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Efficacy of hyperbaric oxygen therapy in bacterial biofilm eradication


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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.
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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:
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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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.
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
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
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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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’-GCTTGACGGGCGGTGT-3’) 16S rDNA primers
(20µM each) were used with a 16S rDNA probe
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.
Samples were amplied for semiconductor sequencing
using a forward and reverse fusion primer. The forward
primer was constructed with the Ion A linker
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
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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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.
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.
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)
p=0.002 p=0.07 p=0.03
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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
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
% of cases
0 10 20 30 40 50 60 70 80 90 100
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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%
n Prevotella bivia
n Enterococcus faecalis
n Veillonella pervula
n Other
n Haemophilus
n Streptococcus angiosus
n Other
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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.
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%
n Corynebacterium
n Staphylococcus
n Xylophilus ampenlinus
n Streptococcus mitis
n Roseateles
n Fusobacterium
n Other
n Finegoldia magna
n Anaerococcus vaginalis
n Porphyromonas levii
n Anaerococcus octavius
n Sporanaerobacter
n Peptoniphilus ivorii
n Peptoniphilus indolicus
n Porphyromonas
n Peptoniphilus harei
n Candidatus
n Brevibacterium
n Anaerococcus prevotii
n Facklamia languida
n Anaerococcus
n Other
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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?
Need to know what new products are available at your  ngertips?
Ever wonder which product is the best for your patient?
The professional’s essential guide to dermatology product selection
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Use for licensed purposes only. No other uses without permission. All rights reserved.
... An early and combined HBOT therapy plus current practices may be crucial as a lifesaving and cost-efficacy therapy, particularly in the most critical patients [73]. Clinical practice agrees on the necessity of HBOT in the event of an anaerobic infection, as anaerobic bacteria are killed by a much higher amount of pressurized O 2 [74,75]. For instance, the use of HBOT in the anaerobic Clostridium perfringens bacteria is specially recommended [76]. ...
... The anti-inflammatory potential of HBOT also aids to lessen tissue damage and infection expansion [72], also explained by a decrease in neutrophil activation, eviting rolling and accumulation of white blood cells (WBCs), hence limiting the production of ROS by neutrophils and avoiding reperfusion injury [45]. Moreover, this is observed in In vitro studies, having been demonstrated the biofilm shrinkage ability with the significant decreases in cellular load of anaerobic bacteria and fungi after HBOT [75]. A sepsis mouse model showed a significant increase in survival rate, >50%, with early HBOT compared to a control group that did not receive the treatment and was associated with lower expression of TNF-α, IL-6 and IL-10 [78]. ...
Full-text available
Hyperbaric oxygen therapy (HBOT) consists of using of pure oxygen at increased pressure (in general, 2–3 atmospheres) leading to augmented oxygen levels in the blood (Hyperoxemia) and tissue (Hyperoxia). The increased pressure and oxygen bioavailability might be related to a plethora of applications, particularly in hypoxic regions, also exerting antimicrobial, immunomodulatory and angiogenic properties, among others. In this review, we will discuss in detail the physiological relevance of oxygen and the therapeutical basis of HBOT, collecting current indications and underlying mechanisms. Furthermore, potential areas of research will also be examined, including inflammatory and systemic maladies, COVID-19 and cancer. Finally, the adverse effects and contraindications associated with this therapy and future directions of research will be considered. Overall, we encourage further research in this field to extend the possible uses of this procedure. The inclusion of HBOT in future clinical research could be an additional support in the clinical management of multiple pathologies.
... Hypoxia is a hallmark of infectious diseases, and HBOT is approved and indicated in severe bacterial infections such as necrotizing soft tissue infection (Wilkinson and Doolette, 2004), chronic wounds (Sanford et al., 2018), refractory osteomyelitis (Mader et al., 1978(Mader et al., , 1990, and intracranial abscesses (Bilic et al., 2012). Although HBOT is a well-established treatment used both for infectious and non-infectious diseases, there is a need for improved clinical documentation. ...
... Together with the documented positive effects of HBOT in clinical studies of other infectious diseases (Korhonen, 2000;Escobar et al., 2005;Kaur et al., 2012;Skeik et al., 2015;Sanford et al., 2018), this supports the concept of using HBOT in biofilm infections and the rationality of setting up a clinical feasibility study in patients with IE. The benefit of HBOT in IE can most likely be extended to most courses, although the effect in patients that do not respond to conservative antibiotic therapy might be significantly limited. ...
