Efficacy of hyperbaric oxygen therapy in bacterial biofilm eradication

Abstract

Objective:
Chronic wounds typically require several concurrent therapies, such as debridement, pressure offloading, and systemic and/or topical antibiotics. The aim of this study was to examine the efficacy of hyperbaric oxygen therapy (HBOT) towards reducing or eliminating bacterial biofilms in vitro and in vivo.

Method:
Efficacy was determined using in vitro grown biofilms subjected directly to HBOT for 30, 60 and 90 minutes, followed by cell viability determination using propidium monoazide-polymerase chain reaction (PMA-PCR). The efficacy of HBOT in vivo was studied by searching our chronic patient wound database and comparing time-to-healing between patients who did and did not receive HBOT as part of their treatment.

Results:
In vitro data showed small but significant decreases in cell viability at the 30- and 90-minute time points in the HBOT group. The in vivo data showed reductions in bacterial load for patients who underwent HBOT, and ~1 week shorter treatment durations. Additionally, in patients' chronic wounds there was a considerable emergence of anaerobic bacteria and fungi between intermittent HBOT treatments.

Conclusion:
The data demonstrate that HBOT does possess a certain degree of biofilm killing capability. Moreover, as an adjuvant to standard treatment, more favourable patient outcomes are achieved through a quicker time-to-healing which reduces the chance of complications. Furthermore, the data provided insights into biofilm adaptations to challenges presented by this treatment strategy which should be kept in mind when treating chronic wounds. Further studies will be necessary to evaluate the benefits and mechanisms of HBOT, not only for patients with chronic wounds but other chronic infections caused by bacterial biofilms.

Full text

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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018
S20
Objective: Chronic wounds typically require several concurrent
therapies, such as debridement, pressure ofoading, and systemic
and/or topical antibiotics. The aim of this study was to examine the
efcacy of hyperbaric oxygen therapy (HBOT) towards reducing or
eliminating bacterial biolms in vitro and in vivo.
Method: Efcacy was determined using in vitro grown biolms
subjected directly to HBOT for 30, 60 and 90 minutes, followed by cell
viability determination using propidium monoazide-polymerase chain
reaction (PMA-PCR). The efcacy of HBOT in vivo was studied by
searching our chronic patient wound database and comparing
time-to-healing between patients who did and did not receive HBOT
as part of their treatment.
Results: In vitro data showed small but signicant decreases in cell
viability at the 30- and 90-minute time points in the HBOT group. The
invivo data showed reductions in bacterial load for patients who
underwent HBOT, and ~1 week shorter treatment durations. Additionally,
in patients’ chronic wounds there was a considerable emergence of
anaerobic bacteria and fungi between intermittent HBOT treatments.
Conclusion: The data demonstrate that HBOT does possess a certain
degree of biolm killing capability. Moreover, as an adjuvant to standard
treatment, more favourable patient outcomes are achieved through a
quicker time-to-healing which reduces the chance of complications.
Furthermore, the data provided insights into biolm adaptations to
challenges presented by this treatment strategy which should be kept
in mind when treating chronic wounds. Further studies will be
necessary to evaluate the benets and mechanisms of HBOT, not only
for patients with chronic wounds but other chronic infections caused
by bacterialbiolms.
Declaration of interest: The authors declare no conict of interest.
No funding was received for this study.
Historically, the cause of chronic non-
healing wounds has been attributed to
diabetes, arterial and venous disease, and
burn and radiation exposure wounds.1,2
However, greater attention has been paid
to wound microbiota, propagating mainly as biolms,
and their contribution to the chronicity of wounds.3–8
Bacterial biolms have been recognised as the primary
cause of wound chronicity.9–11 Several denitions for
bacterial biolms have been proposed in the literature,
but the most widely accepted denition is ‘a coherent
cluster of bacterial cells embedded in a matrix, which
is more tolerant to most antimicrobials and the host
defence than planktonic bacterial cells.’12
Increased resistance to antimicrobials and host
defence systems results from physical factors such as
the extracellular polymeric substance (EPS) matrix. The
EPS encloses a multitude of bacteria in the biolm
superstructure, protecting the bacteria from
environmental factors, such as ultra violet (UV)
radiation and desiccation.13 Additionally, the EPS and
sheer size of the biolm superstructure may hinder the
host immune recognition and phagocytosis,
respectively.14 The EPS also inuences diffusion of
biofilm ● chronic wounds ● hyperbaric oxygen therapy ● HBOT
oxygen and antibiotics into the biolm, factoring into
the durability of biolms. Genetic factors are also
responsible for bacterial biolm resilience. The close
association of bacteria of the same or different species
allows the fast and organised sharing of resistance
plasmids, and enables efficient cell-to-cell
communication throughout the entire biofilm
community.15–17 This communication system facilitates
the dynamic existence of bacteria in either the
planktonic or biolm mode of growth by signalling for
recruitment of bacteria in favourable conditions and,
alternatively, signalling for dispersal in the planktonic
form in unfavourable conditions.18 These factors make
bacterial biolms difcult to eradicate.
The principal method for biolm eradication from
wounds is aggressive and frequent debridement.
Unfortunately, complete removal of the biolm in a
clinical setting is imperfect and, even with local
anaesthetic, can be very painful for the patient, and
allows the biolm to return within 24–48 hours.6,16
Hyperbaric oxygen therapy (HBOT) has been proposed
and, to some extent, researched as an adjuvant therapy
for chronic wound healing.17,19–21 While there are
conicting reports in the literature, there is limited
evidence contradicting the benets of HBOT in the
practice of wound care and healing.
