby bacterial death in patients without shock, whereas in patients with shock in whom the level of oxidative stress is already highly expressed at baseline, HO-1 induction suppresses the deleterious effects of free-heme produced during the course of the infection, rather than from an direct antibacterial effect.60 Thus, it can be show, that the HBO₂ effect is mainly anti-inflammatory in patients with shock but enhances the oxidative stress-mediated immune response and therefore bacterial death in patients without shock.

A possible pathophysiological pathway involving nitric oxide in NSTI has been suggested previously,61 and multiple HBO₂ treatments have shown an increase in nitrite and nitrate in selected cohorts.28 Therefore, we evaluated changes in nitrite+nitrate in patients with NSTI; however, we were unable to observe any changes in nitrite+nitrate levels after one session of HBO₂ treatment. Previous reports have demonstrated high baseline nitrite and nitrate levels in patients with NSTI.61 A theoretical explanation for the absence of a nitrite+nitrate response in our cohort could be that nitric oxide synthase systems were already highly expressed at baseline.

Antibiotics such as those administered to patients with NSTI use different mechanisms of action to cause bacterial death, including bacterial cell wall disruption by β-lactam antibiotics, disruption of protein synthesis by aminoglycosides, or blocking of the bacterial DNA/RNA synthesis by fluoroquinolone. These antibiotics induce metabolic changes of the bacteria's Krebs cycle, leading to an accumulation of toxic hydroxyl oxygen radicals.62 These ROS can subsequently react with bacterial DNA, lipids or proteins, resulting in lethal bacterial effects, in addition to the stated target-specific killing effects. The formation of ROS is dependent on the presence of oxygen. Therefore, bactericidal antibiotics have reduced activity in infectious foci with poor oxygen supply, such as abscesses, biofilm infections or during high consumption of oxygen due to polymorphonuclear leukocytes influx.63 The infected tissue in NSTI is characterized by the presence of local tissue edema and hypoxia at the infection site,1 which may be corrected by HBO₂ treatment.64

Recently, attention has been drawn specifically to the effects of HBO₂ treatment-mediated tissue reoxygenation on bacterial infections with biofilm formation.64 In the biofilm milieu, polymorphonuclear leukocytes that are activated by bacteria consume available oxygen during the process of converting oxygen to ROS and during the formation of RNS by inducible nitric oxide synthases.64 65 This local deprivation of tissue oxygen causes a dormant state of bacterial anaerobic respiration, which in turn will cause the antibiotics targeting metabolic active bacteria to become less efficient because of reduced uptake and downregulation of the antibiotic targets.64 -66 Moreover, amplification of endogenous ROS will increase the bacteria's sensitivity to oxidative attack.65 The effects of HBO₂ treatment with reoxygenation of biofilm-related infections have been demonstrated in a Pseudomonas aeruginosa biofilm model and experimental Staphylococcus aureus endocarditis.67 -69 HBO₂ treatment may have similar reoxygenation capabilities in human tissues with insufficient oxygen partial pressures.70 71 Furthermore, biofilm formation has been documented in human group A streptococcal patients with NSTI.72

We found high baseline levels of SOD to be associated with increased mortality in univariate and age-sex adjusted analyses, with an acceptable diagnostic accuracy of approximately 0.7 AUC and sensitivity of 92%. High levels of MPO are associated with increased risk of dying in patients with sepsis19; however, in the present age-sex-SOFA score-adjusted analysis, we observed a trend to an improved survival in those presenting with high levels of MPO. Although this result did not reach statistical significance, it might indicate a possible pathophysiological difference between patients with sepsis and NSTI. Moreover, it highlights the importance of future research in NSTI as current pathophysiological reasoning and treatment recommendations often relies on available evidence from non-NSTI sepsis.