Full-text available
Infective endocarditis (IE) is a life-threatening infective disease with increasing incidence worldwide. From early on, in the antibiotic era, it was recognized that high-dose and long-term antibiotic therapy was correlated to improved outcome. In addition, for several of the common microbial IE etiologies, the use of combination antibiotic therapy further improves outcome. IE vegetations on affected heart valves from patients and experimental animal models resemble biofilm infections. Besides the recalcitrant nature of IE, the microorganisms often present in an aggregated form, and gradients of bacterial activity in the vegetations can be observed. Even after appropriate antibiotic therapy, such microbial formations can often be identified in surgically removed, infected heart valves. Therefore, persistent or recurrent cases of IE, after apparent initial infection control, can be related to biofilm formation in the heart valve vegetations. On this background, the present review will describe potentially novel non-antibiotic, antimicrobial approaches in IE, with special focus on anti-thrombotic strategies and hyperbaric oxygen therapy targeting the biofilm formation of the infected heart valves caused by Staphylococcus aureus. The format is translational from preclinical models to actual clinical treatment strategies.
... NPWT may assist wound healing by increasing tissue perfusion and help in the production of granulation tissue besides reducing exudates, edema, and bacterial contamination [152]. Recent work suggests that NPWT with the instillation of antimicrobials such as diluted hypochlorous acid contributes to a significant reduction in wound bioburden and thereby shows promising results in wounds with mature biofilms [153]. With the added advantage of absent bacterial resistance development, this technique in combination with topical antiseptics is ideal in the management of difficult-to-heal wounds. ...
Full-text available
The bubbling community of microorganisms, consisting of diverse colonies encased in a self-produced protective matrix and playing an essential role in the persistence of infection and antimicrobial resistance, is often referred to as a biofilm. Although apparently indolent, the biofilm involves not only inanimate surfaces but also living tissue, making it truly ubiquitous. The mechanism of biofilm formation, its growth, and the development of resistance are ever-intriguing subjects and are yet to be completely deciphered. Although an abundance of studies in recent years has focused on the various ways to create potential anti-biofilm and antimicrobial therapeutics, a dearth of a clear standard of clinical practice remains, and therefore, there is essentially a need for translating laboratory research to novel bedside anti-biofilm strategies that can provide a better clinical outcome. Of significance, biofilm is responsible for faulty wound healing and wound chronicity. The experimental studies report the prevalence of biofilm in chronic wounds anywhere between 20 and 100%, which makes it a topic of significant concern in wound healing. The ongoing scientific endeavor to comprehensively understand the mechanism of biofilm interaction with wounds and generate standardized anti-biofilm measures which are reproducible in the clinical setting is the challenge of the hour. In this context of “more needs to be done”, we aim to explore various effective and clinically meaningful methods currently available for biofilm management and how these tools can be translated into safe clinical practice.
... It helps reduce tissue edema, restore venous return, improve microcirculation (113,114), and stimulate angiogenesis (115), Furthermore, it also increases PaO 2 in the fractured area (especially in the callus and medullary cavity), enhances the activities of osteoclasts and osteoblasts, and accelerates bone callus formation (116). In addition, hyperbaric oxygen also enhances the anti-infective ability of local tissues, especially against anaerobic bacteria (117). Clinically, hyperbaric oxygen chambers have been widely used in patients with bone defects to shorten the recovery time. ...
Full-text available
Reaching areas at altitudes over 2,500–3,000 m above sea level has become increasingly common due to commerce, military deployment, tourism, and entertainment. The high-altitude environment exerts systemic effects on humans that represent a series of compensatory reactions and affects the activity of bone cells. Cellular structures closely related to oxygen-sensing produce corresponding functional changes, resulting in decreased tissue vascularization, declined repair ability of bone defects, and longer healing time. This review focuses on the impact of high-altitude hypoxia on bone defect repair and discusses the possible mechanisms related to ion channels, reactive oxygen species production, mitochondrial function, autophagy, and epigenetics. Based on the key pathogenic mechanisms, potential therapeutic strategies have also been suggested. This review contributes novel insights into the mechanisms of abnormal bone defect repair in hypoxic environments, along with therapeutic applications. We aim to provide a foundation for future targeted, personalized, and precise bone regeneration therapies according to the adaptation of patients to high altitudes.