For nearly ve decades, HBOT has been used as an
oxygen delivery system to ameliorate oxygen
deciencies in the blood and ischaemia by diffusing
oxygen into the plasma, allowing cellular production
of appropriate signalling molecules and metabolites
Efcacy of hyperbaric oxygen therapy
in bacterial biolm eradication
Nicholas E. Sanford,1 PhD, Laboratory Manager; Jeremy E. Wilkinson,2 PhD, Director of
Operations; Hao Nguyen,3 Medical Student; Gabe Diaz,1 Certied Hyperbaric Technician;
Randall Wolcott,1 MD, Medical Director
Corresponding author email: nick@randallwolcott.com
1 Southwest Regional Wound Care Center, Lubbock, Texas. 2 RTLGenomics, Lubbock,
Texas. 3 Texas Tech University Health Sciences Center, Lubbock, Texas.
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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018 S21
necessary for cell migration and mitosis.22 At a tissue
level, HBOT enhances vasculogenesis and angiogenesis,
helping to sustain the area of the wound after
healing.21,23–25 Furthermore, HBOT stimulates the
immune system, via white blood cell (WBC) activation,
and enhances phagocytosis.26 In traditional HBOT, the
entire body is placed in a pressurised chamber at 100%
oxygen. At 3 atmospheres of pressure there are enough
oxygen molecules dissolved in the plasma that no red
blood cells are needed to adequately oxygenate the
tissues and keep them viable. While the mechanisms of
wound healing at the host tissue level by HBOT are
moderately well understood, the effects of HBOT on
wound microbiota are underrepresented in the literature.
HBOT is lethal for anaerobic organisms and can
retard bacterial growth at pressures greater than
1.3atmospheres absolute (ATA).26 However, the ability
of oxygen to diffuse into biolms is lower, because the
EPS and high cell density impede convective ow of the
bulk fluid, substantially increasing the diffusion
distance.27 Increased diffusion time prevents oxygen
from reaching the hypoxic core of a bacterial biolm,
leaving anaerobic bacteria unharmed.24 Moreover,
bacterial growth rates are much slower within biolms
compared with their planktonic counterparts,
attenuating the benecial effects of HBOT.28
Nonetheless, HBOT is an excellent adjuvant therapy
for healing chronic wounds, which raises the following
question: what is the bactericidal capacity toward the
chronic wound biolm of HBOT? A low number of
studies of HBOT regarding chronic wound biolm have
limited its use as an adjuvant therapy in wound care,
due to denials for reimbursement by insurance
companies and the Centers for Medicare and Medicaid
Services (CMS). It is our hope that renewing interest in
HBOT as an adjuvant treatment for chronic wounds
will supply the necessary data to make the technology
more accessible to patients.
Methods
Patients who participated in this study provided
consent under a protocol that was approved by the
Western Institutional Review Board (WIRB PRO NUM:
20062425). All elements of this study were considered
to pose less than minimal risk to the patients, and each
patient was fully informed and educated through the
consenting process. All patient identiers were removed
from all study data, and only the clinical research
coordinator securely retained the documentation
linking an individual patient to study data.
Study patients were chosen from our patient database.
Patients presenting with wounds that had persisted for
at least 30 days with no considerable signs of healing
were eligible for HBOT. The control group comprised of
patients with similar wounds who do not receive HBOT.
Reasons for omission of HBOT include the clinician not
deeming HBOT necessary for wound care, the patient
responded to our standard of care (SOC), the patient
was claustrophobic and declined HBOT, the patient
could not afford the prescribed number of treatments,
or the patient could not come in often enough for
HBOT to contribute to wound healing. A second
criterion for study inclusion was that the patient had
molecular diagnostic testing on wound samples at the
initial visit, during the course of treatment, and on the
nal treatment.
Wound sampling
The study wound was cleansed with normal saline as part
of our usual SOC. Next, the patient’s wound was biopsied
under local anaesthesia then subjected to sharp
debridement using sterile curette, scissors, and/or scalpel
to remove slough and devitalised tissue from its surface.
The slough and devitalised tissue were then transferred
to a sterile 2ml tube and stored at room temperature for
no more than two hours before laboratory analysis.
Samples of patient wounds were collected at the
beginning of treatment and after completion of the
HBOT treatment regimen.
Patient HBOT treatments
All patients completed a minimum of 30 HBOT
treatments. Chambers were pressurised to 2.0ATA at a
rate of 1 pound-force per square inch (psi) per minute.
Once a pressure of 2.0ATA was attained, a timer was set
for 90 minutes. Sechrist 3200 monoplace hyperbaric
Table 1. Patient demographics
Number in study Average age Age range Sex
Male/female
Caucasian/
hispanic
Diabetic
SOC 10 56 34–90 5/5 7/ 3 4/10
SOC + HBOT 11 63 49–83 6/5 5/6 8/11
Wound type
Time to healing
(weeks)
DFU NHSW DU VLU CW
SOC 6.6 30133
SOC + HBOT 5.8 71111
SOC—standard of care; HBOT—hyperbaric oxygen therpay; DFU—diabetic foot ulcer; NHSW—non-healing surgical wound; DU—diabetic ulcer; VLU—venous leg ulcer; CW—chronic wound
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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018
S22
polymerase enzyme from binding to and amplifying
the target nucleic acid sequence during qPCR, resulting
in different Ct values for PMA-treated and untreated
samples at the same time point. The untreated Ct value
represents the total DNA in the sample, which includes
extracellular DNA of the biolm and DNA from cells
with damaged membranes (considered unviable). The
PMA-treated Ct value represents the fraction of DNA
within the sample coming only from viable cells.
