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M. Contreras and E. Wang
completed on the right side is then reproduced on the left
side, thus allowing access to bilateral infusion ports, affording an extra port available in the event that one catheter may
fail/thrombose. The neck wound is then closed in three layers with 3-0 Vicryl running suture with care to minimize
dead space and kinking of the catheter. Skin borders are
approximated and closed with 3-0 Vicryl subcuticular running suture. Dressings and bandages are applied to protect
the wound.
In addition to the single dose of s.c. buprenorphine administered at the beginning of the surgical procedure, a transdermal fentanyl (2–4μg/kg) patch is applied to the skin for the
rst 72h. The pigs are then placed back in their respective
cage (individually) and monitored during recovery.
Alloxan Administration: Once animals have recovered
from the initial, JV catheterization and s.c. infusion port
placement and wounds have healed (10–14days), they are
weighed and sedated with Telazol (3–5 mg/kg) i.m. The
alloxan solution is prepared fresh and ltered-sterilized just
prior to animal administration (100–200mg/kg). Alloxan is
dissolved in sterile normal saline to achieve a 5% concentration (W/V) and used right away, since it is known to be
highly unstable. The syringe is then loaded to an automated
infusion pump set up to dispense the total volume over
1–3min, through a 20g angiocatheter previously placed into
the marginal ear vein. In addition, to decrease the risk of
nephrotoxicity (hyperuricemia), an i.v. injection of 0.9%
saline (2–3mL/kg) at an infusion rate of 2mL/min is administered. To mitigate any distress or pain from the alloxan
injection, meloxicam (0.308mg/kgs.c.), a NSAID, is administered to the pigs.
To counteract the hypoglycemic effect of alloxan, 2, 4, 6,
and 8 h after alloxan injection, 10 mL of either 5 or 50%
dextrose i.v. is administered through the i.v. infusion port
(JV catheter) left in place from the previous surgical
implantation.
Twenty-four hours after alloxan injection, pigs receive a
second dose of meloxicam (0.308 mg/kg) s.c. Because
alloxan is also known to cause distress, animals should be
closely observed and monitored throughout the diabetes
induction period for any signs of discomfort and distress
including a rise in temperature.
Personal Protective Equipment (PPE): Any personnel
handling alloxan will require using PPE and following strict
rules for hazardous substance handling and administration
during and during the 72h post-alloxan administration. The
PPE includes a disposable gown, hair bonnet, and face
shield. PPE and the waste collected from the cage are disposed of in a biohazard container for later disposal/
incineration.
Post-alloxan diabetes conrmation: Two days following
alloxan administration, pigs are placed NPO for 4h prior to
BGL determination and Gatorade is switched to regular
water. Pigs exhibiting a BGL>250 are considered diabetic
(this value was chosen based on previous studies). Only pigs
that do not become diabetic receive a second dose of alloxan
(100 mg/kg iv), following the same procedure described
above. In our experience, more than 95% of the animals turn
diabetic with one single alloxan initial dose.
Insulin Administration Considerations: Pigs that have
become diabetic (BGL >250mg/dL) are given insulin once a
day, a combination of Humalog insulin (0.05 U/kg) and
Lantus insulin (0.2 U/kg/100 U/mL), using a small 1 mL
syringe with a 25 g needle. Insulin is administered at the
same time every day, in the morning, between 8 and 9am,
right after animals have been fed. Site of injection should be
rotated between doses (posterior aspect of the neck or
buttocks).
Ear venous needle stick: In the event that a BG determination is needed, a needle stick with a very small needle (25–
26g) on a supercial ear vein to allow collection of a drop of
blood to place on a glucose measurement strip to be read by
the glucometer is recommended. Pressure at the puncture
should be maintained for 2–3min by placing a 8×12 cotton
gauze, making sure that bleeding has stopped/ceased completely, before leaving the animal.
IV injections: Should there be a need to quickly intervene
in the event of hypoglycemia (<125mg/dL), in addition to
oral administration of corn starch syrup, there might be a
need to administer i.v. (through infusion port previously
implanted) 50% dextrose. Volume to be administered will be
determined based on BGL, and it could range from 10 to
15mL.Rectal temperature is measured every day for the rst
week after alloxan and meloxicam are administered PRN (if
temperature is >103°F).