We assessed severity of disease by SOFA score, SAPS II score and blood lactate level, all of which have previously been associated with mortality in patients with NSTI.4 73 74 We observed that all markers were correlated with SOFA score, and SOD, HO-1 and nitrite+nitrate were correlated with SAPS II, whereas MPO, SOD and HO-1 correlated with blood lactate. To our knowledge, these are the first results that demonstrate correlations between markers of oxidative stress and severity of disease and patient outcome in NSTI. In accordance with the presented results, previous studies have demonstrated significantly higher levels of MPO in patients with septic shock compared with patients without septic shock,19 as well as higher levels of nitrite+nitrate in patients with septic shock,75 76 whereas the activity of SOD has been reported to negatively correlate with SOFA score in patients with septic shock.52

We found that patients with group A Streptococcus NSTI had higher baseline MPO. This finding may reflect that a larger fraction of patients with group A Streptococcus NSTI were in septic shock,4 with consequently higher levels of MPO. We observed significant correlation between baseline SOD and nitrite+nitrate, indicating a potential regulatory mechanism between SOD and the nitric oxide pathway as previously reported.31

Our present study has a number of strengths: clinical data and blood were prospectively sampled by dedicated research personnel following a predefined protocol42; the laboratory analysts were blinded for patient outcome; and our follow-up was above 98%. Blood sample selection before and after HBO₂ treatment was performed within a well-defined time span around treatment, and therefore confounding events related to other treatment modalities are unlikely. Still, several limitations need to be taken into consideration. First, the present study did not have a matched non-HBO₂ control group, and therefore our observations may only be reported as associations. Even though the fluctuations observed at the time of the HBO₂ intervention makes the presence of other confounders unlikely, they may simply reflect the overall changes in ROS during disease progression and their associations to NSTI severity (SAPS II, lactate, RRT, amputation and mortality). Second, SOD's wide CIs in the multivariate analyses make the interpretation of the results difficult. Third, we have adjusted for numerous confounders (age, sex and SOFA score) that are among the most likely factors to influence disease severity and mortality. However, a series of other potential confounders, (eg, comorbidity score and blood lactate) were not included in the multivariate models because only 12 patients did not survive until day 90, which limited the number of confounders to consider. Finally, there may have been some level of selection bias favoring the less severe cases, as patients with the highest level of hemodynamic instability may have been too unstable to be moved from the ICU, and therefore have not been offered HBO₂ treatment. Thus, caution is required when extrapolating the present results to other non-selected cohorts.

In conclusion, we found HBO₂ treatment to be associated with an immediate increase in MPO and SOD after the first HBO₂ treatment session and with an increased HO-1 in patients with NSTI with septic shock. Patients with high baseline SOD were found to have increased 90-day mortality. MPO, SOD, HO-1 and nitrite+nitrate correlated with disease severity.

Footnotes

Acknowledgements

We thank biotechnician Lars Christophersen for help with the laboratory analyses.

Contributors Study planning: MH, JV, PØJ, CM, and OH; laboratory analysis: PØJ, CM and Lars Christophersen; data analysis: MH and OH; results interpretation: MH, JV PØJ, CM, and OH; drafting the manuscript: MH and OH; revision and approval of final version of the manuscript: MH, JV, PØJ, CM, and OH.

Funding The study was supported by the projects of PERMIT (grant number 8113-00009B) funded by Innovation Fund Denmark and EU Horizon 2020 under the frame of ERA PerMed (project 2018-151) and PERAID (grant number 8114-00005B) funded by Innovation Fund Denmark and Nordforsk (project no. 90456). The research leading to these results has received funding from the European Union Seventh Framework Programme: (FP7/2007-2013) under the grant agreement 305340 (INFECT project). Moreover, this work was funded by Copenhagen University Hospital (Rigshospitalet) Research Grant (grant number R167-A7352-B3897), which included a research fellowship for MH. CM is supported by The Novo Nordisk Foundation (Borregaard Clinical Scientist Fellowship in translational research; grant no. NNF17OC0025074).

Competing interests None declared.

Patient consent for publication Not required.