Genomic DNA extraction and quantitative
polymerase chain reaction (qPCR)
Genomic DNA was extracted from wound samples and
in vitro biolms using the Roche High Pure PCR
Template Preparation kit (Roche Life Sciences,
Indianapolis, IN, US) according to manufacturer
specications. Sample lysates for DNA extraction were
produced using the Qiagen TissueLyser (Qiagen Inc.,
Valencia, CA, US) and 0.5mm zirconium oxide beads
(Next Advance, Averill Park, NY, US). Semi-quantitative
determination of bacterial load using the universal 16S
rDNA was performed using the LightCycler 480 (Roche
Life Sciences). Forward
(5’-CCATGAAGTCGGAATCGCTAG-3’) and reverse
(5’-GCTTGACGGGCGGTGT-3’) 16S rDNA primers
(20µM each) were used with a 16S rDNA probe
(5’-TACAAGGCCCGGGAACGTATTCACCG-3’) in
Quanta PerfeCTa qPCR ToughMix (Quanta Biosciences,
Beverly, MA, US). The template DNA (2.5µl) was added
to the master mix containing the primers and probe
(10µl each), and the reaction was run with the following
thermal cycling prole: 50ºC for two minutes, 95ºC for
10minutes, 35 cycles at 95ºC for 15 seconds, 60ºC for
one minute, and 40ºC for 30 seconds. 16S rDNA
quantication cycle (Cq) values were used for pre- and
post-HBOT comparisons of bacterial load. Escherichia
coli c600 (ATCC 23724, Manassas, VA, US) genomic
DNA was used as a positive 16S rDNA control and
molecular grade water (Phenix Research Products,
Chandler, NC, US) was used as a no template control.
Sequencing
Samples were amplied for semiconductor sequencing
using a forward and reverse fusion primer. The forward
primer was constructed with the Ion A linker
(5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’),
an 8–10 base pair (bp) barcode, and the 28F primer
(5’-GAGTTTGATCNTGGCTCAG-3’). The reverse fusion
primer was constructed with a biotin molecule, the Ion
P5 linker (5’-CCTCTCTATGGGCAGTCGGTGAT-3’),
and the 388R primer (5’-TGCTGCCTCCCGTAGGAGT-3’).
Amplications were performed in 25µl reactions with
Qiagen HotStarTaq master mix (Qiagen Inc.), 1µl of
each primer (5µM), and 1µl of template. Samples were
amplied with the ABI Veriti thermocycler (Applied
Biosystems, Carlsbad, CA, US) under the following
thermal prole: 95°C for ve minutes, 35 cycles at 94°C
for 30 seconds, 54°C for 40 second, 72°C for one
minute, 72°C for 10 minutes, and 4°C hold.
chambers (Sechrist, Anaheim, CA, US) at the Southwest
Regional Wound Care Center were used for the study.
Cell culture
The Lubbock chronic wound biolm (LCWB) was grown
as described by Sun et al.29 The biolm contained
Pseudomonas aeruginosa, Enterococcus faecalis, and
Staphylococcus aureus. Cells were grown overnight at 37ºC
in tryptic soy broth (TSB) (Sigma Aldrich, St. Louis, MO,
US) to produce planktonic cultures, after which
Pseudomonas aeruginosa culture (100μl), Enterococcus
faecalis culture (150μl), and Staphylococcus aureus (200μl)
cultures were added to Bolton broth (Oxoid Ltd.,
Basingstoke, Hampshire, UK) containing 50% (v/v)
bovine plasma (Innovative Research Inc., Novi, MI, US)
and incubated for 48 hours at 37ºC with rotational
shaking at 200rpm. A pipette tip was used as the scaffold
for biolm formation. Once biolms were formed and
tip-attached, the biolm was transferred from the tip to
tryptic soy agar (TSA) plates (Sigma Aldrich).
HBOT treatment of in vitro biolms
Open TSA plates containing the LCWB were placed
into Sechrist 3200 monoplace hyperbaric oxygen
chambers. Chambers were pressurised to 2.0ATA at a
rate of 1psi per minute. Once a pressure of 2.0ATA was
attained, a timer was set for 30, 60 and 90 minutes.
After HBOT treatment, samples were split for cell
viability determination.
Cell viability
In vitro biolms were divided in two groups, propidium
monoazide (PMA)-treated and untreated, for performing
the live-dead assay. Samples were added to 0.65ml
microtubes, resuspended in 1×phosphate-buffered saline
(PBS), and sonicated in ice using a Bioruptor for 12
minutes (Diagenode, Denville, NJ, US). After sonication,
400µM PMA was added to the PMA-treated group. Both
treated and untreated samples were incubated in the
dark at 4°C for 10 minutes with frequent vortexing.
Samples were exposed to light for 15 minutes using a
PMA-Lite LED photolysis device (Biotium, Hayward, CA,
US) to cross-link the PMA dye to DNA. Percentage
viability was determined by averaging the inverse of the
threshold cycle (Ct) values of the treated and non-treated
groups, and dividing the resulting values of the HBOT
group by the non-treated group.