... The use of hyperbaric oxygen therapy reduces the infection rate due to the bacteriostatic action of oxygen, and also facilitates the neutralisation of exotoxins by increasing the oxygenation of the damaged tissue 55,56 . HBO therapy can effectively destroy bacterial biofilm, which is important in the treatment of chronic infection associated with skin damage 57 . ...
Full-text available
Introduction: Hyperbaric oxygen therapy (HBOT) involves the use of 100% pure oxygen in conditions of increased pressure, exceeding atmospheric pressure. This allows the supply of several times more oxygen to the internal organs and blood serum than when using standard pressure. HBOT has proven to support the treatment of autoimmune skin diseases, complications of metabolic diseases and burns, as confirmed by clinical studies. In addition, this therapy can also be used to improve the physiological condition of the skin after cosmetology procedures. Objectives: The aim of this work is to review information on the therapeutic effects of hyperbaric oxygen therapy in the treatment of skin diseases, especially atopic dermatitis, psoriasis, diabetic foot, 2nd-degree burns and complications following cosmetic procedures. Method: The review was based on the works published in the last 20 years (1999-2019), available in the following databases: PubMed, Google Scholar and PEDro. Results and conclusions: The use of HBOT is becoming more common in the treatment of skin complications related to diabetes, as well as burn wounds. It is estimated that HBOT reduces the risk of foot ulcers and amputation in diabetic foot syndrome. In addition, HBOT promotes faster healing of burn wounds, also with the use of allogenic skin grafts. By increasing the level of reactive oxygen species (ROS), hyperbaric oxygen therapy significantly supports the treatment of psoriasis and atopic dermatitis. Despite this, the exact mechanisms of hyperbaric oxygen are still poorly understood, and the use of HBOT in the treatment of skin diseases has not yet been included in treatment protocols.
Full-text available
Chronic wounds have harmful effects on both patients and healthcare systems. Wound chronicity is attributed to an impaired healing process due to several host and local factors that affect healing pathways. The resulting ulcers contain a wide variety of microorganisms that are mostly resistant to antimicrobials and possess the ability to form mono/poly-microbial biofilms. The search for new, effective and safe compounds to handle chronic wounds has come a long way throughout the history of medicine, which has included several studies and trials of conventional treatments. Treatments focus on fighting the microbial colonization that develops in the wound by multidrug resistant pathogens. The development of molecular medicine, especially in antibacterial agents, needs an in vitro model similar to the in vivo chronic wound environment to evaluate the efficacy of antimicrobial agents. The Lubbock chronic wound biofilm (LCWB) model is an in vitro model developed to mimic the pathogen colonization and the biofilm formation of a real chronic wound, and it is suitable to screen the antibacterial activity of innovative compounds. In this review, we focused on the characteristics of chronic wound biofilms and the contribution of the LCWB model both to the study of wound poly-microbial biofilms and as a model for novel treatment strategies.
Chronic wounds including vascular ulcers, diabetic ulcers, pressure ulcers, and burn wounds show delayed progress through the healing process. Some of their common features are prolonged inflammation, persistent infection, and presence of biofilms resistant to antimicrobials and host immune response. Biofilm formation by opportunistic pathogens is a major problem in chronic wound management. Some of the commonly and traditionally used chronic wound management techniques are physical debridement and cleansing. In recent years, novel techniques based on anti-biofilm agents are explored to prevent biofilm-associated infections and facilitate wound healing. In this chapter, the role of biofilms formed by the ESKAPE pathogens (Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) and Candida species in delayed wound healing have been discussed. The current and emerging techniques in the detection of biofilms for the management of wounds have been focused. The limitations of the existing therapeutics and novel wound management strategies have been deliberated.