Animals are fed once a day (at the same time in the morning) with 750–850g of pig chow. Dietary supplement such
as Glucerna to ensure proper nourishment until the animal is
fully recovered and eating well on its own could be initially
administered. BGL and ketone check: if hyperglycemia
(BG > 500 mg/dL) or hypoglycemia (BG < 125 mg/dL)
occurs, insulin dose should be adjusted (Table19.4). In addition to BGL and ketone check, pigs are closely observed
throughout the study period for any visual signs of discomfort and distress including no or reduced urine output and
feces, reduced water and food intake, and reduced activity.
Table 19.4 Lantus (long-acting) and Humalog (fast-acting) insulin
dosing chart
Blood glucose
level (BGL) Lantus insulin
350–450mg/dL 0.1U/kg <75mg/dL 0U
450–550mg/dL 0.2U/kg 76–150mg/dL 1U
550–600mg/dL 0.3U/kg 151–250mg/dL 2U
600–700mg/dL 0.4U/kg 251–550mg/dL 3U
>700mg/dL 0.5U/kg 551–700mg/dL 4U
Blood glucose
level (BGL)
Humalog
insulin

abc
19 Experimental Animal Models inResearch: Diabetes andImpaired Wound Healing
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357
Once stable hyperglycemia has been established, BGLs are
measured once a day, the same time in the morning, using the
same blood glucometer.
Second Surgical Intervention/Post-alloxan Diabetes
Induction: Skin wound surgical creation—All surgeries are
performed 30 days following alloxan induction and the
hyperglycemia state has been instituted.
Mini pigs are fasted for 12h overnight. The following
morning, animals are prepared for surgery. Anesthetic
induction is performed with Telazol (3–5mg/kg) i.m. followed by isourane via facemask at 4–5%, 100%/liter of
oxygen, followed by endotracheal intubation and placement on an isourane vaporizer at 1–3%, 100%/liter of
oxygen. Pig ears are shaved and a depilatory cream is used
to remove any excess hair:
1. Blood draw: First blood draw (5cc) through the ear cen-
tral artery is done.
2. Blood glucose (BG) check: Using commercially avail-
able glucometer, BG is measured using 1 ul of blood
(generally blood drop left from blood draw). Depending
on the results of blood glycemia is determined, administration of a full or one-half the daily insulin dose is given.
An i.v. catheter (20–22g) is inserted into the marginal ear
vein to establish an i.v. line to administer lactated Ringer’s
or saline solution (500mL and 1g cefazolin) throughout
the length of the surgical procedure. A single dose of s.c.
buprenorphine is administered.
The pig’s dorsum is then shaved and a depilatory cream
(Nair) applied and removed a minute later with warm watersoaked cotton dressing and then padded dry and then transferred from the prep room to the main OR.Once the animal
is on the surgical table, laying over a water-heating blanket,
the animal’s dorsa, both left and right, are thoroughly disinfected, wiping the skin alternating three times with alcohol
and betadine solution, following sterile technique.
An i.v. slow infusion (drip) of 5% dextrose is started to
support the animal while the procedure is ongoing and to
compensate the lack of morning feeding because of presurgical fasting. The dextrose i.v. solution is administered at a rate
of 0.25g/kg/h and not higher (the maximum oxidation rate
of glucose is 0.36g/kg/h), which is a total of 7.5–8.75gm/h
for a 30–35kg animal (150–170mL of 5% dextrose/h). A
sterile-fenestrated drape is then placed over the entire animal, allowing exposure through the fenestration of the previously prepped dorsal area. Using a pre-fabricated acrylic
stencil with the distribution of the total number of wounds
that need to be created, is placed over the prepped dorsum,
using a sterile marking pen location of the wounds are
mapped out and traced, either; 24 (12 individual markings p/
side) or 18 (9 individual markings p/side) equally in size
(1.5×1.5cm2) spaced out 2.5cm from each other. A scalpel
(#11 blade) is used to make full-thickness individual wounds.
Each of the skin squares is excised, thus creating the individual wound, 1.5 by 1.5cm and 6–8mm deep (Fig.19.6).
Either Tegaderm dressing (control), different treatment
dressings, or new therapeutic balms to be tested could then
be applied to ear individual wound.