The in vitro LCWB was used to assess HBOT
bactericidal activity. In our study, in vitro biolms on
TSA were subjected to HBOT, and control biolms on
TSA were placed in an inactive chamber to account for
any chamber effects. Reductions in the amount of
bacterial genomic DNA were determined using the
PMA cell viability assay and quantitative polymerase
chain reaction (qPCR) of the universal 16S rDNA
(ribosomal DNA). PMA binds irreversibly to any
extracellular DNA, and DNA in cells with damaged cell
walls and plasma membranes which are considered
dead. The covalent binding of PMA to DNA inhibits the
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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018 S23
Amplication products were visualised with eGels
(Life Technologies, Grand Island, NY, US). Products
were pooled into equimolar mixtures. Each pool was
size-selected using Agencourt AMPure XP (Beckman
Coulter, Indianapolis, IN, US) following Life
Technologies protocols. Size-selected pools were
quantied using the Qubit 2.0 Fluorometer and the
Qubit dsDNA HS Assay Kit (Life Technologies), and
diluted to 23pM. Diluted pools were subjected to
emulsion PCR (emPCR), enriched using the OneTouch
2 System (Life Technologies), and sequenced using the
Ion Torrent Personal Genome Machine (PGM) (Life
Technologies), following the manufacturer protocols.
Bioinformatics
The sequence data were analysed at RTLGenomics
(Lubbock, TX, US) using its standard microbial diversity
analysis pipeline. The data analysis pipeline consisted of
two major stages, the de-noising and chimera detection
stage, and the microbial diversity analysis stage.
De-noising was performed by various techniques to
remove short sequences, singleton sequences, and noisy
reads. Once the low-quality reads were removed, chimera
detection was performed to aid the removal of chimeric
sequences. Finally, the remaining sequences were
corrected, base by base, to remove noise from within
each sequence. During the diversity analysis stage, each
sample was run through the analysis pipeline to cluster
reads into operational taxonomic units (OTUs), which
went through taxonomic classication down to species-
level identication.
Results
Important considerations when analysing clinical
HBOT data are patient demographics (Table 1) and the
nature of treated wounds, which are characteristically
quite diverse and unique. In this retrospective study
comprised of patients who underwent SOC alone and
SOC with HBOT as an adjuvant, the HBOT group was,
on average, seven years older than the SOC group.
Additionally, patients undergoing HBOT typically have
increased comorbidities, such as diabetes mellitus,
neuropathy, and/or arterial and venous insufciencies,
compared with patients who successfully heal with
SOC alone. In our patient population, sex- and age-
matched acute wounds (controls) are not readily
available. These factors can easily confound
experimental data, suggesting that HBOT only works by
contributing to host healing mechanisms rather than
having any bactericidal activity.
The trends of each treatment over time were very
similar, with both control and HBOT group viability
dropping at the 60-minute time point (Fig 1). Cell
viability of treated biolms, relative to control, was
signicantly different at the 30- and 90-minute time
points and approached signicance (p=0.07) at the
60-minute time point. While these differences are minor,
and perhaps clinically insignicant, the data suggest that
HBOT does bear a degree of biocidal activity towards
bacterial biolms. The differences observed may possibly
be amplied with additional age/sex-matched samples
and/or different bacterial species included in the
biolms. One caveat of the in vitro data is that the
biolms did not have to contend with the host immune
system, were only subjected to a single treatment of 30,
60 and 90 minutes, and did not receive any SOC therapy,
such as antibiotics or antimicrobial dressings.
The rebound in cell viability, observed in both the
control and treatment groups at the 90-minute time
point, may be due to a reorganisation of the community
structure of the biolm by decreasing the competition
in the biolm superstructure or a reversion of some or
all species to the faster growing planktonic phenotype
(Fig 1). The rebound that was observed in the in vitro
biolms was interesting and gave rise to the question
of whether a similar trend is observed in patients with
chronic wounds who have undergone HBOT.
Retrospective data from 2011 to 2016 from patients
who had received molecular diagnostics before and
after their prescribed treatment regimens were selected
from the patient database. To rene the list, patients
who had HBOT during their treatment regimen were
selected. Patients who did not undergo HBOT but had
similar lengths of treatment and wound type were
selected, attempting to balance each group for sex, age,
and race. The patient demographics (Table 1) show the
relevant metric and diabetic status of the patients
included in this study.
To assess if and how HBOT as adjuvant therapy
contributes to wound healing clinically, bacterial
burden determined via qPCR of patient wounds from
initial and nal visits were compared (Fig 2). Both the
HBOT and the SOC group showed reductions in
bacterial burden, as expected for healing wounds.
Fig 1. Response of in vitro biolms to hyperbaric oxygen therapy (HBOT).
The in vitro Lubbock chronic wound biolm model contained three bacterial
species. Biolms were exposed to HBOT for 30, 60, and 90 minutes. Control
biolms (ambient) were placed in inactive closed chambers to account for any
chamber effects. Decreases on the Y-axis correspond to decreases in cell
viability. Cell viability was determined using propidium monoazide (PMA)-PCR.
Statistical signicance was determined using the Welch 2 sample t-test
dCt (untreated–treated)
Treatment and time point (minutes)
Ambient
30
HBOT
30
Ambient
60
HBOT
60
Ambient
90
HBOT
90
2
1
0
-1
-2
p=0.002 p=0.07 p=0.03
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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018
S24
However, the data for the HBOT group showed an
appreciably smaller spread and higher median Ct at the
nal visit, indicating a greater reduction in bacterial
burden for the HBOT group than for the SOC group.
This, coupled with the time-to-healing information
shown in Table 1 (~1 week shorter for HBOT patients)
provides additional evidence that HBOT not only
contributes to host healing processes, but may also
facilitate reduction of bacterial burden.
