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Update on Managing Serious Wound Infections in Horses 4. Wounds Involving Soft Tissues

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Abstract

This fourth and final article of the series continues the discussion of advances in the management of serious wound infections in horses, this time with the focus on wounds involving soft tissues. The preceding articles focused on wounds involving joints and other synovial structures 1,2 and wounds involving bone. 3 Wound infections can be a considerable challenge in this era of multidrug-resistant bacteria. Examples include these variants of common equine wound pathogens: 4 • methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE) • vancomycin-resistant Enterococcus spp. (VRE) • heteroresistant vancomycin-intermediate S. aureus (hVISA) • penicillin-resistant Actinobacillus spp. • aminoglycoside-resistant Escherichia coli • Pseudomonas aeruginosa strains that are resistant to most commonly used antibiotics Note that resistance is now documented in equine isolates for vancomycin5-8 and imipenem7,9,10—drugs we are advised in veterinary medicine to reserve for use only with documented multidrug-resistant pathogens. Multidrug-resistant strains of bacterial pathogens are still relatively uncommon in equine practice, but their increasing prevalence and diversity—particularly in hospitalized patients but also on-farm—spurs us to seek treatment approaches that extend beyond a reliance on antibiotics.4 The usual response to a serious wound infection is to change or add antibiotics, increase the antibiotic dosage(s), add local or regional delivery to systemic administration of antibiotics—in other words, to continue to rely on antibiotic drugs primarily. Certainly, if culture and sensitivity results indicate that a change of drug is needed or a polymicrobial infection is present which requires the addition of a different class of antibiotic drug, then these measures must be implemented immediately. In addition, local and regional modes of antibiotic delivery have undoubtedly improved our ability to manage difficult infections in horses, such as septic arthritis1,2 and osteomyelitis.3 However, because of the nature of serious wound infections, other factors beyond antibiotic sensitivity and effective delivery become crucial to a successful outcome. A healthy body has the circulatory and immunological resources to deal with most instances of bacterial invasion, so in most wounds bacterial infection remains localized and is dealt with quickly and effectively by the patient, either alone or with the aid of veterinary treatment. So with a serious wound infection, it is wise to begin with a question: Why has this infection persisted and progressed?
Update on Managing Serious Wound Infections in Horses
4. Wounds Involving Soft Tissues
Christine King BVSc, MANZCVS, MVetClinStud
This fourth and final article of the series continues the discussion of advances in the management
of serious wound infections in horses, this time with the focus on wounds involving soft tissues.
The preceding articles focused on wounds involving joints and other synovial structures1,2 and
wounds involving bone.3
Wound infections can be a considerable challenge in this era of multidrug-resistant bacteria.
Examples include these variants of common equine wound pathogens:4
methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE)
vancomycin-resistant Enterococcus spp. (VRE)
heteroresistant vancomycin-intermediate S. aureus (hVISA)
penicillin-resistant Actinobacillus spp.
aminoglycoside-resistant Escherichia coli
Pseudomonas aeruginosa strains that are resistant to most commonly used antibiotics
Note that resistance is now documented in equine isolates for vancomycin5-8 and
imipenem7,9,10—drugs we are advised in veterinary medicine to reserve for use only with
documented multidrug-resistant pathogens.
A recent study from Utrecht University in The Netherlands illustrates the problem we all now
face. The researchers examined in vitro antimicrobial susceptibility patterns in 1,091 bacterial
isolates from 588 foals with sepsis between 1979 and 2010, and found the following trends:10
gentamicin—decrease in percentage of susceptible enterobacteria (E. coli, Klebsiella spp.,
Salmonella spp., etc.), Actinobacillus spp., and β-hemolytic Streptococcus spp.
amikacin—increase in minimum inhibitory concentrations (MIC) in those same groups (i.e.,
higher concentrations needed for bactericidal effect)
ceftiofur—decrease in percentage of susceptible enterobacteria, and increase in MIC values
in Enterococcus spp. and Pseudomonas spp.
ceftizoxime—increase in MIC values in enterobacteria
ticarcillin-clavulanate—increase in MIC values in Enterococcus spp.
imipenem—increasing resistance in Enterococcus spp. in recent years
In a companion study,11 the prevalence of particular genera also changed over time. Notably,
there was a significant increase in Gram-positive isolates and a decrease in enterobacteria,
particularly Klebsiella spp., over time. In addition, Enterococcus spp. were isolated more often in
recent years. It is likely that patterns of antimicrobial use influenced all of these changes and
created somewhat of a vicious cycle by driving the changes in resistance and prevalence, and
then by responding to these changes with different patterns of antimicrobial use.
Multidrug-resistant strains of bacterial pathogens are still relatively uncommon in equine
practice, but their increasing prevalence and diversity—particularly in hospitalized patients but
also on-farm—spurs us to seek treatment approaches that extend beyond a reliance on
antibiotics.4 The usual response to a serious wound infection is to change or add antibiotics,
increase the antibiotic dosage(s), add local or regional delivery to systemic administration of
antibiotics—in other words, to continue to rely on antibiotic drugs primarily. Certainly, if culture
and sensitivity results indicate that a change of drug is needed or a polymicrobial infection is
present which requires the addition of a different class of antibiotic drug, then these measures
must be implemented immediately. In addition, local and regional modes of antibiotic delivery
have undoubtedly improved our ability to manage difficult infections in horses, such as septic
arthritis1,2 and osteomyelitis.3 However, because of the nature of serious wound infections, other
factors beyond antibiotic sensitivity and effective delivery become crucial to a successful
outcome.