A BGL is done at the end of the surgery. If BGL is <150mg/
dL, then 5 mL of 50% dextrose is administered through the
20–22 g angiocatheter previously placed in the marginal ear
vein. When pigs recover from anesthesia and are awake, the ET
tube is removed so they can breathe on their own. The 20–22g
i.v. catheter is then removed. A single dose of buprenorphine
(0.01–0.05mg/kg) is administered s.c., and a fentanyl dermal
patch (4μg/kg) is applied to a previously shaved portion on the
back, providing analgesia for the next 72h, maintaining the
Fig. 19.6 Schematic representation of surgical procedures. (a) Skin wound mapping, (b) surgical skin excision, (c) wound dressing application

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M. Contreras and E. Wang
animal comfortable and without experiencing any pain. In
addition, the animals are given a s.c. dextrose (50%) bolus
injection at the end of the surgical procedure. The rationale
behind a s.c. bolus injection is that the dextrose solution can be
absorbed slowly/gradually over time to provide caloric support
for several hours while the animal is fully awake and can
resume eating/drinking on its own. Pigs have xed skin and
therefore s.c. injections are not as easy to administer as in other
animals with loose skin (such as the rabbit or dog). S.c. injections in pigs are still possible to do, particularly if the animals
are under anesthesia. Preferred locations in small pigs include
the axillary area caudal to the elbow or in the inguinal region in
the ank area and the loose skin behind the ear in larger pigs. It
is recommended that not more than 3 mL per injection site
should be administered while the animal is under anesthesia at
the end of the surgical procedure.
A custom-made jacket is then tted into the animal to further protect the skin wounds with their individual dressings
and also to prevent the possibility of skin infection. It is
highly recommended that several jackets are available;
should one of them become dirty/soiled, the jacket can then
be removed and replaced.
Once vital signs are stable, animals are returned to their
individual cage and continued to be monitored for the duration of the study.
Euthanasia and Tissue Harvest: At the end of the experimental study, animals are initially sedated with Telazol
(3–5 mg/kg) i.m. followed by either isourane via face
mask at 4–5%, 100% per liter of oxygen, or by endotracheal intubation and placement on an isourane vaporizer
at 1–3%, 100%/liter of oxygen. Final wound measurements
and imaging (photographs, ultrasound, etc.) capturing are
performed. A nal blood sample (5cc) is taken to determine BG and HbA1C levels, from a 20 g angiocatheter
placed in the marginal ear vein. A lethal dose of Fatal-Plus
(pentobarbital sodium/1 mL/10 lb) is then administered
i.v., and once the animal has expired, collection of the pig’s
skin wounds take place, so all individual healing wounds
can be assessed for histology, immunohistochemistry, and
other additional studies (gene expression, single-cell analysis, proteomics, etc.).
Finally, Table 19.5 shows a comprehensive summary of
USDA covered species models of diabetic induction and
their advantages and limitations.
Table 19.5 Summary of USDA covered species
Induction
method Animal model Description Advantages and limitations
Alloxan (ALX) Nonhuman
primates
(NHP)
ALX Swine Single high-dose ALX injection i.v. thus,
ALX Rabbit Single high-dose ALX injection i.v.
Streptozotocin
(STZ)
STZ Swine High-dose (200mg/kg) STZ injection i.v.
STZ Rabbit STZ 65mg/kg b.w., as single i.v. dose in
NHP Single high-dose STZ injection i.v. to
Alloxan injection i.v. to destroy beta cells
through free radicals and induce T1D state
chemical ablation to destroy beta cells and
induce T1D
requiring fast and constant infusion rate to
be effective in destroying beta cells
destroy beta cells through DNA damage to
replicate T1D
induces DNA strand breaks and ultimately
leads to cell death
1mL citrate buffer, pH4.6, to destroy beta
cells through DNA damage to replicate
T1D
Useful in preclinical trials of novel therapeutic approaches
for T1D treatment. However, alloxan has high toxicity to host
and small window of efcacy and is difcult to work with
due to chemical instability
Used to develop novel therapeutics for T1D.Porcine models
have been widely considered as one of the best wound
healing models due to their anatomical, physiological, and
metabolic similarities to human skin
Challenges: requires additional surgical vascular (arterial and
venous access) support and well-trained and experienced
personnel for post-op monitoring and care
Useful in preclinical trials of novel therapeutic approaches
for T1D treatment. However, alloxan has high toxicity to
host; thus, a high mortality is associated with the model.