Neutrophils are important to the defense of the host against bacterial infection. Pathogens and the immune system cells create via respiration, a hypoxic environment in infected regions. Hypoxic conditions affect both the neutrophil’s ability to eradicate the infection and also change the behavior of the bacterial-pathogens by eliciting the production of various virulence factors, the creation of bacterial biofilm and the initialization of anaerobic metabolism. In this work interactions of bacterial biofilm and neutrophils are studied in a domain where oxygen is diffusing into the environment and is being consumed by biofilm. Within a hypoxic environment, bacteria grow anaerobically and secrete higher levels of toxin that diffuses and lyses neutrophils. A mathematical model explicitly representing the biofilm volume fraction, oxygen, and diffusive virulence factors (toxin) as well as killing of bacteria by neutrophils is developed and studied first in 1D and then in 2D. Stability analysis and numerical simulations showing the effects of oxygen and toxin concentration on neutrophil-bacteria interactions are presented to identify different possible scenarios that can lead to elimination of the infection or its persistence as a chronic infection. Specifically, when bacteria are allowed to utilize anaerobic breathing and or to produce toxin, their fitness is enhanced against neutrophils attacks. A possible insight on how virulent bacterial colonies can synergistically resist neutrophils and survive is presented.
The ageing world population, the rise in immunocompromised individuals, prolonged hospitalizations, the overuse of antibiotics, and others led to an increase in the incidence of diseases associated with chronic biofilm infections (e.g., periodontitis and periodontal disease, chronic skin diseases, chronic upper and lower respiratory tract diseases, urinary tract infections, nosocomial infections secondary to indwelling medical devices). While biofilm-associated infections are more resistant to conventional therapy, nanotechnology-based approaches have recently achieved visible advances in their management by an improved targeted delivery of medicines. In this chapter, we aimed to review the main diseases associated with biofilm chronic infections as well as their nano-targeted therapeutic approach. Nanomedicine may establish novel standards in the therapy of pathogenic biofilms in everyday clinical practice. More research is needed to clarify the complex interactions between nano-therapeutics, microbial biofilms, and the host, to better understand their efficacy, toxicity, and long-term effects.
Full-text available
Implant-associated infections caused by bacterial biofilms are difficult to treat. Surgical intervention is often necessary to cure the patient, as the antibiotic recalcitrance of biofilms renders them untreatable with conventional antibiotics. Intermittent hyperbaric oxygen treatment (HBOT) has been proposed as an adjuvant to conventional antibiotic treatment and it has been speculated that combining HBOT with antibiotics could improve treatment outcomes for biofilm infections. In this study we addressed whether HBOT could improve treatment outcomes of daptomycin and rifampicin combination therapy. The effect of HBOT on the treatment outcomes of daptomycin and rifampicin against implant-associated osteomyelitis was quantified in a murine model. In total, 80 mice were randomized into two groups receiving antibiotics, either alone or in combination with daily intermittent HBOT (304 kPa for 60 min) following injection of antibiotics. Treatment was initiated 11 days after animals were infected with Staphylococcus aureus and treatment duration was 14 days. We found that HBOT did not improve the cure rate and did not reduce the bacterial load on the implant surface or in the surrounding tissue. Cure rates of daptomycin + rifampicin were 40% in infected tibias and 75% for implants while cure rates for HBOT-daptomycin + rifampicin were 50% and 85%, respectively, which were not significantly higher (Fisher’s exact test). While it is encouraging that the combination of daptomycin and rifampicin is very effective, our study demonstrates that this efficacy cannot be improved by adjuvant HBOT.
Full-text available
Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterised by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leucocyte activity. Whilst the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficacy of several bactericidal antibiotics such as ciprofloxacin is enhanced by stimulation of the aerobic respiration of pathogens, and that lack of O2 increases their tolerance. Reoxygenation of O2-depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. We tested such a strategy using reoxygenation of O2-depleted P. aeruginosa strain PAO1 agarose-embedded biofilms by hyperbaric oxygen treatment (HBOT) (100% O2, 2.8 bar), enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. This proof-of-principle study demonstrates that biofilm reoxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections.
Full-text available
Antibiotic resistance has become a significant and growing threat to public and environmental health. To face this problem both at local and global scales, a better understanding of the sources and mechanisms that contribute to the emergence and spread of antibiotic resistance is required. Recent studies demonstrate that aquatic ecosystems are reservoirs of resistant bacteria and antibiotic resistance genes as well as potential conduits for their transmission to human pathogens. Despite the wealth of information about antibiotic pollution and its effect on the aquatic microbial resistome, the contribution of environmental biofilms to the acquisition and spread of antibiotic resistance has not been fully explored in aquatic systems. Biofilms are structured multicellular communities embedded in a self-produced extracellular matrix that acts as a barrier to antibiotic diffusion. High population densities and proximity of cells in biofilms also increases the chances for genetic exchange among bacterial species converting biofilms in hot spots of antibiotic resistance. This review focuses on the potential effect of antibiotic pollution on biofilm microbial communities, with special emphasis on ecological and evolutionary processes underlying acquired resistance to these compounds.