A healthy body has the circulatory and immunological resources to deal with most instances of
bacterial invasion, so in most wounds bacterial infection remains localized and is dealt with
quickly and effectively by the patient, either alone or with the aid of veterinary treatment. So
with a serious wound infection, it is wise to begin with a question: Why has this infection
persisted and progressed?
ORIGINS OF WOUND INFECTION
Of the many possible scenarios, most serious wound infections involve at least one, and usually a
combination, of the following factors:4
extensive contamination, or bacterial burden that overwhelms the patient’s resources
§ fecal contamination—most likely with wounds involving the lower limb and with
penetrating wounds to the abdomen or perineum that involve compromised bowel
(including surgical wounds)
§ penetrating wounds to the oral cavity, pharynx, esophagus, or upper airway—these sites
have a good blood supply, but they also have an extensive resident microflora that are
readily disseminated by mucus secretions
§ environmental contamination—dirt, plant debris, or insect activity (e.g., flies) may
introduce large numbers of bacteria into any open wound
refugia which protect the bacteria from host defenses and antibiotic drugs
§ foreign bodies—most often pieces of metal, wood, or other plant material (thorns, awns,
etc.)
§ surgical implants—metal plates, screws, pins, wire; surgical mesh; prostheses; embedded
suture, etc.
§ devitalized tissue—bone, muscle, tendon, ligament, fascia, or skin
§ inflammatory/necrotic debris—pockets or pools of free purulent material, abscessation
§ mucoid biofilms produced by the bacteria themselves
immunocompromise (see below)
poor perfusion (see below)
antibiotic insensitivity
§ inherent antibiotic resistance—i.e., inappropriate drug choice for the pathogen(s)
involved
§ acquired antibiotic resistance—e.g., MRSA, aminoglycoside-resistant E. coli; unlike
inherent resistance, acquired resistance is unpredictable
§ inappropriate drug dosage, route, or duration of treatment—each may result in
subtherapeutic antibiotic concentrations at the site of infection, even when the pathogen
is susceptible to the drug in vitro
§ poor perfusion—it, too, may result in subtherapeutic antibiotic concentrations at the site
of infection, even with an appropriate drug choice and dosage
§ protection from inhibitory or lethal antibiotic concentrations by refugia, particularly
purulent material, necrotic tissue, and bacterial biofilms
Successful treatment of serious wound infections must recognize and address each of the factors
involved in the specific case.
GENERAL TREATMENT APPROACH
The fundamentals of wound management apply across the board, whether dealing with
inherently well vascularized tissues such as skin, subcutis, muscle, and adipose tissue or with
dense connective tissues such as tendons, ligaments, and fascia that are relatively not well
vascularized.
Lavage
Thoroughly lavage the wound with sterile isotonic fluid until all visible contamination, slough,
and purulent discharge are removed:4
use sterile 0.9% saline or balanced polyionic solution (e.g., lactated Ringer's solution)
use copious quantities, as much as necessary to thoroughly cleanse the wound
use moderate delivery pressure (e.g., forcefully depress the plunger of a 30- or 60-ml syringe,
with 18-ga needle attached)
repeat as necessary during the course of treatment
Debridement
Remove all devitalized or irreparably damaged tissue, such as necrotic skin, fascial tags, frayed
tendon or ligament, and soft, discolored bone:4
open the wound as much as necessary to allow thorough lavage and debridement and
subsequent drainage
if in doubt, leave suspect tissue in place and re-evaluate in 24 hours
§ thorough debridement is particularly important with infections involving tendons,
ligaments, or fascia, as their vascularity is inherently low
§ at the same time, take care to avoid compromising structural integrity any further
repeat lavage to remove any residual debrided tissue
repeat debridement as necessary, monitoring the wound closely during initial treatment
Maggot debridement therapy
Consider maggot debridement therapy (MDT) as an alternative or adjunct when complete
surgical debridement would be difficult or impossible without damaging viable tissues. Maggot
therapy may be particularly useful in contaminated wounds with deep or multiple tracts. It has
several benefits for infected wounds:3
debridement—both physical and chemical (larval alimentary secretions/excretions)
disinfection—physical ingestion and digestion of bacteria and broad-spectrum antibacterial
secretions/excretions reduce the number and variety of bacteria in the wound
§ MDT is effective even against multidrug-resistant pathogens
§ Proteus spp. may be a unique exception (may be symbionts in the larval gut?)3
potentiation of some antibiotic drugs (e.g., gentamicin and synthetic penicillins)
biofilm inhibition and degradation
wound healing—larval activities affect local cytokine, cellular, and growth factor expression
to...