Requires well-trained and experienced personnel for post-op
monitoring and care
Used to develop novel therapeutics for T1D; STZ is more
chemically stable than alloxan. However, single high doses
have severe side effects: weight loss, liver damage, and renal
failure
Zanosar STZ induces diabetes relatively safely in pigs;
however, nephrotoxicity and hepatotoxicity are disadvantages
of STZ.It does require highly skilled and experienced
personnel to execute and monitor
STZ induces a characteristic multiphasic immediate response
in rabbits similar to one reported in other rodents. Behavioral
changes are characteristic of hypoglycemia warranting early
management in order to avoid fatalities. Post-treatment
followed by initial i.v. and oral glucose and followed by
insulin therapy

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Table 19.5 (continued)
Induction
method Animal model Description Advantages and limitations
ALX and STZ Canine ALX and STZ are both cytotoxic causing
Surgical
(SURG)
SURG Canine Removal of the pancreas, complex
SURG Swine Removal of the pancreas, complex
NHP Removal of the pancreas, complex
necrosis of pancreatic β-cells and therefore
halt the production of insulin
procedure requiring skillful and
experienced surgeon, as well as supportive
OR staff
procedure requiring skillful and
experienced surgeon, as well as supportive
OR staff
procedure requiring skillful and
experienced surgeon, as well as supportive
OR staff
Induction of T1D diabetes mellitus. Persistent, insulindependent hyperglycemia. Used to develop novel
therapeutics for T1D treatments. ALX and STZ combined
allows for the use of lower doses and a single i.v. injection.
Social and ethical issues using canines in research
Used to study T1D outcomes. Affects the digestive system
due to pancreatic insufciency. Additional postoperative
nursing care is needed as the animal is insulin-dependent and
it shows abnormal digestive system
First USDA covered species model for induction of T1D
diabetes mellitus and discovery of insulin (Isletin).
Affects the digestive system due to pancreatic insufciency
Social and ethical issues using canines in research remain
today
Total pancreatectomy results in severe hyperglycemia. This
method should be considered only when other methods are
not feasible. A major disadvantage of pancreatectomy is that
it includes removal of both exocrine and endocrine tissues,
which is not characteristic of diabetes in humans
359
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Part III
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Management of the Diabetic Foot

Microbiology andTreatment ofDiabetic
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Foot Infection
MaryT.LaSalvia andAdolfW.Karchmer
20
Abstract
The diagnosis and treatment of diabetic foot infection
should be based on an assessment of the severity of infection using a well-studied approach such as the International
Working Group on the Diabetic Foot (WGDF)/Infectious
Diseases Society of America (IDSA) classication
scheme. The major pathogens causing diabetic foot infection can be anticipated based on the severity of infection
and further informed by additional patient specic factors
such as failed prior antibiotic treatment or extensive
health-care exposure. Establishing a microbiologic diagnosis is key to informing antibiotic therapy, particularly in
those patients with limb threatening infection or risk factors for infection caused by antibiotic resistant pathogens.
Surgical resection of infected bone has historically been
the standard treatment of osteomyelitis; however, in carefully selected patients, medical therapy may also be an
effective strategy. The duration of antibiotic therapy for
soft tissue infection is based on the severity disease and
for osteomyelitis on whether treatment utilizes medical
therapy alone or includes complete surgical resection of
involved bone. Long-term healing of foot ulcers to prevent future episodes of infection and the preservation of a
weight-bearing foot remain core goals requiring collaboration across a multidisciplinary team.
Introduction
bers supplying muscles of the foot causes asymmetric muscle strength, which in turn results in foot deformities and
maldistribution of weight (or pressure) on the foot plantar
surface. Dysfunction of the sensory bers supplying the skin
and deeper structural elements of the foot allows minor and
major injury to these tissues to proceed without appreciation
by the patient. As a result of neuropathy, the foot may be
dramatically deformed, ulcerate in areas of unperceived
trauma (mal perforans), and on occasion be warm and hyperemic in response to deep structural injury (acute Charcot’s
disease). This warmth and hyperemia may be misinterpreted
as cellulitis and an ulceration, while a major portal of entry
for infection may be uninfected. In the patient with diabetes,
peripheral neuropathy may develop in isolation or commonly
in parallel with atherosclerotic peripheral vascular disease.