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Recommendations Classification/diagnosis Diabetic foot infection must be diagnosed clinically, based on the presence of local or systemic signs or symptoms of inflammation (strong; low). Assess the severity of any diabetic foot infection using the Infectious Diseases Society of America/International Working Group on the Diabetic Foot classification scheme (strong; moderate). Osteomyelitis For an infected open wound, perform a probe‐to‐bone test; in a patient at low risk for osteomyelitis, a negative test largely rules out the diagnosis, while in a high‐risk patient, a positive test is largely diagnostic (strong; high). Markedly elevated serum inflammatory markers, especially erythrocyte sedimentation rate, are suggestive of osteomyelitis in suspected cases (weak; moderate). A definite diagnosis of bone infection usually requires positive results on microbiological (and, optimally, histological) examinations of an aseptically obtained bone sample, but this is usually required only when the diagnosis is in doubt or determining the causative pathogen's antibiotic susceptibility is crucial (strong; moderate). A probable diagnosis of bone infection is reasonable if there are positive results on a combination of diagnostic tests, such as probe‐to‐bone, serum inflammatory markers, plain X‐ray, magnetic resonance imaging (MRI) or radionuclide scanning (strong; weak). Avoid using results of soft tissue or sinus tract specimens for selecting antibiotic therapy for osteomyelitis as they do not accurately reflect bone culture results (strong; moderate). Obtain plain X‐rays of the foot in all cases of non‐superficial diabetic foot infection (strong; low). Use MRI when an advanced imaging test is needed for diagnosing diabetic foot osteomyelitis (strong; moderate). When MRI is not available or contraindicated, consider a white blood cell‐labelled radionuclide scan, or possibly single‐photon emission computed tomography (CT) and CT (SPECT/CT) or fluorine‐18‐fluorodeoxyglucose positron emission tomography/CT scans (weak; moderate). Assessing severity At initial evaluation of any infected foot, obtain vital signs and appropriate blood tests, debride the wound and probe and assess the depth and extent of the infection to establish its severity (strong; moderate). At initial evaluation, assess arterial perfusion and decide whether and when further vascular assessment or revascularization is needed (strong; low). Microbiological considerations Obtain cultures, preferably of a tissue specimen rather than a swab, of infected wounds to determine the causative microorganisms and their antibiotic sensitivity (strong; high). Do not obtain repeat cultures unless the patient is not clinically responding to treatment, or occasionally for infection control surveillance of resistant pathogens (strong; low). Send collected specimens to the microbiology laboratory promptly, in sterile transport containers, accompanied by clinical information on the type of specimen and location of the wound (strong; low). Surgical treatment Consult a surgical specialist in selected cases of moderate, and all cases of severe, diabetic foot infection (weak; low). Perform urgent surgical interventions in cases of deep abscesses, compartment syndrome and virtually all necrotizing soft tissue infections (strong; low). Consider surgical intervention in cases of osteomyelitis accompanied by spreading soft tissue infection, destroyed soft tissue envelope, progressive bone destruction on X‐ray or bone protruding through the ulcer (strong; low). Antimicrobial therapy While virtually all clinically infected diabetic foot wounds require antimicrobial therapy, do not treat clinically uninfected wounds with antimicrobial therapy (Strong; Low) Select specific antibiotic agents for treatment based on the likely or proven causative pathogens, their antibiotic susceptibilities, the clinical severity of the infection, evidence of efficacy of the agent for diabetic foot infection and costs (strong; moderate). A course of antibiotic therapy of 1–2 weeks is usually adequate for most mild and moderate infections (strong; high). Administer parenteral therapy initially for most severe infections and some moderate infections, with a switch to oral therapy when the infection is responding (strong; low). Do not select a specific type of dressing for a diabetic foot infection with the aim of preventing an infection or improving its outcome (strong; high). For diabetic foot osteomyelitis, we recommend 6 weeks of antibiotic therapy for patients who do not undergo resection of infected bone and no more than a week of antibiotic treatment if all infected bone is resected (strong; moderate). We suggest not using any adjunctive treatments for diabetic foot infection (weak; low). When treating a diabetic foot infection, assess for use of traditional remedies and previous antibiotic use and consider local bacterial pathogens and their susceptibility profile (strong; low).