§ moderate the inflammatory response
§ improve tissue perfusion
§ stimulate granulation (angiogenesis, fibroblast proliferation and migration)
Maggot debridement therapy in horses is discussed in detail in the third article of this series.3
Drainage
Create or encourage gravity-assisted (dependent) ventral/distal drainage from the wound:4
take advantage of the location, orientation, and shape of the wound where possible
avoid opening the wound any more than is necessary to allow thorough lavage, debridement,
and continued drainage
§ avoid exposing the interior of the wound to further contamination and causing damage to
the vasculature and nerves supplying the wound
§ take care when using Penrose drains and setons, as they may suck or wick contaminated
material into the wound
exception—when there is gas accumulation in the deeper tissues, create as large an egress as
possible
§ wounds that act as one-way valves, such as deep wounds to the axilla or inguinal area,
suck and trap air within the tissues, causing subcutaneous emphysema that may be
extensive
§ clostridial myonecrosis is often accompanied by extensive subcutaneous and
intramuscular gas accumulation that may extend some distance along fascial planes
§ these wounds are best left open, and enlarged if necessary, to prevent air trapping in the
first instance and facilitate treatment of the anaerobic infection in the second
§ intensive antibiotic therapy (penicillin ± metronidazole) is also required for clostridial
myonecrosis
Movement
Controlled movement may be used to further encourage wound drainage. Specific techniques
depend on the nature of the wound, the stage of healing, and the potential for harm with
excessive or inappropriate movement:4
massage and/or passive range-of-motion exercises may be appropriate for horses restricted to
a stall
§ to avoid complications, manual therapy may be best directed by a physical therapist or
veterinary rehabilitation specialist when treating serious wound infections
hand-grazing or hand-walking, and even limited turnout in a pen or small pasture, may be
appropriate for more ambulatory patients
§ avoid unattended turnout when wound dressings include bandages or leg wraps that may
loosen and either leave the wound exposed or cause injury during unrestricted activity
movement may have additional benefits...
§ helps optimize blood flow to, from, and within the wound
§ limits or prevents fibrosis
§ prevents or resolves digestive and musculoskeletal problems associated with inactivity
§ relieves the stress, social isolation, and boredom of confinement
§ otherwise improves the patient's physical and psychological health and well-being
These aspects of nursing care are particularly important when the nature of the wound
necessitates stall confinement for weeks or months.
Vacuum-assisted closure
Vacuum-assisted closure (VAC), or negative-pressure wound therapy, provides active wound
drainage. Subatmospheric pressure (typically, 125 mmHg) is applied to the wound using a
small vacuum pump and occlusive dressing. It offers several benefits for infected wounds:3
evacuates wound exudate, which...
§ moderates the inflammatory response
§ reduces edema in and around the wound
removes bacteria from the wound, including multidrug-resistant pathogens
improves microcirculation and thus tissue perfusion within the wound bed
disrupts bacterial biofilm formation and maturation
prevents further contamination by keeping the wound covered
protects the wound bed from desiccation and maceration
speeds wound closure...
§ limits the retraction phase in acute wounds (mechanically draws the wound edges closer
together)
§ facilitates the contraction phase by the same means
§ upregulates the expression of cytokines and growth factors involved in granulation and
epithelialization
Vacuum-assisted closure in horses is discussed in detail in the third article of this series.3
Dressing
In addition, the wound bed must be protected from further contamination and from desiccation or
maceration until the infection is resolved and epithelialization is almost complete. The optimal
wound dressing and protective or supportive wrap depends on the wound type, site, and stage. In
the early stages of wound infection, ready access to the wound bed for frequent monitoring and
treatment may be a primary consideration in the choice of dressing and bandage. With very
effusive wounds, keeping the coverings clean and dry is also an important factor.
Antibiotic Therapy
Culture-guided antibiotic therapy is an essential component of management for serious wound
infections—with the emphasis equally on essential and component. Culture-guided antibiotic
therapy is only one component of effective treatment for serious wound infections.
Never has bacterial culture and antibiotic sensitivity testing been more important, given the rise
in frequency and diversity of antibiotic resistance in common equine pathogens. Culture of the
wound exudate and tissues should be performed as soon as possible in the course of wound
infection and repeated as often as necessary, based on response to treatment.
That said, over-reliance on antibiotic therapy, even when guided by culture, may be the most
common reason why treatment fails to completely resolve the infection. The other factors that
contribute to the persistence of infection must be given serious consideration in the diagnostic
and therapeutic plan.
The first three articles in this series focused on specific aspects of treatment, including these:
synovial (endoscopic or catheter/needle) lavage1
local and regional modes of antibiotic delivery2,3
§ indwelling intrasynovial catheter for infection involving a synovial structure
§ indwelling venous cephalic/saphenous catheter for regional perfusion of the distal limb
§ antibiotic-impregnated depot materials, including polymethylmethacrylate (PMMA)
beads and biodegradable/absorbable materials such as plaster of Paris, gentamicin-
impregnated collagen sponges, and ground bone or hydroxyapatite
maggot debridement therapy3
vacuum-assisted closure3
As those techniques also address the soft-tissue component of the wound infection, and some
(e.g., regional limb perfusion, maggots, and VAC) are applicable to wound infections that
primarily or exclusively involve soft tissues, the rest of this article focuses on immunological
defenses and perfusion.
IMMUNOCOMPROMISE
With serious wound infections, it is important to bear in mind that antibiotic therapy alone is
inadequate in the face of an incompetent immune system. Antibiotic drugs are only ever
adjunctive therapy, albeit a very important component of the management for serious wound
infections. A competent immune system is essential in the prevention and resolution of wound
infections.