The latter involves major in-ow vessels to the lower extremity but commonly is associated with occlusive lesions of the
tibial and peroneal arteries between the knee and ankle. The
resulting arterial insufciency can alter the appearance of the
foot and obscure infection. Rubor may reect vascular insufciency rather than inammation and conversely ischemic
pallor may mute the erythema of acute infection. Gangrene
and necrosis may be primarily ischemic or may reect accelerated ischemia due to elevated foot compartment pressures
and capillary occlusion in the setting of infection. In sum, the
diagnosis of infection involving the foot in patients with diabetes requires a careful detailed examination of the lower
extremity and its blood supply.
The foot of patients with diabetes mellitus is affected by several processes that not only contribute to the development
and progression of infection but on occasion alter the appearance of the foot in ways that may obscure the clinical features of local infection. Neuropathy involving the motor
M. T. LaSalvia (*) · A. W. Karchmer
Division of Infectious Diseases, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, MA, USA
e-mail: mlasalvi@bidmc.harvard.edu
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
A. Veves et al. (eds.), The Diabetic Foot, Contemporary Diabetes, https://doi.org/10.1007/978-3-031-55715-6_20
The Diagnosis ofFoot Infections
The initial step in the diagnosis of a foot infection in a patient
with diabetes is to recognize those patients at greatest risk and
to maintain a suspicion for infection. Foot infections often
present with more subtle ndings in patients with diabetes
because of impaired leukocyte function, ischemia, and peripheral neuropathy; thus, clinicians should evaluate any foot
wound for the possibility of infection [1, 2]. Suspicion for
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M. T. LaSalvia and A. W. Karchmer
infection should be heightened if additional clinical factors
that have been signicantly associated with foot infection are
present. These include peripheral arterial disease with absent
pulses or an ankle brachial index of <0.9; peripheral neuropathy leading to a loss of protective sensation; a history of recurrent foot ulcers or prior amputation; foot ulcers of >30days of
duration; a wound that extends to bone, i.e., a positive probe to
bone test (see Osteomyelitis); and a traumatic wound [3–5].
Thereafter, infection is diagnosed clinically and to varying
degrees supported by test results. Finding purulent drainage
(pus) or two or more signs or symptoms of inammation (erythema, induration, swelling, pain, tenderness, or warmth) is
indicative of infection. Clinical signs on occasion belie the
signicance and severity of infection. A minimally inamed
but deep ulceration may be associated with underlying osteomyelitis [6]. Serious limb-threatening infection may not result
in systemic toxicity. For example, among patients hospitalized
for limb- threatening infection, only 12–35% have signicant
fever [7, 8]; however, its presence may predict increased risk
of amputation [9]. Fever higher than 102°F suggests infection
involving deeper spaces in the foot with tissue necrosis and
undrained pus, extensive cellulitis, or bacteremia with the
potential for hematogenous seeding of remote sites. Laboratory
studies may be supportive of the diagnosis of these infections
but must be interpreted in the context of clinical ndings.
White blood cell count may be normal in up to 50% of patients
with deep foot infection [10–12]. Erythrocyte sedimentation
rate (ESR) and C-reactive protein (CRP) may be normal in
infected patients, and in those with peripheral neuropathy,
ESR may have reduced utility in predicting osteomyelitis
compared to CRP [13]. Elevated concentration of CRP and
procalcitonin can help distinguish mild or moderately infected
ulcers from those that are uninfected [14]. In addition to the
presence of classic pathogens, foot ulcers are often contaminated or colonized by commensal organisms that on occasion
become pathogens. Consequently, cultures, while essential in
the assessment of the microbiology of foot infections, do not
in isolation establish the presence of infection. Unless the cultured material is obtained from deep tissue planes by surgical
biopsy or percutaneous aspiration, the results of cultures must
be interpreted in the clinical context.
