Journal of

Clinical Medicine

Review
Bacterial Diversity of Diabetic Foot Ulcers: Current
Status and Future Prospectives
Fatemah Sadeghpour Heravi 1 , Martha Zakrzewski 2 , Karen Vickery 1 , David G. Armstrong 3
and Honghua Hu 1, *
1 Surgical Infection Research Group, Faculty of Medicine and Health Sciences, Macquarie University,
Sydney 2109, Australia; [email protected] (F.S.H.); [email protected] (K.V.)
2 QIMR Berghofer Medical Research Institute, Brisbane QLD 4006, Australia;
[email protected]
3 Southwestern Academic Limb Salvage Alliance (SALSA), Keck School of Medicine of University of Southern
California (USC), Los Angeles, CA 90007, USA; [email protected]
* Correspondence: [email protected]; Tel.: +61-2-98502709; Fax: +61-2-98502701




Received: 17 October 2019; Accepted: 8 November 2019; Published: 10 November 2019


Abstract: Diabetic foot ulcers (DFUs) and diabetic foot infections (DFIs) are associated with reduced
patient quality of life, lower-extremity amputation, hospitalization, and high morbidity and mortality.
Diverse bacterial communities have been identified in DFUs/DFIs, playing a significant role in
infection prognosiS. However, due to the high heterogeneity of bacterial communities colonized in
DFUs/DFIs, culture-based methods may not isolate all of the bacterial population or unexpected
microorganismS. Recently, high sensitivity and specificity of DNA (metagenomics) and RNA
(metatranscriptomics) technologies have addressed limitations of culture-based methods and have
taken a step beyond bacterial identification. As a consequence, new advances obtained from DNA-
and RNA-based techniques for bacterial identification can improve therapeutic approacheS. This
review evaluated the current state of play in aetiology of DFUs/DFIs on culture and molecular
approaches, and discussed the impact of metagenomic and metatranscriptomic methods in bacterial
identification approaches.

Keywords: diabetic foot ulcers; diabetic foot infections; microbiology; culture; culturomics; 16S rRNA
sequencing; microbiota; metagenomics; metatranscriptomics

1. Introduction
The number of people with diabetes is expected to increase rapidly—from 425 million in 2017 to a
predicted value of 600 million by 2030. More than one-third of people with diabetes develop diabetic
foot ulcers (DFUs) during their lifetime, with half of these becoming infected and causing diabetic
foot infections (DFIs). Fifteen percent of patients with DFIs require lower limb amputation to prevent
progression of the infection [1,2].
Diabetic foot care is very expensive, with an estimated US $8659 annual cost per patient,
thus emphasizing the importance of early diagnosis and treatment of DFUs/DFIs [3]. Treatment consists
of improving patient intrinsic factors, such as improving glucose control, as well as targeting extrinsic
factors, the principal being the removal of bacterial contamination/infection. However, DFUs/DFIs
harbor diverse bacterial communities, which increase the difficulty in treatment choice.
There are several laboratory techniques available with different sensitivities and specificities to
determine the bacterial composition of DFUs/DFIS. Nonetheless, the characterization of the entire
polymicrobial community at different severity stages ranging from mild to severe is still a major
challenge [4].

J. Clin. Med. 2019, 8, 1935; doi:10.3390/jcm8111935 www.mdpi.com/journal/jcm

J. Clin. Med. 2019, 8, 1935 2 of 18

Although culture-based methods are the principal method of bacterial identification, they often
produce false-negative results in patients who have received antibiotics; fail to identify slow-growing,
fastidious, anaerobic, and unknown pathogens; and are time-consuming, hindering proper and
early detection of the bacterial community in DFUs/DFIS. Recent advances in molecular technologies
overcome many of the mentioned inadequacies and provide new insights into the bacterial diversity of
DFUs/DFIS. These advancements have important implications for the identification of so far unknown
and uncultivable bacteria in DFUs/DFIs.
This manuscript will review the current state of play in culture and molecular methods to
assess the bacterial diversity in DFUs/DFIs, and analyze the future impact of metagenomics and
metatranscriptomic approaches on bacterial identification and treatment.