The molecular diagnostics not only take into account
bacterial load but also microbial diversity. Analysis of
the microbial subpopulations (aerobe versus anaerobe
versus fungi) in the HBOT group, upon completion of
the treatment regimen, revealed peculiarities about
these biolm communities. Fig 3 shows how the
microbial diversity shifted after completion of
treatment. Unsurprisingly, aerobes were the most
persistent subpopulation of the wound microbiota and,
during treatment, some of the original species were
eradicated, leaving a niche for others to emerge as more
dominant members of the community. It is important
to note that commensal aerobes likely make up for the
majority of the detected microbiota in this analysis.
In approximately 35% of cases, anaerobes were not
present before or after treatment with HBOT. However,
in another approximately 35% of cases, anaerobes
emerged as dominant members of the wound microbiota
after the treatment regimen. Lastly, in approximately
17% of cases, anaerobes persisted throughout HBOT,
while only approximately 13% were eradicated by the
treatment. A similar outcome was observed for fungal
species within the wound microbiota, with an increased
prevalence of fungal emergence after treatment (48% of
cases), likely due to the use of antibiotics decreasing
bacterial competition. These data suggest that, while
HBOT does have bactericidal effects against microbial
biolms, a collapse of the dominant species in the
biolm community may take place. This allows the
expansion of rarer and less competitive species which
may not be as recalcitrant as the original dominant
species, allowing SOC treatment practices, such as
debridement and antibiotics (systemic and topical), to
be more effective in promoting wound healing.
To better understand how the microbial diversity of
the biolms shifted in this particular cohort with
higher resolution, exemplar patients were chosen for
further analysis (Figs 4 and 5). The patient described
in Fig 4 (control group) showed a signicant reduction
in bacterial load and complete wound closure using
SOC treatment alone within four months. Fig 5
describes a patient with a chronic wound that
underwent SOC+HBOT, and had complete wound
closure in less than three months. However, at the last
molecular testing, the patient had many more
microbial species compared with the initial testing
event, suggesting a massive community disruption
and reorganisation that led to an increase in microbial
diversity perhaps more susceptible to SOC methods.
These data suggest that HBOT is benecial as an
adjuvant therapy by disrupting the microbiota in the
biolm phenotype, likely interfering with many
processes that are nely tuned for certain groups of
microbiota within the biolm. Disruption appears to
play a major, if not primary, role in eradicating
biolms, providing a window of susceptibility to
SOC treatments.
Fig 2. Microbial response to hyperbaric oxygen therpay (HBOT) in vivo. The
reduction in bacterial load for the treatment and control group was determined
by comparing the initial and nal molecular diagnostics. Reductions in
bacterial load are evident but not signicant for the HBOT and standard of
care (SOC) groups (p=0.06 and p=0.1641 respectively). Statistical signicance
was determined using the Welch 2 sample t-test
Ct Value
HBOT initial HBOT nal SOC initial SOC nal
26
24
22
10
18
Fig 3. Response of wound microbiota to hyperbaric oxygen therapy (HBOT).
16S rDNA sequencing of patient samples at the initial and nal visits revealed
how biolm communities were altered after standard of care (SOC) treatment
protocols with adjuvant HBOT therapy. On the Y-axis, ‘none’ indicates that no
microbes of the specied type were detected at either sequencing event.
‘Emerge’ indicates that microbes not present in the initial sample were
detected at the second sequencing event at the end of the treatment protocol.
‘Persist’ indicates that microbes were present at both sequencing time points.
‘Eradicated’ indicates microbes that were present in the rst sequencing
sample but not in the second. Bacteria were grouped according to oxygen-
dependence, and fungi were categorised separately
Aerobes
None
Emerge
Persist
Eradicted
Anaerobes
None
Emerge
Persist
Eradicted
Fungi
None
Emerge
Persist
Eradicted
% of cases
0 10 20 30 40 50 60 70 80 90 100
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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018
S26
Discussion
The efcacy of HBOT on angiogenesis, bone formation,
and skin rejuvenation has been well
documented.24–27,30,31 However, few studies focused on
the bactericidal activity relative to the bacterial biolm
phenotype, despite many reports that described HBOT
expediting healing of chronic wounds.24–27,30,31 Recent
research in our laboratory demonstrated that wound
microbiota was the primary cause of pathogenesis in
chronic wounds.11 That nding raised the question:
does HBOT have bactericidal activity against wound
microbiota-forming biolm?
Typically, patients undergoing SOC alone have acute
infections inhabited by microbiota, which can be
treated quickly and efciently. To qualify for HBOT,
patients must have had a persistent wound being treated
for at least 30 days, indicative of a chronic wound
inhabited by recalcitrant multispecies bacterial biolms.
Biolm recalcitrance to antibiotics is compounded by
low diffusion rates into biolms. Low diffusion rates
may contribute to lessened antibiotic delivery and
limits oxygenation of the biolm which suppresses
bacterial metabolism, further limiting antibiotic
targets.32,33 HBOT drastically increases oxygenation of
host cells and very likely the biolm as well. This
oxygenation probably occurs at the host/biolm
interface, as well as at the biolm/environment
interface, which would increase oxygen diffusion into
the biolm. This scenario could possibly lead to
upregulation of metabolic genes and proteins
enhancing antibiotic susceptibility. Moreover, increased
oxygenation and metabolism could potentially trigger
a detachment and dispersion event within the biolm,
reverting biofilm cells to the more susceptible
planktonic phenotype. While this explanation is
attractive, much work remains to be done to fully
elucidate the mechanisms involved.