A number of conditions or circumstances may compromise the patient's ability to prevent or
resolve bacterial infection, including these:4
vulnerable age—especially neonatal foals (<2 weeks of age) and old horses (>20 years)
antibody deficiency, such as…
§ failure of passive transfer (FPT) of maternal antibodies in neonatal foals
§ congenital deficiencies of immunoglobulin production
§ protein-losing enteropathy, nephropathy, or dermatopathy (e.g., extensive third-degree
burns, large open wounds)
malnutrition
§ macronutrientsparticularly protein-calorie malnutrition, resulting in a low body
condition score ( 3/9)
§ micronutrients—profound or prolonged trace mineral, essential fatty acid, or amino acid
deficiencies
intense or chronic stress (physical or psychological)
§ intense athletic training, competitive events
§ long-distance transport
§ social isolation
§ sustained hypothermia (cold stress)
§ hospitalization for illness or injury
pituitary pars intermedia dysfunction (PPID, equine Cushing's disease)
§ PPID causes hypercortisolemia ± hyperglycemia (a risk factor for wound infections and
biofilm formation in humans)
§ theoretically, diabetes mellitus (DM) would also increase the risk of serious wound
infections, but DM is rarely reported in horses
corticosteroid administration, other immunosuppressive drugs
sepsis—causes early hyperinflammation then severe immunosuppression
other concurrent systemic infection or debilitating disease (e.g., severe parasitism)
General Treatment Approach
Depending on the signalment and history, including the nature of the wound and response to
initial treatment, the following steps may be advisable:4
review or further investigate the patient's...
§ signalment
§ history, including recent medications and preventive care program
§ physical examination findings
§ routine blood work (complete blood count, serum chemistry panel)
§ repeat physical examination and/or blood work as needed
if possible, treat any condition or circumstance identified that may be compromising immune
function, for example...
§ administer IV plasma to a neonatal foal with partial or complete FPT
§ begin or review the dosage of pergolide for a senior horse with clinical signs of PPID
§ ensure optimal nutrition and internal parasite control
§ provide compatible company in a low-stress environment (an isolation stall or barn may
be indicated for a potentially zoonotic infection such as MRSA or VRE, but such housing
may be an additional stressor unless the patient can at least see other horses nearby)
avoid immunosuppressive therapies, such as corticosteroids and cryotherapy (therapeutic
hypothermia)
§ both are highly effective anti-inflammatory therapies, but they are also
immunosuppressive
§ if corticosteroids are considered necessary, such as for initial treatment of sepsis/systemic
inflammatory response syndrome, then use a short-acting drug (e.g., dexamethasone) and
a short treatment course (<48 hours)
optimize tissue perfusion (discussed later)
Plasma Therapy
Passive local immunotherapy involving the administration of polyclonal antibodies (pooled
human IgG) directly to the wound has been shown to reduce the morbidity and mortality of
experimental wound infections in mice. Disease models have included lethal E. coli-induced
peritonitis12 and sepsis resulting from infection of burn wounds with antibiotic-resistant strains of
P. aeruginosa13,14 or Klebsiella sp.12 These studies make two clinically useful points:
local control of infection using high-titer polyclonal antibodies (commercial pooled human
IgG) works synergistically with systemic antibiotic therapy to control wound infection and
prevent systemic dissemination12,14
this approach may be particularly useful with antibiotic-resistant wound pathogens12,14
§ the antimicrobial effect of polyclonal antibodies is broad-spectrum and appears to be
independent of bacterial antibiotic-resistance mechanisms12
Plasma therapy has not been well studied for this purpose in horses, but two in vitro equine
studies suggest that this approach might be of value in the treatment of equine wound infections
as well, including those caused by multidrug-resistant pathogens such as MRSA:
compared with balanced polyionic solution (Plasmalyte™), hyperimmune equine plasma
(Polymune™) reduced the adhesion of S. aureus to equine bone15
§ samples of periosteum, intact and cut cortical bone, and endosteum from the third
metacarpus were immersed for 1 hour in hyperimmune plasma or polyionic solution
§ the explants were then incubated with a clinical isolate of S. aureus (from a human
orthopedic infection) for 6 hours at 37° C
§ fewer bacteria were adhered to any of the bone surfaces in the explants treated with
hyperimmune plasma than in those treated with polyionic solution
equine plasma inhibited the growth of methicillin-sensitive S. aureus (MSSA) and MRSA,
unless the plasma was heat-inactivated16
§ whole blood was collected from healthy adult horses into citrated tubes and centrifuged
for plasma harvest
§ the bacteriostatic effects of various plasma products were then evaluated against MSSA
and MRSA—platelet-rich plasma (PRP), platelet-rich gel (PRG), leukocyte-poor plasma
(LPP), leukocyte-poor gel (LPG), plasma, and heat-inactivated plasma (IP)
§ after incubation for 6 and 24 hours at 37° C, bacterial growth of both isolates was
significantly inhibited by all products except IP
§ after 6 hours, plasma caused 100% inhibition of MSSA and 98% inhibition of MRSA
§ after 24 hours, plasma caused 97% inhibition of MSSA and 88% inhibition of MRSA
§ in a companion study,17 the authors concluded that complement was mainly responsible
for the bacteriostatic effect of these various plasma products
§ in both studies,16,17 the bacteriostatic effect of all plasma products was lower at 24 hours
than at 6 hours, suggesting consumption or inactivation of the bacteriostatic agent(s) over
time
The practical implications of these various animal studies include the following:
topically applied, fresh plasma may be a simple, inexpensive, and readily available means of
augmenting local defenses and directly inhibiting bacterial adhesion and growth
§ however, supplemental plasma may need to be applied more often than once a day for
optimal bacteriostatic effect
fresh plasma from a healthy horse living on the same premises (and thus exposed to the same
pathogens) may be a suitable alternative to autogenous plasma if the patient is severely
compromised
§ although xenogeneic (other species) antibodies have been used with success in
experimental studies, allogeneic (other individual, same species) antibodies probably are
preferable
commercial hyperimmune plasma is likely to be more effective than plain plasma, although
comparative studies are needed
antigen-specific, hyperimmune plasma may be of particular value when the wound pathogen
is known
§ commercial hyperimmune equine plasma is available in the US for E. coli, Salmonella
spp., Streptococcus equi, Rhodococcus equi, and various Clostridium spp.a
there is currently no commercial hyperimmune equine plasma for S. aureus in the US, but it
is encouraging to know that plain equine plasma significantly inhibited MRSA growth in
vitro16
Until more specific products are developed and tested in controlled clinical trials, local
application of fresh plasma or a plasma product to the wound may fill the present gap in
immunotherapy for the adjunctive treatment of serious wound infections in horses.