2. Sample Collection from DFIs

The first and most critical step, not only in culture-based methods but also in advanced
molecular-based approaches, is sample collection. Historically, curettage, biopsies, swabs, and wound
aspirations have been the principal routine samples taken by wound care providers [5]. As the Infection
Disease Society of America (IDSA) advises that samples be taken from the base of wounds, tissue
biopsies have been proposed as a gold standard method [6]. Swab cultures of the wound surface
are also commonly used, but due to a high number of commensal microflora inhabiting healthy skin,
swab culture results may not be as reliable as tissue samples [5]. For instance, coagulase-negative
staphylococci (CoNS), Micrococcus, Bacillus spp., and Corynebacterium, which are a part of normal skin
flora and have been frequently isolated from DFIs swabs, are not usually considered as pathogenic
bacteria, unless the samples are taken from deep tissues [7]. Even though the collection of swab
samples is easier than tissue samples, some studies have shown that swab culture results are less
specific and sensitive [7,8].
Although culturing of superficial swabs and deep tissue specimens from infected ulcers provided
identical results in 62% of cases, the swabs only identified 91% of the organisms isolated from tissue
samples [9]. Similar results were obtained by Mutluoglu in 69.2% of wounds, but superficial swabs
failing to detect all organisms in 9% of caseS. The positive predictive value of swabs relative to tissue
was 84.4% [10].
Swab samples are less reliable in isolating Gram-negative bacteria such as E. coli and Citrobacter [11].
A higher concordance rate of 80% was found by Huang et al. in deep ulcers; however, when abscess
osteomyelitis or gangrene was present, significantly different results were obtained by swabs and
tissue biopsy with only around 30% concordance. Moreover, some Gram-negative bacteria, such as
Ralstonia pickettii and Serratia, were only identified in deep tissue samples [11].
Deep tissue samples also showed higher sensitivity for the monitoring of bacterial species that
have been previously reported as antibiotic-resistant strains [8]. Similarly, percutaneous bone biopsy
identified a higher number of organisms causing diabetic foot osteomyelitis compared to swab
samples [12,13]. Significantly more bacteria were isolated from tissue samples compared to 247 paired
swab samples with a 42% concordance [14].
Based on the aforementioned studies that compared the efficiency of bacterial culture using tissue
and swab samples, it can be stated that tissue samples provide more reliable results for bacterial
identification and monitoring of bacterial population in DFIs.

3. Microbiology of DFUs/DFIs
According to bacterial culture and molecular approaches, DFUs/DFIs can be colonized by different
aerobes and anaerobeS. DFIs of a shorter duration seem to have a simpler microbiota and are mainly
colonized by Gram-positive cocci (Staphylococcus and Streptococcus spp.). In contrast, chronic DFIs may
have polymicrobial infections colonizing by different types of aerobic bacteria, such as Staphylococcus,
Streptococcus, Enterococcus, Pseudomonas spp., and anaerobic pathogens (Figure 1) [15]. Bacteroides fragilis
has also been reported in several studies as the most abundant anaerobic bacteria in DFIs [16,17].
J. Clin. Med. 2019, 8, x FOR PEER REVIEW 9 of 18
J. Clin. Med. 2019, 8, 1935 3 of 18

Geographical features, infection duration, patient’s metadata (e.g., smoking habits), and
antibiotic
Based use
on can also influence
these the bacterial
explicitlydistribution
designed toinculture
patients. For example, a Korean study
J. Clin. Med. 2019, 8,studies which
x FOR PEER were
REVIEW anaerobes, anaerobic bacteria9 were
of 18
conducted
reported on 737 abundance
in low patients found
withthat
lowsmoking
impact on can increaseprogress.
infection the risk of DFIs with Pseudomonas spp.
[18]. Geographical features, infection duration, patient’s metadata (e.g., smoking habits), and
antibiotic use can also influence the bacterial distribution in patients. For example, a Korean study
conducted on 737 patients found that smoking can increase the risk of DFIs with Pseudomonas spp.
[18].

FigureFigure 1. Bacterial
1. Bacterial diversity
diversity of diabetic
of diabetic footfoot infectionsinininfection
infections infection progression.
progression.