Taken together, the data from this study provide
compelling evidence that HBOT possesses bactericidal
activity towards wound biolms in vitro and in vivo. The
efficacy of HBOT on wound healing is
documented;24–27,30,31 however, the reimbursement for
treatment of chronic wounds is severely limited to
patients with only a few indications, such as diabetic
foot wounds or osteomyelitis. It is the hope of the
authors that this study will spark interest in the
community to conduct further research on the efcacy
of HBOT, which may hopefully lead to a more
widespread use of the technology in wound care.
Interestingly, sequencing of wound microbiota
before and after HBOT revealed that anaerobes and
fungi become more prevalent in wounds after HBOT.
This may be due to the combination of targeted
treatments for common wound microbiota such as
Pseudomonas aeruginosa and Staphylococcus aureus, serial
debridement, and HBOT decreasing competition in the
wound for less prevalent microbes. This nding suggests
that molecular diagnostics should be used in
conjunction with HBOT to determine if certain
antibiotics/antifungal treatments are necessary during
and post-HBOT for wound healing and closure. It is also
reasonable to speculate that such increased oxygen
concentrations within the capillary may diffuse out of
the host, potentiating antibiotics and host counter-
measures to aid in removal of the wound microbiota.
While the exact mechanism of bactericidal action has
not yet been elucidated, it can be speculated that
cellular responses to hyperoxygenation are involved.
Hyperoxygenation leads to increased accumulation of
reactive oxygen species (ROS) in cells which, at certain
thresholds, overwhelm the antioxidant defense and
repair systems of the cell.34 The biological targets of
ROS are widespread, including DNA, RNA, proteins,
and lipids. Immune cells exploit ROS production via
the reduced nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase enzyme as a weapon
during invasion of pathogenic bacteria.35 Increased
ROS production under hyperbaric conditions may
produce the same effect and serve to temporarily
destabilise the biolm community, allowing a window
for improved efcacy of SOC treatment protocols. It is
clear that if there is any signicant planktonic load,
3ATA of pure oxygen will have bactericidal activity
regardless of the species; however, it is less certain how
hyperoxygenation affects bacterial biolms. The results
presented here demonstrate in real patient wounds that
total wound microbiota decreased when using HBOT as
an adjuvant relative to that using SOC alone.
Fig 4. Patient progression with standard-of-care (SOC)
protocol. A 51-year-old male with a diabetic foot ulcer
(DFU) who underwent SOC alone. The initial bacterial
load was quite high for this patient (threshold cycle
(Ct)=17.39) but was able to be effectively treated with
SOC alone (nal Ct=21.78). The sequencing data showed
that the biolm community in this wound was completely
disrupted, having changed species composition by the
time of wound closure. While the 92.77% reduction in
bacterial load was very important, this patient spent
approximately four months in treatment
92.7 7%
reduction
n Prevotella bivia
n Enterococcus faecalis
n Veillonella pervula
n Other
n Haemophilus
parainuenzae
n Streptococcus angiosus
n Other
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JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018 S27
Limitations of the study
The in vitro biolms used in this study were grown in
broth media and transferred to TSA plates upon
maturation to undergo the HBOT treatment. In such an
experimental setup, metabolic waste products may
accumulate and nutrient shortage within the biolm
may signal a dispersal event and cause bacterial cells to
revert to the planktonic phenotype. Additionally,
handling the in vitro biolms excessively will damage
the cells resulting in misleading cell viability data. This
prevented in vitro biolms from undergoing multiple
HBOT treatments over the course of several days. It is
possible that additional HBOT treatments may have
produced more robust reductions in viable bacterial
cells strengthening the evidence presented. The in vivo
data was collected retrospectively from the patient
database which limited the ability to match HBOT
patients and SOC patients by sex, age and wound type.
Retrospective data collection was deemed appropriate
for the purpose of this study because patients at our
facility receive targeted treatments based on severity of
the wound, various comorbidities, etc. which would
prohibitively increase time needed to collect an
appropriate number of samples for the study.
Conclusions
Previous works on the efcacy of HBOT as an adjuvant
therapy has produced varying evidence regarding its
use as an adjuvant therapy in treating chronic
wounds.36,37 This may result from differences in choice
of experimental systems, antibiotic usage, and/or
duration of HBOT treatments. Recently, Jorgensen et
al.37 studied the efcacy of daptomycin and rifampicin
with intermittent HBOT to treat Staphylococcus aureus
biolms and did not observe any signicant benet in
treating implant-associated osteomyelitis in a murine
model. Kurt et al.,37 also using a murine model,
observed signicant reductions in bacterial load using
vancomycin or tigecycline in combination with HBOT
compared with using antibiotic alone in post-
sternotomy mediastinitis. In addition, HBOT as an
adjuvant to ciprooxacin resulted in increased bacterial
killing in vitro Pseudomonas aeruginosa biolms and in
humans with malignant otitis externa.38,39 The
evidence here supports the hypothesis that
hyperoxygenation of wound biolms using HBOT as an
adjuvant to SOC treatment contributes to the reduction
of total bacterial load. CMS only covers HBOT for a very
small subset of patients suffering from chronic wounds.