POOR PERFUSION
An effective immune response to bacterial invasion relies not just on local tissue resources but
also on the systemic delivery of white blood cells, complement, antibodies, oxygen, and nutrients
to the site of infection. Any circumstance that limits optimal blood flow to, or at, the site of
infection inevitably limits the host's immune response, as well as its wound repair capabilities—
which renders the wound vulnerable to re-infection. Examples include the following:4
sustained hypovolemia or hypotension (e.g., endotoxemia)
thrombotic states (e.g., disseminated intravascular coagulation, vasculitis)
extensive tissue trauma, particularly crushing, tearing, or strangulating injuries that result in
local ischemia
fibrosis at or proximal to the site of infection
location of a foreign body, surgical implant, or devitalized tissue that impedes blood flow
pressure from an improperly applied bandage or cast, or from prolonged recumbency
severe local or regional edema (inflammatory or dependent)
Edema is so common with infected wounds that it is easily overlooked as a potentially
problematic factor, other than its adverse effect on patient comfort and client perception of
treatment efficacy. However, severe edema, whether inflammatory or merely dependent, may
adversely affect blood flow at the site of infection by creating local interstitial pressures that may
exceed the perfusion pressure of the microvasculature. The result may be decreased tissue
perfusion in the wound bed.18
General Treatment Approach
Blood flow is tightly regulated, so there is a limit to how much blood flow can be increased in
any tissue. Even so, wound infections that are complicated by poor perfusion are difficult to
manage unless any impediments to optimal blood flow are identified and addressed. Depending
on the wound, the following measures may be indicated:4
ensure good hydration
provide specific treatment for systemic disorders as needed
remove any impediments to blood flow at the wound site as needed
§ in particular, debride traumatized tissue that has been ischemic for longer than a few
hours and is becoming, or already, necrotic
control edema
§ use nonsteroidal anti-inflammatory drugs (phenylbutazone, flunixin meglumine, etc.) to
address any vasculitis and to control inflammatory edema
§ use manual and movement therapy (see DRAINAGE, above) to control dependent edema
and otherwise optimize blood flow
§ use compression bandages where appropriate, applying the bandage from distal to
proximal (but avoid creating excessive or focal pressure)
consider using VAC therapy (see DRAINAGE, above); benefits include...
§ reduction or resolution of edema
§ improved microcirculation
consider using one of the oxygen therapies (see below)
Hyperbaric Oxygen Therapy
Wound infections are one of the indications for hyperbaric oxygen therapy (HBOT) in human
medicine.19,20 Some of the data on HBOT are compelling from a clinical standpoint; for
example:20
when breathing normobaric air, arterial oxygen tension (PaO2) is ~100 mmHg and tissue
oxygen tension (PtO2) is ~55 mm Hg
when breathing 100% O2 at 3 atmospheres pressure, PaO2 increases to ~2,000 mmHg and
PtO2 to ~500 mmHg
§ in addition, O2 delivery to tissues is increased from ~3 ml/liter of blood to ~60 ml/l
As HBOT increases the amount of oxygen in solution (i.e., in plasma and tissue fluids), it
improves tissue oxygenation even when circulation or oxygen delivery via red blood cells is
impaired (ischemia, anemia, etc.).
Specifically in regard to infected wounds, HBOT:19,20
increases the generation of oxygen radicals, which are broadly antimicrobial, as they...
§ oxidize proteins and membrane lipids
§ damage DNA
§ inhibit bacterial metabolism
generates tissue O2 concentrations that are lethal to anaerobes
potentiates the oxygen-dependent peroxidase system by which neutrophils and other white
cells kill all types of bacteria, including multidrug-resistant strains
stimulates cellular mechanisms necessary for wound healing
However, HBOT is not widely available for equine use. Neither is it well studied in horses.
Anecdotal reports indicate that HBOT is useful in the treatment of injuries complicated by poor
perfusion in horses,19 but clinical and experimental studies of HBOT for the treatment of equine
wounds are lacking.