Ethics approval The present study abided by the principles of the Declaration of Helsinki and was approved by the Regional Ethical Committee of the Capital Region (H-19016085) and the Danish Data Protection Agency (VD-2019-179). All patients or their legal surrogates gave oral and written informed consent.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement The data that support the findings of this study are partly available from the corresponding author upon reasonable request.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

References

Stevens

, Bryant

. Necrotizing soft-tissue infections. N Engl J Med 2017;377:2253–65.doi:10.1056/NEJMra1600673http://www.ncbi.nlm.nih.gov/pubmed/29211672

Hedetoft

, Madsen

, . Incidence, comorbidity and mortality in patients with necrotising soft-tissue infections, 2005-2018: a Danish nationwide register-based cohort study. BMJ Open 2020;10:e041302. doi:10.1136/bmjopen-2020-041302http://www.ncbi.nlm.nih.gov/pubmed/33067303

Das

, Baker

, Venugopal

. Increasing incidence of necrotizing fasciitis in New Zealand: a nationwide study over the period 1990 to 2006. J Infect 2011;63:429–33.doi:10.1016/j.jinf.2011.07.019http://www.ncbi.nlm.nih.gov/pubmed/21864570

Madsen

, Skrede

, Perner

, . Patient's characteristics and outcomes in necrotising soft-tissue infections: results from a Scandinavian, multicentre, prospective cohort study. Intensive Care Med 2019;45:1241–51.doi:10.1007/s00134-019-05730-xhttp://www.ncbi.nlm.nih.gov/pubmed/31440795

Endorf

, Supple

, Gamelli

. The evolving characteristics and care of necrotizing soft-tissue infections. Burns 2005;31:269–73.doi:10.1016/j.burns.2004.11.008http://www.ncbi.nlm.nih.gov/pubmed/15774280

Pham

, Moore

, Costa

, . Assessment of functional limitation after necrotizing soft tissue infection. J Burn Care Res 2009;30:301–6.doi:10.1097/BCR.0b013e318198a241http://www.ncbi.nlm.nih.gov/pubmed/19165118

Motoyama

, Okamoto

, Kukita

, . Possible role of increased oxidant stress in multiple organ failure after systemic inflammatory response syndrome. Crit Care Med 2003;31:1048–52.doi:10.1097/01.CCM.0000055371.27268.36http://www.ncbi.nlm.nih.gov/pubmed/12682471

Ware

, Fessel

, May

, . Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis. Shock 2011;36:12–17.doi:10.1097/SHK.0b013e318217025ahttp://www.ncbi.nlm.nih.gov/pubmed/21372753

von Dessauer

, Bongain

, Molina

, . Oxidative stress as a novel target in pediatric sepsis management. J Crit Care 2011;26:103.e1–103.e7.doi:10.1016/j.jcrc.2010.05.001http://www.ncbi.nlm.nih.gov/pubmed/20646907

10.

Mathieu

, Marroni

, Kot

. Tenth European consensus conference on hyperbaric medicine: recommendations for accepted and non-accepted clinical indications and practice of hyperbaric oxygen treatment. Diving Hyperb Med 2017;47:24–32.doi:10.28920/dhm47.1.24-32http://www.ncbi.nlm.nih.gov/pubmed/28357821

11.

Anderson

, Jacoby

. Necrotizing soft tissue infections. In: Moon

, ed. Hyperbaric oxygen therapy indications. Best Publishing Company, 2019: 239–63.

12.

Thom

. Oxidative stress is fundamental to hyperbaric oxygen therapy. J Appl Physiol 2009;106:988–95.doi:10.1152/japplphysiol.91004.2008http://www.ncbi.nlm.nih.gov/pubmed/18845776

13.

Memar

, Ghotaslou

, Samiei

, . Antimicrobial use of reactive oxygen therapy: current insights. Infect Drug Resist 2018;11:567–76.doi:10.2147/IDR.S142397http://www.ncbi.nlm.nih.gov/pubmed/29731645

14.

Hsiao

S-Y

, Kung

C-T

, Su

C-M

, . Impact of oxidative stress on treatment outcomes in adult patients with sepsis. Medicine 2020;99:e20872. doi:10.1097/MD.0000000000020872http://www.ncbi.nlm.nih.gov/pubmed/32590792

15.

Huet

, Dupic

, Harriois

, . Oxidative stress and endothelial dysfunction during sepsis. Front Biosci 2011:1986–95.

16.

Bochkov

, Kadl

, Huber

, . Protective role of phospholipid oxidation products in endotoxin-induced tissue damage. Nature 2002;419:77–81.doi:10.1038/nature01023http://www.ncbi.nlm.nih.gov/pubmed/12214235

17.