Objective: Diverse microorganisms present on the surface of chronic wounds have been established to constitute wound microbiota. The aims of this study were to quantify the viability of wound microbiota, classify dispersal of viable microbes from the wound, and determine if human wound microbiota can produce a chronic wound in an animal model. Method: Wound microbiotas as units (multiple microbial species acting as one infectious agent) were obtained from well-defined human chronic wounds and seeded onto mouse surgical excision wounds to produce chronically infected wounds that closely resembled the chronic wounds observed in the original hosts. Results: We found the wound microbiota harvested from 35 out of 43 (81%) patients could produce similar chronic wounds (producing slough and exudate) in a murine chronic wound model. The top 30 species present in patient wounds were identified in the mouse wounds by molecular sequencing. Koch's postulates could therefore be applied to establish wound microbiota as the cause of the original human chronic wound infections. Evidence-based medicine criteria such as Hill's criteria for causation can all be satisfied by what is currently known about wound microbiota. Conclusion: This study demonstrates that wound microbiota actively disseminates from the chronic wound by forces and mechanisms intrinsic to the wound. Koch's postulates and Hill's criteria for causation together suggest chronic wound microbiota to be the main cause underlying the pathogenesis of chronic wounds. Declaration of interest: RW has an equity interest in PathoGenius Labs. No funding was received for this study.
There has been an increasing interest and emphasis on the sessile bacterial lifestyle biofilms since it was discovered that the vast majority of the total microbial biomass exists as biofilms. Leeuwenhoek first described the aggregation of bacteria in 1677, but it was only recently recognized as being important in chronic infection. In 1993, the American Society for Microbiology (ASM) recognized that the biofilm mode of growth was relevant to microbiology. This book covers both the evidence for biofilms in many chronic bacterial infections as well as the problems facing these infections such as diagnostics, pathogenesis, treatment regimes, and in vitro and in vivo models for studying biofilms. This is the first scientific book on biofilm infections, with chapters written by world- renowned scientists and clinicians. The intended audience of this book includes scientists, teachers at the university level, as well as clinicians. About the Editors: Thomas Bjarnsholt, Ph.D., Technical University of Denmark, Lyngby, Denmark Peter Oestrup Jensen, Dept. of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark Claus Moser, Ph.D., Dept. of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark Niels Hoeby, Dept. of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark. © Springer Science+Business Media, LLC 2011. All rights reserved.
Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
Background Malignant external otitis is a rapidly spreading bacterial infection that is aggressive in nature and may be fatal if left untreated. Hyperbaric oxygen therapy (HBOT) is a medical treatment in which the entire body is placed in an airtight chamber at increased atmospheric pressure and has been proven to be effective for a number of different medical conditions. Objective The aim of this study was to assess the usefulness of HBOT as an adjunctive treatment in patients with malignant otitis externa. Patients and methods Forty-three diabetic patients, who had malignant otitis externa, underwent control of diabetes mellitus and were treated with ciprofloxacin. HBOT was administered to 15 patients as an adjunctive treatment. All the patients were evaluated clinically (in terms of ear discharge, granulations, and pain severity) and radiologically by a temporal bone computed tomography scan. The minimum follow-up duration in both groups was 2 months. HBOT was administered in one session every other day for 2 months, resulting in a total of 30 sessions. Patient factors analyzed included age, sex, ear discharge, and pain severity. Results A total of 43 patients (28 men, 15 women) were divided into two groups: group A (28 patients) only received the antibiotic ciprofloxacin and group B (15 patients) was treated with ciprofloxacin and hyperbaric oxygen. The severity of pain improved considerably and the pain score decreased markedly from score 3 (severe) to score 0 (no pain) after 1 month in 46.7 and 93.3% of the patients by the end of the second month in comparison with patients treated only with the antibiotic: 0% after 1 month and 28.5% after 2 months. On clinical and microscopic examination, both ear discharge and granulations in the external canal had improved considerably. There was no ear discharge in 80% of patients in group B after one month treathent, 93.3% after 2 months, in comparison with 0% after 1 month, 28.5% after 2 months treatment in group A, highly statistically significant ( P Conclusion The addition of HBOT to medical treatment is highly effective and has facilitated considerable improvement in patients.