One in vivo equine study examined the effects of HBOT on clean, full-thickness skin wounds
that were surgically created on the limbs and subsequently mesh-grafted with skin from the
pectoral region.21 The authors concluded that HBOT is not indicated after full-thickness skin
grafting for uncompromised fresh (day 0) or granulating (day 7) wounds. The grafts treated with
HBOT (1 hour at 2.6 atmospheres, daily for 7 days) developed less edema and granulation tissue;
however, they also had less neovascularization and more inflammation, and the superficial
portion of the graft was less viable than the grafts not treated with HBOT. Clearly, more research
is needed on the clinical value and specific indications for HBOT in horses, particularly for the
treatment of wound infections.
Ozone Therapy
Ozone (O3) therapy is more practical than HBOT and offers some of the same benefits:22
broadly antimicrobial—inactivates bacteria, viruses, fungi, and protozoa
§ disrupts bacterial cell wall integrity via oxidation of phospholipids and lipoproteins
§ effective against aerobes and anaerobes, including multidrug-resistant strains such as
MRSA and Clostridium difficile
stimulates oxygen metabolism in the patient's tissues
§ enhances the release of oxygen from red blood cells
§ stimulates ATP production via activation of the Krebs cycle
§ stimulates the production of free-radical scavengers and cell-wall protectors such as
glutathione peroxidase, catalase, and superoxide dismutase
§ causes vasodilation via the production of prostacyclin
induces the immune activation
§ increases the production of interferon, tumor necrosis factor, and interleukin-2
analgesic
§ downregulates the genes involved in allodynia
There is ample clinical and experimental evidence that ozone therapy is useful in the treatment of
wound infections in humans and laboratory animals.22-33 Studies of ozone therapy for wound
infections in horses are lacking, but studies in other species indicate the following:
ozone is an effective antibacterial agent alone, but in infected wounds its efficacy may be
enhanced by combination with...
§ pressurized lavage, using fine-stream dispersion of ozonized water23
§ antibiotic drugs (see below)31
ozone therapy is effective against multidrug-resistant pathogens such as MRSA31,32
§ in an in vivo study in rats, intraperitoneal ozone therapy significantly decreased bacterial
numbers in the sternal and mediastinal layers following experimental infection of a
median sternotomy wound with MRSA31
§ the combination of vancomycin and ozone therapy decreased bacterial numbers more
than either treatment alone31
ozone is effective against bacterial biofilms at concentrations tolerated by mammalian cells34
§ in an in vitro study, S. aureus biofilms were generated on surgical titanium and then
exposed to ozonized saline
§ within 15 minutes of exposure, ozonized saline caused a 99% reduction in the number of
planktonic S. aureus
§ S. aureus biofilms were significantly more resistant to ozone but were completely
eradicated within 5 hours of exposure
§ additionally, ozonized saline was not cytotoxic to ovine primary osteoblasts
Ozone therapy may be delivered to the wound by ozonizing:
medical-grade oxygen28,30,33
§ the wound/limb is encased in a sterile plastic bag, wrap, or cup which is then inflated
with the O2-O3 mixture (typically, 70–80 µg O3/ml for purulent wounds) for 20–30
minutes; the procedure is repeated once or twice a day as needed
§ the wound must be damp with either exudate or distilled water/saline solution, as the O3
must be dissolved in water at the wound surface to be effective
distilled water23,28,29 or physiologic saline solution34,35
§ used as irrigation/lavage fluid or as a damp compress or wound dressing
§ some authors advise using double-distilled ("ultrapure") water instead of single
distillation, to maximize O3 concentration and longevity in solution28
vegetable oil (e.g., sesame oil)28,36
§ more suitable for chronic wounds, particularly when healing has stalled
§ other vegetable oils (e.g., olive, sunflower) may also be used
Intralesional injection of ozone must be used with care, as it may cause tissue necrosis.37
Intravenous infusion of ozonized physiologic saline should also be used with care. In an in vivo
canine study, IV ozonized saline stimulated neutrophil phagocytosis, but repeated infusion 48
hours later caused a significant decrease in phagocytic capacity.35
Small ozone generators that ozonize room air are widely available and relatively inexpensive.
However, ozonizing air yields lower and less consistent amounts of O3 than ozonizing medical-
grade oxygen, and it also generates potentially toxic nitrates, dry air being 78% nitrogen and
only 21% oxygen at sea level.28 Ozone generators designed specifically for medical use with
medical-grade oxygen provide higher concentrations and more controlled delivery of O3 as pure
O2-O3 mixtures.
Ozone therapy is used in horses and other domestic animals by a small but enthusiastic group of
veterinarians, based on very little clinical research in veterinary medicine. The list of purported
benefits or indications is long,38 but the more focused clinical reports describe the use of ozone
therapy for pain relief,39-42 the treatment of intervertebral disc disease,40 and postoperative wound
infection (Klebsiella pneumoniae infection after total hip replacement in two dogs).41 Hopefully,
in the near future we will have some clinical studies of its use in the treatment of wound
infections in horses.
CONCLUDING THOUGHTS
The situation with ozone therapy in veterinary medicine is a good place to conclude, as it is
rather exemplary of the point at which we find ourselves with serious wound infections in horses.