The Diagnosis ofOsteomyelitis
The evaluation of the wound should also focus on the presence of possible bone infection. The diagnosis of osteomyelitis is often difcult because of the confounding effect of
bone injury due to Charcot neuro-osteoarthropathy and adjacent soft tissue infection. In the diabetic foot, osteomyelitis
almost always results from direct extension through an overlying infected chronic ulcer. Clinical features that increase
the probability of osteomyelitis are an ulcer larger than
2 cm2, an ulcer extending down to bone, and an ESR of
greater than 70mm/h [15]. The depth of an ulcer should be
explored by gentle probing of the ulcer base with a sterile,
blunt metallic probe. The probe-to-bone (PTB) test, which is
performed prior to extensive debridement, can identify bone
that is exposed (no longer covered by soft tissue) but not visible on examination of the base of a pedal ulcer [1, 2, 16].
Probing is generally tolerated without pain due to the nearly
universal presence of marked sensory neuropathy. The positive and negative predictive values of the PTB test are dependent on the prevalence of osteomyelitis in the population
studied. When performed on moderate or severely infected
foot ulcers, a positive PTB test is highly suggestive of osteomyelitis; however, a negative test does not exclude the diagnosis. In an uninfected wound, a positive PTB is not specic
for osteomyelitis, but the diagnosis is made less likely with a
negative test result. In a prospective study of 75 patients with
76 clinically infected foot ulcers, palpating bone on probing
the pedal ulcer had a sensitivity of 66%, a specicity of 85%,
a positive predictive value (PPV) of 89%, and a negative predictive value (NPV) of 56% for diagnosing osteomyelitis
[17]. An additional prospective study including 210-foot
lesions evaluated clinical and radiographic signs of infection, ulcer culture, and the probe-to-bone test. The probe-tobone test was of greatest diagnostic value with a sensitivity
of 94%, a specicity of 78%, a PPV of 95%, and a NPV of
91% [18]. A systematic review of the accuracy of the PTB
test to diagnose diabetic foot osteomyelitis demonstrated a
pooled sensitivity of 87% and specicity of 83%, further
supporting the ability of the PTB test to accurately diagnose
osteomyelitis in the diabetic foot [19].
Plain radiographs of the foot are a reasonable rst imaging study to assess for osteolytic bone changes and periosteal elevation, suggestive of osteomyelitis. The combined
use of serial PTB test and plain radiography has been found
to increase agreement among clinicians and positive predictive value of the diagnosis of osteomyelitis [20, 21]. The
low sensitivity of plain radiography early in infection may
lead to consideration for advanced modalities of imaging
such as magnetic resonance imaging (MRI), leukocyte
scintigraphy, or 18F-FDG positron emission tomography
(PET)/computed tomography (CT). MRI remains the study
of choice when further imaging is required due to its
enhanced sensitivity and the accessibility of the modality
[1, 2, 16, 22, 23]. The use of imaging in the diagnosis of
osteomyelitis is reviewed in detail in Chap. 5, “Radiographic
Changes of the Diabetic Foot.”
Osteomyelitis can be conrmed at bone biopsy by histopathologic ndings or bone culture. However, the histopathology of biopsied bone may be falsely negative because of
the patchy distribution of infection, and the yield of bone
culture may be reduced by prior antibiotic therapy or alternatively may represent false-positive results [24, 25]. In a study

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of the diagnosis of osteomyelitis, bone culture obtained from
surgical debridement was compared to bone histopathology
on 44 bone specimens; the two tests performed similarly but
were concordant with one another only 54% of the time, suggesting both are required for optimal assessment [26].
After surgical debridement, the use of bone culture and
pathology from retained bone or a “proximal margin” to guide
management remains an area of uncertainty. Studies have demonstrated variable inter-rater agreement on the histopathologic
diagnosis of osteomyelitis, and poor concordance of bone culture with histopathology is also seen [27, 28]. If a proximal
margin is sent, the results should be interpreted in the setting of
the clinical context and suspicion for residual infection.
If surgical debridement is not undertaken, percutaneous
bone biopsies have been shown to be safe and superior to
supercial wound swabs for detecting organisms causing
osteomyelitis and have a high yield of positive results to
guide treatment [29, 30]. Bone biopsy through healthy skin
rather than through the ulcer is the optimal approach where
feasible. Culture results from specimens taken on biopsy
through the wound do not correlate well (<50%) with those
obtained by transcutaneous bone biopsy [31]. Swab cultures
of ulcers overlying bone have a low rate of concordance with
cultures of bone itself. In a retrospective study of 76 patients
with pathology-conrmed osteomyelitis, culture of bone and
wound swab were fully concordant in only 17% of patients,
and bacteria present on bone culture were isolated from the
corresponding wound swab in only 30% of patients [29].