This is likely due to a lack of research on the topic and
conicting results reported from different groups
working on disparate models. It is our hope that the
literature on HBOT and its use as an adjuvant therapy
for chronic wound healing will continue to grow, so
that this technology will be better understood and can
potentially be more widely available to patients with
chronic non-healing wounds and other afictions,
which HBOT may be able to relieve. JWC
Fig 5. Patient progression – standard of care (SOC) + hyperbaric oxygen
therapy (HBOT). A 74-year-old male with a diabetic foot ulcer who underwent
HBOT. This patient began treatment with a medium-high bacterial load
(threshold cycle (Ct)=23.82) and opted for SOC with adjuvant HBOT (nal
Ct=26.18). The sequencing data for this patient showed a complex
polymicrobial community at the initial sequencing event, which became more
diverse as treatment progressed to the nal sequencing event. Interestingly,
the initial biolm community was composed of aerobic microbes, and the nal
sequencing event contained several anaerobic species. This suggested that
the original wound biolm was successfully disrupted by SOC with adjuvant
HBOT, promoting successful wound healing within approximately 2.5 months
of treatment
67. 01%
reduction
n Corynebacterium
tuberculosis
n Staphylococcus
haemolyticus
n Xylophilus ampenlinus
n Streptococcus mitis
n Roseateles
depoymerans
n Fusobacterium
nucleatum
n Other
n Finegoldia magna
n Anaerococcus vaginalis
n Porphyromonas levii
n Anaerococcus octavius
n Sporanaerobacter
acetigenes
n Peptoniphilus ivorii
n Peptoniphilus indolicus
n Porphyromonas
somerae
n Peptoniphilus harei
n Candidatus
peptoniphilus
n Brevibacterium
paucivorans
n Anaerococcus prevotii
n Facklamia languida
n Anaerococcus
lactolyticus
n Other
References
1 Mekkes JR, Loots MA, Van Der Wal AC, Bos JD. Causes, investigation
and treatment of leg ulceration. Br J Dermatol 2003; 148(3)388–401
2 Tsourdi E, Barthel A, Rietzsch H et al. Current aspects in the
pathophysiology and treatment of chronic wounds in diabetes mellitus.
Biomed Res Int 2013; 2013:385641. https://doi.org/10.1155/2013/385641
3 Costerton JW, Stewart PS, Greenberg EP. Bacterial biolms: a common
cause of persistent infections. Science 1999; 284(5418):1318–1322.
4 Kirketerp-Møller K, Jensen PO, Fazli M et al. Distribution, organization,
and ecology of bacteria in chronic wounds. J Clin Microbiol 2008;
46(8):2717–2722. https://doi.org/10.1128/JCM.00501-08
5 Wolcott RD, Ehrlich GD. Biolms and chronic infections. JAMA 2008;
299(22):2682–2684. https://doi.org/10.1001/jama.299.22.2682
6 Wolcott RD, Rhoads DD. A study of biolm-based wound management
in subjects with critical limb ischaemia. J Wound Care 2008;
17(4):145–155. https://doi.org/10.12968/jowc.2008.17.4.28835
7 Wolcott R, Costerton JW, Raoult D, Cutler SJ. The polymicrobial nature
of biolm infection. Clin Microbiol Infect 2013; 19(2):107–112. https://doi.
© 2018 MA Healthcare ltd
© MA Healthcare Ltd. Downloaded from magonlinelibrary.com by Randall Wolcott on January 23, 2018.
Use for licensed purposes only. No other uses without permission. All rights reserved.

research
JOURNAL OF WOUND CARE NORTH AMERICAN SUPPLEMENT, VOL 27, NO 1, JANUARY 2018
S28
org/10.1111/j.1469-0691.2012.04001.x
8 Attinger C, Wolcott R. Clinically addressing biolm in chronic wounds.
Adv Wound Care 2012; 1(3):127–132. https://doi.org/10.1089/
wound.2011.0333
9 Zhao G, Usui ML, Lippman SI et al. Biolms and inammation in chronic
wounds. Adv Wound Care 2013; 2(7):389–399. https://doi.org/10.1089/
wound.2012.0381
10 Ganesh K, Sinha M, Mathew-Steiner SS et al. Chronic wound biolm
model. Adv Wound Care 2015; 4(7):382–388. https://doi.org/10.1089/
wound.2014.0587
11 Wolcott R, Sanford N, Gabrilska R et al. Microbiota is a primary cause
of pathogenesis of chronic wounds. J Wound Care 2016; 25 Sup10:S33–
S43. https://doi.org/10.12968/jowc.2016.25.Sup10.S33
12 Bjarnsholt T, Jensen PØ, Moser C, Høiby, N (Eds). Biolm infections.
Springer-Verlag New York 2011
13 Chadha T. Bacterial biolms: survival mechanisms and antibiotic
resistance. J Bacteriol Parasitol 2014; 5(3):5–8. https://doi.org/
10.4172/2155-9597.1000190
14 Rabin N, Zheng Y, Opoku-Temeng C et al. Biolm formation
mechanisms and targets for developing antibiolm agents. Future Med
Chem 2015; 7(4):493–512. https://doi.org/10.4155/fmc.15.6
15 Aminov RI. Horizontal gene exchange in environmental microbiota.
Front Microbiol 2011; 2 158. https://doi.org/10.3389/fmicb.2011.00158
16 Cook LC, Dunny GM. The inuence of biolms in the biology of
plasmids. Microbiol Spectr 2014; 2(5). https://doi.org/10.1128/
microbiolspec.PLAS-0012-2013
17 Balcázar JL, Subirats J, Borrego CM. The role of biolms as
environmental reservoirs of antibiotic. Front Microbiol 2015; 6:1216.
https://doi.org/10.3389/fmicb.2015.01216
18 Costerton JW, Cheng KJ, Geesey GG, et al. Bacterial biolms in nature
and disease. Ann Rev Microbiol 1987;(41):435–464. https://doi.
org/10.1146/annurev.mi.41.100187.002251
19 Kim PJ, Steinberg JS. Wound care: biolm and its impact on the latest
treatment modalities for ulcerations of the diabetic foot. Semin Vasc Surg
2012; 25(2):70–74. https://doi.org/10.1053/j.semvascsurg.2012.04.008
20 Löndahl M, Katzman P, Nilsson A, Hammarlund C. Hyperbaric oxygen
therapy facilitates healing of chronic foot ulcers in patients with diabetes.