Winterbourn

, Kettle

, Hampton

. Reactive oxygen species and neutrophil function. Annu Rev Biochem 2016;85:765–92.doi:10.1146/annurev-biochem-060815-014442http://www.ncbi.nlm.nih.gov/pubmed/27050287

18.

Klebanoff SJ: myeloperoxidase: friend and foe. J Leukoc Biol 2005;77:598–625.

19.

Schrijver

, Kemperman

, Roest

. Myeloperoxidase can differentiate between sepsis and non-infectious SIRS and predicts mortality in intensive care patients with SIRS. Intensive Care Med Exp 2017;5:1–9.

20.

Sureda

, Batle

, Martorell

, . Antioxidant response of chronic wounds to hyperbaric oxygen therapy. PLoS One 2016;11:1–14.doi:10.1371/journal.pone.0163371http://www.ncbi.nlm.nih.gov/pubmed/27654305

21.

Thom

, Bhopale

, Mancini

, . Actin S-nitrosylation inhibits neutrophilβ2 integrin function. J Biol Chem 2008;283:10822–34.doi:10.1074/jbc.M709200200http://www.ncbi.nlm.nih.gov/pubmed/18283105

22.

Huang

T-Y

, Tsai

P-S

, Wang

T-Y

, . Hyperbaric oxygen attenuation of lipopolysaccharide-induced acute lung injury involves heme oxygenase-1. Acta Anaesthesiol Scand 2005;49:1293–301.doi:10.1111/j.1399-6576.2005.00812.xhttp://www.ncbi.nlm.nih.gov/pubmed/16146466

23.

Chang

K-Y

, Tsai

P-S

, Huang

T-Y

, . HO-1 mediates the effects of HBO pretreatment against sepsis. J Surg Res 2006;136:143–53.doi:10.1016/j.jss.2006.06.004http://www.ncbi.nlm.nih.gov/pubmed/16930622

24.

Rothfuss

, Radermacher

, Speit

. Involvement of heme oxygenase-1 (HO-1) in the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO) treatment. Carcinogenesis 2001;22:1979–85.doi:10.1093/carcin/22.12.1979http://www.ncbi.nlm.nih.gov/pubmed/11751428

25.

Speit

, Dennog

, Eichhorn

, . Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment. Carcinogenesis 2000;21:1795–9.doi:10.1093/carcin/21.10.1795http://www.ncbi.nlm.nih.gov/pubmed/11023535

26.

Körpınar

Şefika

, Uzun

, Körpınar

. The effects of hyperbaric oxygen at different pressures on oxidative stress and antioxidant status in rats. Medicina 2019;55:1–8.doi:10.3390/medicina55050205http://www.ncbi.nlm.nih.gov/pubmed/31137620

27.

Bai

, Li

, Chen

, . Protective effects of hyperbaric oxygen preconditioning against LPS-induced acute lung injury in rats. Undersea Hyperb Med 2018;45:323–33.http://www.ncbi.nlm.nih.gov/pubmed/30028919

28.

Gurdol

, Cimsit

, Oner-Iyidogan

, . Collagen synthesis, nitric oxide and asymmetric dimethylarginine in diabetic subjects undergoing hyperbaric oxygen therapy. Physiol Res 2010;59:423–9.doi:10.33549/physiolres.931702http://www.ncbi.nlm.nih.gov/pubmed/19681664

29.

Gasier

, Fothergill

. Oxidative stress, antioxidant defenses and nitric oxide production following hyperoxic exposures. Undersea Hyperb Med 2013;40:125–34.http://www.ncbi.nlm.nih.gov/pubmed/23682544

30.

Uusijärvi

, Eriksson

, Larsson

, . Effects of hyperbaric oxygen on nitric oxide generation in humans. Nitric Oxide 2015;44:88–97.doi:10.1016/j.niox.2014.12.002http://www.ncbi.nlm.nih.gov/pubmed/25498903

31.

Allen

, Demchenko

, Piantadosi

. Two faces of nitric oxide: implications for cellular mechanisms of oxygen toxicity. J Appl Physiol 2009;106:662–7.doi:10.1152/japplphysiol.91109.2008http://www.ncbi.nlm.nih.gov/pubmed/18845774

32.