Almost all of the clinical and experimental research on wound infections in horses has focused
on antibiotic therapy and the development of new drugs or new delivery methods, all of which
are mere variations of the same theme: antibiotics are the answer. That may have been true (or
true in most cases) at one time, but we can no longer rely on there being an antibiotic solution to
every bacterial problem.
The tremendous adaptability of bacteria to our rather myopic use of antibiotic drugs is forcing us
to look for solutions in two different directions: (1) forward, to the development of novel
antibacterial substances (e.g., isolated or synthesized maggot secretions/excretions),3 and (2)
back, to the fundamentals of physiology and microbiology—which lead us (back) to oxygen as
therapy and the importance of simple evacuation (lavage, debridement, drainage, dressing
changes) diligently applied.
Some of the greatest leaps forward in the management of serious wound infections occurred in
times of war and they pre-date modern antibiotic drugs. The current rise of multidrug-resistant
pathogens is similarly forcing us to get creative with this very agile "enemy" and scan both
horizons for equally smart solutions.
Author’s note: This article was written by me (Christine M. King) on commission in 2016 and published under
another person’s name. This article is some of my best work to date, yet it is credited to someone elsean
act of intellectual dishonesty, the ramifications of which I did not appreciate at the time. This article is
entirely my own original work, exactly as I submitted it. I retain the copyright.
Manufacturers
aLake Immunogenics, Inc., Ontario, New York.
References
1. Orsini JA. Update on managing serious wound infections in horses: 1. Wounds involving
joints and other synovial structures (part A). J Equine Vet Sci 2016; XX: XX–XX.
2. Orsini JA. Update on managing serious wound infections in horses: 2. Wounds involving
joints and other synovial structures (part B). J Equine Vet Sci 2016; XX: XX–XX.
3. Orsini JA. Update on managing serious wound infections in horses: 3. Wounds involving
bone. J Equine Vet Sci 2016; XX: XX–XX.
4. Orsini JA, Elce YA, Kraus E. Management of severely infected wounds. In: Equine Wound
Management, 3rd edn. Theoret CL, Schumacher J, eds. Wiley-Blackwell. 2015; [in press].
5. de Niederhäusern S, Sabia C, Messi P, et al. VanA-type vancomycin-resistant enterococci in
equine and swine rectal swabs and in human clinical samples. Curr Microbiol 2007; 55: 240–
246.
6. Lauková A, Simonová M, Strompfová V, et al. Potential of enterococci isolated from horses.
Anaerobe 2008; 14: 234–236.
7. Singh BR. Prevalence of vancomycin resistance and multiple drug resistance in enterococci in
equids in North India. J Infect Dev Ctries 2009; 3: 498–503.
8. Moura I, Radhouani H, Torres C, et al. Detection and genetic characterisation of vanA-
containing Enterococcus strains in healthy Lusitano horses. Equine Vet J 2010; 42: 181–183.
9. Erol E, Williams NM, Sells SF, et al. Antibiotic susceptibility patterns of Crossiella equi and
Amycolatopsis species causing nocardioform placentitis in horses. J Vet Diagn Invest 2012;
24: 1158–1161.
10. Theelen MJ, Wilson WD, Edman JM, et al. Temporal trends in in vitro antimicrobial
susceptibility patterns of bacteria isolated from foals with sepsis: 1979–2010. Equine Vet J
2014; 46: 161–168.
11. Theelen MJ, Wilson WD, Edman JM, et al. Temporal trends in prevalence of bacteria
isolated from foals with sepsis: 1979–2010. Equine Vet J 2014; 46: 169–173.
12. Barekzi NA, Felts AG, Poelstra KA, et al. Locally delivered polyclonal antibodies potentiate
intravenous antibiotic efficacy against gram-negative infections. Pharm Res 2002; 19: 1801–
1807.
13. Felts AG, Giridhar G, Grainger DW, et al. Efficacy of locally delivered polyclonal
immunoglobulin against Pseudomonas aeruginosa infection in a murine burn wound model.
Burns 1999; 25: 415–423.
14. Felts AG, Grainger DW, Slunt JB. Locally delivered antibodies combined with systemic
antibiotics confer synergistic protection against antibiotic-resistant burn wound infection. J
Trauma 2000; 49: 873–878.
15. Bauer SM. Santschi EM, Fialkowski J, et al. Quantification of Staphylococcus aureus
adhesion to equine bone surfaces passivated with Plasmalyte and hyperimmune plasma. Vet
Surg 2004; 33: 376–381.
16. López C, Alvarez ME, Carmona JU. Temporal bacteriostatic effect and growth factor loss in
equine platelet components and plasma cultured with methicillin-sensitive and methicillin-
resistant Staphylococcus aureus: a comparative in vitro study. Vet Med Int 2014; 2014:
525826. doi: 10.1155/2014/525826.
17. López C, Carmona JU, Giraldo CE, et al. Bacteriostatic effect of equine pure platelet-rich
plasma and other blood products against methicillin-sensitive Staphylococcus aureus. An in
vitro study. Vet Comp Orthop Traumatol 2014; 27: 372–378.
18. Bowering CK. Use of layered compression bandages in diabetic patients. Experience in
patients with lower leg ulceration, peripheral edema, and features of venous and arterial
disease. Adv Wound Care 1998; 11: 129–135.
19. Slovis NM. Review of hyperbaric medicine. Proc Am Assoc Equine Practit 2005; 51: 153–
157.