Diabetes Care 2010; 33(5):998–1003. https://doi.org/10.2337/dc09-1754
21 Goldstein LJ. Hyperbaric oxygen for chronic wounds. Dermatol Ther
2013; 26(3):207–214. https://doi.org/10.1111/dth.12053
22 Gill AL, Bell CN. Hyperbaric oxygen: its uses, mechanisms of action
and outcomes. QJM 2004; 97(7):385–395. https://doi.org/10.1093/qjmed/
hch074
23 Ueno T, Omi T, Uchida E et al. Evaluation of hyperbaric oxygen therapy
for chronic wounds. J Nippon Med Sch 2014; 81(1):4–11. https://doi.
org/10.1272/jnms.81.4
24 Eggleton P, Bishop A, Smerdon G. Safety and efcacy of hyperbaric
oxygen therapy in chronic wound management: current evidence. Chronic
Wound Care Management and Research 2015; 2:81–93. https://doi.
org/10.2147/CWCMR.S60319
25 Barnes RC. Point: hyperbaric oxygen is benecial for diabetic foot
wounds. Clin Infect Dis 2006; 43(2):188–192. https://doi.
org/10.1086/505207
26 Thackham JA, McElwain DL, Long RJ. The use of hyperbaric oxygen
therapy to treat chronic wounds: a review. Wound Repair Regen 2008;
16(3):321–330. https://doi.org/10.1111/j.1524-475X.2008.00372.x
27 Vishwanath G, Bhutani S. Hyperbaric oxygen and wound healing.
Indian J Plast Surg 2012; 45(2):316–324. https://doi.
org/10.4103/0970-0358.101309
28 Heffernan B, Murphy CD, Casey E. Comparison of planktonic and
biolm cultures of Pseudomonas uorescens DSM 8341 cells grown on
uoroacetate. Appl Environ Microbiol 2009; 75(9):2899–2907. https://doi.
org/10.1128/AEM.01530-08
29 Sun Y, Dowd SE, Smith E, Rhoads DD, Wolcott RD. In vitro
multispecies Lubbock chronic wound biolm model. Wound Repair Regen
2008; 16(6):805–813. https://doi.org/10.1111/j.1524-475X.2008.00434.x
30 Grassmann JP, Schneppendahl J, Hakimi AR et al. Hyperbaric oxygen
therapy improves angiogenesis and bone formation in critical sized
diaphyseal defects. J Orthop Res 2015; 33(4):513–520. https://doi.
org/10.1002/jor.22805
31 Sahni T, Singh P, John MJ. Hyperbaric oxygen therapy: current trends
and applications. J Assoc Physicians India 2003; 51:280–284.
32 Stewart PS. Diffusion in Biolms. J. Bacteriol 2003; 185(5):1485–1491
33 Walters MC 3rd, Roe F, Bugnicourt A et al. Contributions of antibiotic
penetration, oxygen limitation, and low metabolic activity to tolerance of
Pseudomonas aeruginosa biolms to ciprooxacin and tobramycin.
Antimicrob Agents Chemother 2003; 47(1):317–323. https://doi.
org/10.1128/AAC.47.1.317-323.2003
34 Godman CA, Joshi R, Giardina C, Perdrizet G, Hightower LE.
Hyperbaric oxygen treatment induces antioxidant gene expression. Ann N
Y Acad Sci 2010; 1197(1):178–183. https://doi.
org/10.1111/j.1749-6632.2009.05393.x
35 Cabiscol E, Tamarit J, Ros J. Oxidative stress in bacteria and protein
damage by reactive oxygen species. Int Microbiol 2000; 3(1):3–8.
36 Jørgensen N, Hansen K, Andreasen C et al. Hyperbaric oxygen
therapy is ineffective as an adjuvant to daptomycin with rifampicin
treatment in a murine model of staphylococcus aureus in. Microorganisms
2017; 5(2):21. https://doi.org/10.3390/microorganisms5020021
37 Kurt T, Vural A, Temiz A et al. Adjunctive hyperbaric oxygen therapy or
alone antibiotherapy? Methicillin resistant Staphylococcus aureus
mediastinitis in a rat model. Braz J Cardiovasc Surg 2015; 30(5): 538–543.
38 Kolpen M, Mousavi N, Sams T et al. Reinforcement of the bactericidal
effect of ciprooxacin on Pseudomonas aeruginosa biolm by hyperbaric
oxygen treatment. Int J Antimicrob Agents 2016; 47(2):163–167. https://
doi.org/10.1016/j.ijantimicag.2015.12.005
39 Sabra RM, Taha MS, Khafagy AG, Elsamny T. Value of hyperbaric
oxygen therapy in the management of malignant otitis externa patients.
The Egyptian Journal of Otolaryngology 2015; 31(3):143–148. https://doi.
org/10.4103/1012-5574.161598
Reective questions
● What are th e methods used by microbiota that cause
wound chronicity?
● What are the molecular mechanisms involved in the biocidal
activit y of Hyperbaric oxygen therapy (HBOT)?
● Does HBOT trigger a dispe rsion event of the biolm,
reverti ng biolm cells to the planktoni c phenotype, which
may be easier to treat with st andard care therapies?
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