Srisook

, Cha

Y-N

. Super-induction of HO-1 in macrophages stimulated with lipopolysaccharide by prior depletion of glutathione decreases iNOS expression and NO production. Nitric Oxide 2005;12:70–9.doi:10.1016/j.niox.2004.12.002http://www.ncbi.nlm.nih.gov/pubmed/15740980

33.

Koakutsu

, Nomura

, Yamanouchi

, . Oxygen free radical and antioxidant status in necrotizing soft tissue infection of the lower extremity; a report of two cases with opposite outcomes. Ups J Med Sci 2013;118:46–50.doi:10.3109/03009734.2012.733738http://www.ncbi.nlm.nih.gov/pubmed/23163625

34.

Dennog

, Gedik

, Wood

, . Analysis of oxidative DNA damage and HPRT mutations in humans after hyperbaric oxygen treatment. Mutat Res 1999;431:351–9.doi:10.1016/s0027-5107(99)00178-5http://www.ncbi.nlm.nih.gov/pubmed/10636000

35.

Dennog

, Hartmann

, Frey

, . Detection of DNA damage after hyperbaric oxygen (HBO) therapy. Mutagenesis 1996;11:605–9.doi:10.1093/mutage/11.6.605http://www.ncbi.nlm.nih.gov/pubmed/8962431

36.

Narkowicz

, Vial

, McCartney

. Hyperbaric oxygen therapy increases free radical levels in the blood of humans. Free Radic Res Commun 1993;19:71–80.doi:10.3109/10715769309056501http://www.ncbi.nlm.nih.gov/pubmed/8225040

37.

Gröger

, Oter

, Simkova

, . DNA damage after long-term repetitive hyperbaric oxygen exposure. J Appl Physiol 2009;106:311–5.doi:10.1152/japplphysiol.90737.2008http://www.ncbi.nlm.nih.gov/pubmed/19023023

38.

Yogaratnam

, Laden

, Madden

, . Hyperbaric oxygen: a new drug in myocardial revascularization and protection? Cardiovasc Revasc Med 2006;7:146–54.doi:10.1016/j.carrev.2006.04.006http://www.ncbi.nlm.nih.gov/pubmed/16945821

39.

Thom

, Bhopale

, Yang

, . Neutrophil beta2 integrin inhibition by enhanced interactions of vasodilator-stimulated phosphoprotein with S-nitrosylated actin. J Biol Chem 2011;286:32854–65.doi:10.1074/jbc.M111.255778http://www.ncbi.nlm.nih.gov/pubmed/21795685

40.

von Elm

, Altman

, Egger

, . The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet 2007;370:1453–7.doi:10.1016/S0140-6736(07)61602-Xhttp://www.ncbi.nlm.nih.gov/pubmed/18064739

41.

Hedetoft

, Moser

, Jensen

Peter Østrup

, . Soluble ICAM-1 is modulated by hyperbaric oxygen treatment and correlates with disease severity and mortality in patients with necrotizing soft-tissue infection. J Appl Physiol 2021;130:729–36.doi:10.1152/japplphysiol.00844.2020http://www.ncbi.nlm.nih.gov/pubmed/33444122

42.

Madsen

, Skrede

, Bruun

, . Necrotizing soft tissue infections - a multicentre, prospective observational study (INFECT): protocol and statistical analysis plan. Acta Anaesthesiol Scand 2018;62:272–9.doi:10.1111/aas.13024http://www.ncbi.nlm.nih.gov/pubmed/29082520

43.

Line

, Alhede

, Kolpen

, . Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum. Front Microbiol 2014;5:554. doi:10.3389/fmicb.2014.00554http://www.ncbi.nlm.nih.gov/pubmed/25386171

44.

Fluss

, Faraggi

, Reiser

. Estimation of the Youden index and its associated cutoff point. Biom J 2005;47:458–72.doi:10.1002/bimj.200410135http://www.ncbi.nlm.nih.gov/pubmed/16161804

45.