Geographical features, infection duration, patient’s metadata (e.g., smoking habits), and antibiotic
3.1. Bacterial Identification of DFIs Based on Culture
use can also influence the bacterial distribution in patientS. For example, a Korean study conducted on
737 patientsFigure
found1.that
Bacterial diversity
smoking of diabetic
can increase footofinfections
the risk DFIs withinPseudomonas
infection progression.
spp. [18].
3.1.1. Gram-Positive Bacteria
3.1.Firmicutes
Bacterial Identification of DFIs Based
is the main bacterial on Culture
phylum, comprising Streptococcus spp. (Streptococcus agalactiae,
Streptococcus pyogenes, and Streptococcus mitis), Staphylococcus spp. (Staphylococcus aureus), and
3.1.1. Gram-Positive Bacteria
3.1.1. Gram-Positive Bacteria
Enterococcus spp. (Enterococcus faecalis) (Figure 2). S. aureus has been reported as the most common
pathogenic speciesis
Firmicutes
Firmicutes isinthe
DFIs
the main
mainin several
bacterial
bacterialstudies.
phylum,
phylum, In acomprising
study conducted
comprising on 342 patients
Streptococcus
Streptococcus with diabetic
spP. (Streptococcus
spp. (Streptococcus foot
agalactiae,
agalactiae,
infections, S.
Streptococcus aureus (20.2%
pyogenes, andof isolates) was
Streptococcus the most
mitis), common
Staphylococcus Gram-positive
Streptococcus pyogenes, and Streptococcus mitis), Staphylococcus spp. (Staphylococcus aureus), and
spP. bacteria
(Staphylococcus [19]. These
aureus), and
results are
Enterococcusrelatively
spP. similar
(Enterococcus to the number
faecalis) of
(Figure Gram-positive
2). S. aureus bacteria
has been in Jneid’s
reported
Enterococcus spp. (Enterococcus faecalis) (Figure 2). S. aureus has been reported as the most common as study
the most(54.7% of
common
isolates) [20] and
pathogenic
pathogenic Al Benwan’s
species
species in
in DFIs
DFIs ininstudy
several
several (32.3%
studieS.of isolates)
studies. In aa study
In study [21], which applied
conducted
conducted on 342
on culture and
342 patients
patients withculturomic
with diabetic foot
diabetic foot
methods to isolate
infections, S.
infections, bacterial
S.aureus
aureus(20.2%
(20.2% species,
of of respectively.
isolates)
isolates)waswasthe most Staphylococcus
the most commoncommon epidermidis
Gram-positive was also
bacteria
Gram-positive isolated
[19]. These
bacteria in one
[19]. results
These
study
are conducted
relatively on
similar 454
to the DFIs
number samples
of as the
Gram-positive most dominant
bacteria in bacterial
results are relatively similar to the number of Gram-positive bacteria in Jneid’s study (54.7%[20]
Jneid’s study species.
(54.7% of Although
isolates) of
Staphylococcus
and
isolates) [20]epidermidis
Al Benwan’s andstudy is part of
(32.3%
Al Benwan’s ofstudy
the normal
isolates) [21],skin,
(32.3% which
of it applied
can cause
isolates) severe
culture
[21], which infections
and culturomic
applied in the presence
methods
culture and of
to isolate
culturomic
foreign
methodsbodies,
bacterial suchrespectively.
species,
to isolate asbacterial
prosthetic devices
species, and wound
Staphylococcus epidermidis
respectively. infections [22,23].
was also
Staphylococcus isolated in was
epidermidis one study conducted
also isolated on
in one
454 DFIs samples as the most dominant bacterial specieS. Although
study conducted on 454 DFIs samples as the most dominant bacterial species. Although Staphylococcus epidermidis is part of
the normal skin, it
Staphylococcus epidermidis can cause severe infections in the presence of foreign bodies, such
is part of the normal skin, it can cause severe infections in the presence of as prosthetic
600
devices and wound infections
553
foreign bodies, such as prosthetic [22,23].
devices and wound infections [22,23].
500

400
600
Number

553 322
294
300 273
500

200
400
Number

322 95
100 294
300 273
31 20
6
0
200

95
100
31 20
6
0

Figure 2. Commonly isolated bacteria from DFIs using culture-based methods. The number
on the bar is the sum of the total number of isolated bacterial genus in 10 studies from 2004
to 2018.
Figure2.2.Commonly
Figure Commonly isolated
isolated bacteria
bacteria fromfrom
DFIs DFIs
usingusing culture-based
culture-based methodS. methods.
The numberTheon
number
the bar
onthe
is thesum
barofisthe
thetotal
sumnumber
of the of
total number
isolated of isolated
bacterial bacterial
genus in genus
10 studies from in 10 to
2004 studies
2018. from 2004
to 2018.
J. Clin. Med. 2019, 8, 1935 4 of 18
J. Clin. Med. 2019, 8, x FOR PEER REVIEW 10 of 18