The Severity ofFoot Infections
Clinicians should routinely utilize a validated classication
system when assessing the severity of diabetic foot infection
[1, 2]. Multiple classication schema have been designed to
dene the severity of foot wounds with or without infection in
patients with diabetes. Some such as the widely used Wagner
system include infection only in one grade [32], while others
are too complex for routine clinical use. Well- studied systems
to classify the severity of infection have been developed by
the Infectious Diseases Society of America (IDSA) and the
International Working Group on the Diabetic Foot (IWGDF)
[1, 2, 33, 34]. The schema has been broadly used since 2004
and is updated quadrennially. The IWGDF/IDSA schema
classies wounds from having no infection to being severely
infected. Infected wounds are then subdivided into mild,
moderate, and severe infection by using the depth of a wound,
presence of ischemia, presence and extent of infection, and
presence of systemic toxicity (Table 20.1) [1, 2]. Increased
severity in the IDSA/IWGDF classication schema (e.g.,
moderate and severe infection) correlates with the need for
hospitalization and amputation [35, 36]. The IWGDF 2019
guideline includes an update to categorize moderate and
severe infection including osteomyelitis with “(O)” after the
grade number [1]. This reects the important diagnostic and
treatment considerations for osteomyelitis as well as prognostic factors including increased risk of prolonged hospitalization and amputation when present [37, 38].
In addition to assessing the wound, the affected limb and
foot should be assessed for signs of arterial ischemia, venous
insufciency, neuropathy, and biomechanical factors which
promote infection [1, 2]. Systemic signs and symptoms of
infection include fever, chills, alteration in mental status,
hemodynamic instability, and metabolic derangements such
as hyperglycemia, acidosis, or renal failure. Of note, hyperglycemia occurs almost universally in patients with both
non-limb-threatening and limb-threatening infections. Fever
is found primarily in patients with extensive cellulitis and
lymphangitis, infection (abscesses) loculated in the deep
spaces of the foot, bacteremia, or hematogenously seeded
remote sites of infection [7, 8, 39].
After adjusting for prior failed medical therapy, which is
likely to result in resistant organisms, these classication
schemes allow one to anticipate the organisms causing
wound infection and thus are an excellent basis from which
to plan initial empiric antimicrobial therapy.
Table 20.1 Classication of severity of diabetic foot infection
Clinical manifestation of infection Infection severity
Wound lacking purulence of any manifestations of inammation Uninfected
Presence of ≥2 manifestations of inammation (purulence, or erythema, pain, tenderness, warmth, or induration),
but any cellulitis/erythema extends ≤2cm around the ulcer, and infection is limited to the skin or supercial
subcutaneous tissues; no other local complications or systemic illness
Infection (as above) in a patient who is systemically well and metabolically stable but which has ≥1 of the
following characteristics: cellulitis extending >2cm, lymphangitic streaking, spread beneath the supercial fascia,
deep tissue abscess, gangrene, and involvement of muscle, tendon, joint, or bone
Infection in a patient with systemic toxicity or metabolic instability (e.g., fever, chills, tachycardia, hypotension,
confusion, vomiting, leukocytosis, acidosis, severe hyperglycemia, or azotemia)
Infection involving bone (osteomyelitis) Add “(O)” after
Adapted from reference [33] with permission
a
In the setting of severe ischemia, all infections are considered severe
a
Mild
Moderate
Severe
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Microbiology
It is possible to anticipate the major pathogens causing diabetic foot infection based on the severity of infection and
informed by patient-specic risk factors and prior available
culture data. Interpretation of culture results requires an
adjustment for the recovery of organisms of known low invasive potential that are likely to be commensals or colonizers.
In non-limb-threatening infections, particularly those occurring in patients who have not previously received antimicrobial therapy, Staphylococcus aureus and streptococci,
particularly group B streptococci, are the predominant
pathogens [1, 40–42]. Although there are differences between
geographic areas, S. aureus causing infections in the feet of
diabetics are, as in other skin and soft tissue infections, frequently methicillin-resistant (MRSA) [42–45]. Risk factors
for MRSA diabetic foot infection include prolonged wound
duration, history of MRSA colonization, prior inpatient
management, and chronic kidney disease [44–46].