Hampton

, Kettle

, Winterbourn

. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils. Infect Immun 1996;64:3512–7.doi:10.1128/IAI.64.9.3512-3517.1996http://www.ncbi.nlm.nih.gov/pubmed/8751892

46.

Hampton

, Kettle

, Winterbourn

. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 1998;92:3007–17.http://www.ncbi.nlm.nih.gov/pubmed/9787133

47.

Naegelen

, Beaume

, Plançon

, . Regulation of neutrophil degranulation and cytokine secretion: a novel model approach based on linear fitting. J Immunol Res 2015;2015:817038. doi:10.1155/2015/817038http://www.ncbi.nlm.nih.gov/pubmed/26579547

48.

Lacy

. Mechanisms of degranulation in neutrophils. Allergy Asthma Clin Immunol 2006;2:98–108.doi:10.1186/1710-1492-2-3-98http://www.ncbi.nlm.nih.gov/pubmed/20525154

49.

Kettle

, Anderson

, Hampton

. Reactions of superoxide with myeloperoxidase and its products. Biochemistry 2007;46:4888–97.

50.

Kettle

, Winterbourn

. Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid. Biochem J 1988;252:529–36.doi:10.1042/bj2520529http://www.ncbi.nlm.nih.gov/pubmed/2843172

51.

Wang

, Branicky

, Noë

, . Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling. J Cell Biol 2018;217:1915–28.doi:10.1083/jcb.201708007http://www.ncbi.nlm.nih.gov/pubmed/29669742

52.

Kumar

, Gupta

, Kaushik

, . Evaluation of oxidative stress and antioxidant status: correlation with the severity of sepsis. Scand J Immunol 2018;87:1–11.doi:10.1111/sji.12653http://www.ncbi.nlm.nih.gov/pubmed/29484685

53.

Chung

, Hall

. Parrella MA: role of heme oxygenase-1 in microbial host defense. Cell microbiol 2009;11:199–207.

54.

Chung

, Liu

, Macias

, . Heme oxygenase-1-derived carbon monoxide enhances the host defense response to microbial sepsis in mice. J Clin Invest 2008;118:239–47.doi:10.1172/JCI32730http://www.ncbi.nlm.nih.gov/pubmed/18060048

55.

Buras

, Holzmann

, Sitkovsky

. Animal models of sepsis: setting the stage. Nat Rev Drug Discov 2005;4:854–65.doi:10.1038/nrd1854http://www.ncbi.nlm.nih.gov/pubmed/16224456

56.

Shaw

, Psoinos

, Emhoff

, . Not just full of hot air: hyperbaric oxygen therapy increases survival in cases of necrotizing soft tissue infections. Surg Infect 2014;15:328–35.doi:10.1089/sur.2012.135http://www.ncbi.nlm.nih.gov/pubmed/24786980

57.

Devaney

, Frawley

, . Necrotising soft tissue infections: the effect of hyperbaric oxygen on mortality. Anaesth Intensive Care 2015;43:685–92.doi:10.1177/0310057X1504300604http://www.ncbi.nlm.nih.gov/pubmed/26603791

58.

Ryter

. Therapeutic potential of heme oxygenase-1 and carbon monoxide in acute organ injury, critical illness, and inflammatory disorders. Antioxidants 2020;9:1153. doi:10.3390/antiox9111153http://www.ncbi.nlm.nih.gov/pubmed/33228260

59.

Takaki

, Takeyama

, Kajita

, . Beneficial effects of the heme oxygenase-1/carbon monoxide system in patients with severe sepsis/septic shock. Intensive Care Med 2010;36:42–8.doi:10.1007/s00134-009-1575-4http://www.ncbi.nlm.nih.gov/pubmed/19787335

60.

Larsen

, Gozzelino

, Jeney

. A central role for free heme in the pathogenesis of severe sepsis (supplementary materials). Sci Transl Med 2010;2:51ra71.

61.

Hansen

, Rasmussen

, Garred

, . Associations of plasma nitrite, L-arginine and asymmetric dimethylarginine with morbidity and mortality in patients with necrotizing soft tissue infections. Shock 2018;49:667–74.doi:10.1097/SHK.0000000000000975http://www.ncbi.nlm.nih.gov/pubmed/28863028

62.