3.1.2. Gram-Negative Bacteria

3.1.2. Gram-Negative Bacteria
The predominance of the Enterobacteriaceae family (Escherichia coli, Klebsiella pneumoniae,
The predominance
Morganella morganii,ofand
theProteus
Enterobacteriaceae family
mirabilis) has (Escherichia
recently coli, Klebsiella
been reported as the pneumoniae,
largest group Morganella
of aerobic
morganii, and Proteus mirabilis) has recently been reported as the largest group of
Gram-negative rods in DFIS. For instance, an average of 1.8 bacterial pathogens per diabetic wound aerobic Gram-
negativewas
sample rodsreported
in DFIs. in
Forone
instance,
study, an
of average of 1.8 bacterial
which, 51.2% pathogens per
were Gram-negative diabetic
bacteria wound
[21], which was
sample
quite was
high reported to
compared in the
onenumber
study, of
ofwhich, 51.2% were
Gram-negative Gram-negative
species bacteria
in Jneid’s study [21],ofwhich
(26.4% was[20].
isolates)
quite high compared to the number of Gram-negative species in Jneid’s study (26.4%
This discrepancy might be due to previous antibiotic use in patients, long duration of hospitalization, of isolates)
[20]. wound
and This discrepancy
chronicity. might be due tocoli
Escherichia previous antibiotic
was also reported useasin the
patients,
most long
commonduration of
Gram-negative
hospitalization,
bacteria and wound
in 342 patients withchronicity. Escherichia
diabetic foot coli[19].
infections was Entrobacter,
also reported as the most common
Pseudomonas, Citrobacter, and
Gram-negative
Provetella bacteria
spP. have in 342reported
also been patientsinwith diabetic
lesser foot infections [19]. Entrobacter, Pseudomonas,
numbers.
Citrobacter, and Provetella spp. have also been reported in lesser numbers. 3.1.3. Polymicrobial
3.1.3. Polymicrobial Infection
Infection
DFIs
DFIs are
are composed
composed of of aa mixture
mixture ofof bacteria. Consequently, the
bacteria. Consequently, the production
production of of many
many different
different
virulence
virulence factors,
factors, such
such as
as proteases,
proteases, collagenases, and hemolysins
collagenases, and hemolysins contribute
contribute toto infection chronicity [23].
infection chronicity [23].
Polymicrobial infections were estimated to occur in 75% of DFI cases in Al Benwan’s
Polymicrobial infections were estimated to occur in 75% of DFI cases in Al Benwan’s study conducted study conducted
on
on 440
440 DFIs
DFIs samples
samples [21].
[21]. In Citron’s study, 83.8%
83.8% ofof positive
positive cultures
cultures were
were polymicrobial
polymicrobial withwith aa
mixed population
population of of Gram-positive
Gram-positiveand andGram-negative
Gram-negativespecies species[23].
[23].In In another study conducted
another study conducted on
on
473473 specimens,
specimens, 56.87%
56.87% of of
thethe DFIswere
DFIs werepolymicrobial
polymicrobialwith withaa high
high abundance
abundance of Gram-negative
Gram-negative
isolates
isolates (76.27%)
(76.27%)[24].
[24]. The
Theoccurrence
occurrenceofofpolymicrobial
polymicrobialinfections
infectionswas
was reported
reported to to
bebe
relatively lower
relatively in
lower
Sánchez’s study (48.3%) [25]. Polymicrobial infections are more challenging to treat
in Sánchez’s study (48.3%) [25]. Polymicrobial infections are more challenging to treat compared to compared to DFIs
with
DFIs simpler bacterial
with simpler composition
bacterial and are
composition andlikely to develop
are likely complex
to develop and chronic
complex infections
and chronic [4]. [4].
infections
Figures
Figures 2 and 3 show the total number of commonly isolated bacterial genus and species in DFIs
from 2004 to to 2018.
2018.