Limb-threatening foot infections, which often involve
deeper tissues, are typically chronic, as well as previously
treated, and are commonly polymicrobial (Table20.2). Both
gram-positive cocci and gram-negative rods are commonly
isolated from a single lesion, and the recovery of both aerobic and anaerobic organisms is frequent [8, 47–53]. S. aureus
(including methicillin-sensitive and methicillin-resistant isolates), streptococci (particularly group B streptococci),
Enterobacterales, and Pseudomonas aeruginosa are the pre-
dominant pathogens in these infections. Among the anaerobes, Peptostreptococcus species, Prevotella species, and
Bacteroides species are recovered frequently [52, 54–56]. Of
note, Clostridium species are recovered infrequently. The
reported isolation rates of anaerobes are variable across studies due to varying specimen collection techniques; however,
with optimal methods, these organisms can be recovered
from 75 to 85% of limb-threatening infections [54]. The frequency of isolating anaerobic bacteria is greatest in those
patients with the most severe infections, particularly those
where infection involves necrotic gangrenous tissue and
amputation is often required. Fetid infections suggest the
presence of anaerobes; however, anaerobes including B. fra-
gilis may be recovered from infections that are not particularly foul smelling. Hence, clinical clues beyond the major
categorization of infections as non-limb-threatening or limbthreatening are not sufcient to predict the microbiology of
foot infections.
The spectrum of bacterial species recovered from foot
infections, especially those that are limb-threatening, can be
dramatically altered by prior failed antimicrobial therapy or
contact with the healthcare system. While antibiotic resistance in gram-negative bacilli is increasingly complex and
methods for susceptibility testing vary by medical center,
clinicians need to be attuned to the risk of common betalactamases such as AmpC found among Enterobacter cloa-
Table 20.2 Microbiology of moderate or severe limb-threatening infections in patients with diabetes
Number of isolates (% of isolates)
Grayson [8]
Organisms
Aerobic
S. aureus (all) 54 (20) 25 (14) 214 (13) 20 (35) 152 (44) 779 (64)
Methicillin-resistant S. aureus NR 14 (56) 50 (24) 7 (12) 27 (8) 321 (26)
Coagulase-negative staphylococci 12 (4) 12 (7) 175 (11) 17 (29) 9 (12) 389 (32)
Enterococcus spp. 28 (10) 21 (11) 155 (10) 6 (7) 25 (15) 219 (18)
Streptococcus spp. 35 (13) NR 177 (11) 2 (3) 48 (17) 373 (31)
Corynebacterium spp. NR NR 116 (7) 3 (7) 4 (9) 345 (28)
E. coli 6 (2) 22 (12) 20 (1) 2 (3) NR 18 (1)
Klebsiella spp. 5 (2) 12 (7) 25 (2) NR NR 11 (1)
Proteus mirabilis 7 (3) 23 (13) 24 (1) NR NR 63 (5)
Enterobacter spp. 9 (3) 1 (0.5) 20 (1) NR NR 34 (3)
P. aeruginosa 7 (3) 18 (10) 40 (2) 2 (3) 26 (9) 127 (10)
Acinetobacter spp. 7 (3) 17 (9) NR NR NR 8 (1)
Other aerobic gram-negative bacilli or not specied 20 (7) 1 (0.5) 96 (6) 14 (24) 164 (63) 194 (16)
Anaerobic
Gram-positive cocci 12 (4) 13 (7) NR NR NR 29 (2)
Bacteroides fragilis 6 (2) 3 (2) 19 (1) NR NR NR
Other Bacteroides spp. or not specied 24 (9) 7 (4) 28 (2) NR NR 79 (6)
Clostridium spp. NR 2 (1) NR NR NR NR
Other anaerobes or not specied 14 (5) 3 (2) 415 (26) 1 (2) 48 (24) 61 (5)
Number of isolates/infection 2.84 2.28 3.8 1.45 1.0–1.5 2.98
NR not reported
a
Specimens obtained by various routes, including deep ulcer swabs, curettage of the ulcer base, aspiration, or tissue biopsy
(n=96)
Gadepalli [47]
(n=80)
Citron [48]
(n=427)
a
Tone [49]
(n=40)
Nelson [50]
(n=400)
Chen [51]
(n=819)
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