Kohanski

, Dwyer

, Hayete

, . A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007;130:797–810.doi:10.1016/j.cell.2007.06.049http://www.ncbi.nlm.nih.gov/pubmed/17803904

63.

Brochmann

, Toft

, Ciofu

, . Bactericidal effect of colistin on planktonic Pseudomonas aeruginosa is independent of hydroxyl radical formation. Int J Antimicrob Agents 2014;43:140–7.doi:10.1016/j.ijantimicag.2013.10.015http://www.ncbi.nlm.nih.gov/pubmed/24315789

64.

PØ Sa

J J

, Lerche

, . Improving antibiotic treatment of bacterial biofilm by hyperbaric oxygen therapy : Not just hot air. Biofilm 2019;1:1–4.

65.

Van Acker

, Coenye

. The role of reactive oxygen species in antibiotic-mediated killing of bacteria. Trends Microbiol 2017;25:456–66.doi:10.1016/j.tim.2016.12.008http://www.ncbi.nlm.nih.gov/pubmed/28089288

66.

Sønderholm

, Bjarnsholt

, Alhede

. The consequences of being in an infectious biofilm: microenvironmental conditions governing antibiotic tolerance. Int J Mol Sci 2017:18.

67.

Møller

, Jensen

Peter Østrup

, Høiby

, . Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients. J Cyst Fibros 2019;18:657–64.doi:10.1016/j.jcf.2019.01.005http://www.ncbi.nlm.nih.gov/pubmed/30711384

68.

Lerche

, Christophersen

, Kolpen

, . Hyperbaric oxygen therapy augments tobramycin efficacy in experimental Staphylococcus aureus endocarditis. Int J Antimicrob Agents 2017;50:406–12.doi:10.1016/j.ijantimicag.2017.04.025http://www.ncbi.nlm.nih.gov/pubmed/28669832

69.

Kolpen

, Mousavi

, Sams

, . Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment. Int J Antimicrob Agents 2016;47:163–7.doi:10.1016/j.ijantimicag.2015.12.005http://www.ncbi.nlm.nih.gov/pubmed/26774522

70.

Korhonen

, Kuttila

, Niinikoski

. Tissue gas tensions in patients with necrotising fasciitis and healthy controls during treatment with hyperbaric oxygen: a clinical study. Eur J Surg 2000;166:530–4.doi:10.1080/110241500750008583http://www.ncbi.nlm.nih.gov/pubmed/10965830

71.

Wang

T-L

, Hung

C-R

. Role of tissue oxygen saturation monitoring in diagnosing necrotizing fasciitis of the lower limbs. Ann Emerg Med 2004;44:222–8.doi:10.1016/j.annemergmed.2004.03.022http://www.ncbi.nlm.nih.gov/pubmed/15332062

72.

Siemens

, Chakrakodi

, Shambat

. Biofilm in group A streptococcal necrotizing soft tissue infections. JCI insight 2016;1:1–13.

73.

Jabbour

, El-Menyar

, Peralta

. Pattern and predictors of mortality in necrotizing fasciitis patients in a single tertiary hospital. World J Emerg Surg 2016;11:1–10.

74.

Yaşar

, Uylaş

, Badak

, . Can we predict mortality in patients with necrotizing fasciitis using conventional scoring systems? Ulus Travma Acil Cerrahi Derg 2017;23:383–8.doi:10.5505/tjtes.2016.19940http://www.ncbi.nlm.nih.gov/pubmed/29052823

75.

Mitaka

, Hirata

, Yokoyama

, . Relationships of circulating nitrite/nitrate levels to severity and multiple organ dysfunction syndrome in systemic inflammatory response syndrome. Shock 2003;19:305–9.doi:10.1097/00024382-200304000-00002http://www.ncbi.nlm.nih.gov/pubmed/12688539

76.

Doughty

, Carcillo

, Kaplan

, . Plasma nitrite and nitrate concentrations and multiple organ failure in pediatric sepsis. Crit Care Med 1998;26:157–62.doi:10.1097/00003246-199801000-00032http://www.ncbi.nlm.nih.gov/pubmed/9428559