20. Theoret C. Effects of hyperbaric oxygen therapy on wound healing: how and why it works.
Proc. Eur Soc Vet Orthop Traumatol 2008; 14: 281–282.
21. Holder TE, Schumacher J, Donnell RL, et al. Effects of hyperbaric oxygen on full-thickness
meshed sheet skin grafts applied to fresh and granulating wounds in horses. Am J Vet Res
2008; 69: 144–147.
22. Elvis AM, Ekta JS. Ozone therapy: a clinical review. J Nat Sci Biol Med 2011; 2: 66–70.
23. Bulynin VI, Ermakova AI, Glukhov AA, et al. Wound treatment using the flow of an
ozonized solution under high pressure. [Article in Russian] Khirurgiia (Mosk) 1998; 8: 23–24.
24. Grigor'ian AS, Grigor'iants LA, Guchetl' MN. Experimental-morphological study of the anti-
inflammatory action of ozone-perfluorane complex application. [Article in Russian]
Stomatologiia (Mosk) 2008; 87: 4–9.
25. Zagirov UZ, Isaev UM, Salikhov MA. Clinicopathologic basis of ozonomagnetophoresis in
treatment of festering wounds. [Article in Russian] Khirurgiia (Mosk) 2008; 12: 24–26.
26. Dmitrieva NA, Zyrianova NV, Grigor'ian AS, et al. Microflora dynamics in purulent skin
wound in rats after ozonized perftorane applications. [Article in Russian] Stomatologiia (Mosk)
2009; 88: 14–16.
27. Khriakov AS, Kozlov KK, Dolgikh VT, et al. Treatment of patients with purulent wound
infection under conditions of extreme north. [Article in Russian] Vestn Khir Im I I Grek 2009;
168: 78–81.
28. Travagli V, Zanardi I, Valacchi G, et al. Ozone and ozonated oils in skin diseases: a review.
Mediators Inflamm 2010; 2010: 610418.
29. Huang HJ, Yu B, Lin QR, et al. Effect of ozone water on the inflammation and repair in
infected wounds. [Article in Chinese] Nan Fang Yi De Da Zue Zue Bao 2010; 30: 515–518.
30. Shah P, Shyam AK, Shah S. Adjuvant combined ozone therapy for extensive wound over
tibia. Indian J Orthop 2011; 45: 376–379.
31. Gulmen S, Kurtoglu T, Meteoglu I, et al. Ozone therapy as an adjunct to vancomycin
enhances bacterial elimination in methicillin resistant Staphylococcus aureus mediastinitis. J
Surg Res 2013; 185: 64–69.
32. Tamai M, Matsushita S, Miyanohara H, et al. Antimicrobial effect of an ultrasonic levitation
washer disinfector with silver electrolysis and ozone oxidation on methicillin-resistant
Staphylococcus aureus. J Dermatol 2013; 40: 1020–1026.
33. Vinnik IuS, Salmina AB, Tepliakova OV, et al. Dynamics of local expression of connexin-43
and basic fibroblast growth factor receptors in patients with skin and soft-tissue infections
against the background of diabetes mellitus type II. [Article in Russian] Vestn Khir Im I I Grek
2014; 173: 47–52.
34. Al-Saadi H, Potapova I, Rochford ET, et al. Ozonated saline shows activity against
planktonic and biofilm growing Staphylococcus aureus in vitro: a potential irrigant for infected
wounds. Int Wound J 2015; Jan 14 [Epub ahead of print] doi: 10.1111/iwj.12412.
35. Volkhovskaya NB, Tkachenko SB, Belopolsky AA. Modulation of phagocytic activity of
blood polymorphonuclear leukocytes with ozonized physiological saline. Bull Exp Biol Med
2008; 146: 559–561.
36. Valacchi G, Lim Y, Belmonte G, et al. Ozonated sesame oil enhances cutaneous wound
healing in SKH1 mice. Wound Repair Regen 2011; 19: 107–115.
37. Uzun G, Mutluoğlu M, Karagöz H, et al. Pitfalls of intralesional ozone injection in diabetic
foot ulcers: a case study. J Am Coll Clin Wound Spec 2014; 4: 81–83.
38. Roman M. Ozone therapy: beyond oxygen. The most needed adjunct to veterinary medicine.
Proc N Am Vet Conf 2008; pp. 78–80.
39. Garcia Liñeiro JA, Argibay T, Scipioni H, et al. Eco guide oxygen peroxidum injection
(ozone therapy) as treatment of intervertebral lesions in horses. Description of the evolution of
15 cases. Proc. World Equine Vet Assoc 2009; 11: 2–3.
40. Han HJ, Kim JY, Jang HY, et al. Fluoroscopic-guided intradiscal oxygen-ozone injection
therapy for thoracolumbar intervertebral disc herniations in dogs. In Vivo 2007; 21: 609–613.
41. Maugeri S, Leonardi P. 2 cases of ultrasound-guided ozone therapy treatment. Proc. Eur Vet
Conf 2008, p. 3.
42. Teixeira LR, Luna SP, Taffarel MO, et al. Comparison of intrarectal ozone, ozone
administered in acupoints and meloxicam for postoperative analgesia in bitches undergoing
ovariohysterectomy. Vet J 2013; 197: 794–799.
ResearchGate has not been able to resolve any citations for this publication.
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