600
551

500

400
Number

300
236
223

200
132
87
100

37
18
0
S. aureus P. aeruginosa E. coli K. pneumoniae S. epidermidis A. baumannii M. morganii

Figure3.3.Commonly
Figure Commonly isolated
isolated bacterial
bacterial species
species in DFIsinusing
DFIsculture-based
using culture-based methods.
methodS. The numberTheon
number
the on sum
bar is the the bar istotal
of the the number
sum of of the
thetotal number
isolated of species
bacterial the isolated bacterial
in 10 studies fromspecies
2004 to in 10
2018.
studies from 2004 to 2018.
3.1.4. Uncommon Bacterial Species
3.1.4.Compared
Uncommon to Bacterial Species of S. aureus, the role of uncommon isolated bacteria in DFIs is less
the pathogenicity
Finegoldia magna
clear.Compared is an anaerobicofGram-positive
to the pathogenicity coccus,
S. aureus, the role of which
uncommonis partisolated
of the normal
bacteriaflora of the
in DFIs is
genitourinary and gastrointestinal tract and can be isolated from the oral cavity and skin.
less clear. Finegoldia magna is an anaerobic Gram-positive coccus, which is part of the normal flora of
Based on Citron’s
the genitourinary findings, F. magna
and gastrointestinal wasand
tract isolated
can beasisolated
the most predominant
from anaerobe
the oral cavity (37.4%) [23],
and skin.
whichBased
has not
on been commonly
Citron’s reported
findings, F. magnaby previous
was isolatedstudieS.
as theOne possible
most reason can
predominant be the different
anaerobe (37.4%)
selective bacterial media that have been used in other studieS. For instance,
[23], which has not been commonly reported by previous studies. One possible reason Brucella agar is commonly
can be the
used for anaerobes
different cultivation,
selective bacterial media which is also
that have beena selective culture
used in other mediaFor
studies. B. fragilisBrucella
forinstance, and Prevotella
agar is
species,
commonly butused
not for
forGram-positive anaerobes
anaerobes cultivation, which F. magna).
(suchisasalso Thisculture
a selective explanation
mediamay describe
for B. fragiliswhy
and
Prevotella species, but not for Gram-positive anaerobes (such as F. magna). This explanation may
describe why several studies have reported B. fragilis as the predominant anaerobes [23].
J. Clin. Med. 2019, 8, 1935 5 of 18

several studies have reported B. fragilis as the predominant anaerobes [23]. Furthermore, Porphyromonas
and Prevotella, Clostridium, Aerococcus, Helcococcus, and Gemella spP. as fastidious and slow-growing
bacteria have been rarely isolated from DFIs [26].
Although culture-based methods have been the gold standard for bacterial identification for many
years, this approach may not necessarily reflect all the clinically important pathogenic bacteria in DFIs,
particularly anaerobes and uncommon species.

3.2. Bacterial Identification of DFIs Based on Culturomic Method

Culturomics involves optimization of culture conditions using matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS) to overcome culture-based method
limitationS. MALDI-TOF MS produces specific mass spectral fingerprints from bacterial proteins,
which is a unique signature for bacteria. All the bacterial protein fingerprints are compared to a protein
database in order to facilitate accurate bacterial identification to genus, and even species level [27].
Culturomics revealed that 88.3% of DFIs were polymicrobial, identifying 53 known and 19 unknown
bacterial specieS. Based on culturomic findings, S. aureus, Enterococcus faecalis, Enterobacter cloacae,
Staphylococcus lugdunensis, Staphylococcus epidermidis, Proteus mirabilis, and F. magna in descending order
were the most abundant bacterial species isolated from the 43 patients [20].
On the downside, fresh bacterial colonies and a fair amount of bacterial biomass are needed to be
detected by mass spectrometer, which may limit the use of culturomics for fastidious and slow-growing
bacteria. Further evaluation of microbiota in DFIs using this method on a larger scale is required to
identify the superiority of this method to other bacterial identification tools.
High specificity and sensitivity of molecular approaches have provided a more reliable insight into
DFIs’ microbiota than traditional methodS. The bacterial 16S ribosomal RNA (rRNA) gene presented
in all bacteria is a widely used phylogenetic marker for bacterial taxonomic classification and is
particularly effective for anaerobes, slow-growing, and fastidious bacteria [28].

4. Molecular DNA-Based Techniques

Depending on the phenotypic and genotypic characteristics of pathogenic bacteria, the initial
assessment of bacterial isolation using a culture-based method may require one week or longer.
Regardless of bacterial characteristics in a heterogeneous sample, molecular approaches can minimize
this time frame to a few days and provide more reliable resultS. DNA-based approaches have cast
new light on the multiplex bacterial community in DFUs/DFIs and have played a significant role in
accurate bacterial identification and the evaluation of new bacterial species, and even new genes that
play a significant role in infection progression. DNA-based approaches can provide not only bacterial
taxonomic classification, but also antibiotic susceptibility pattern (resistome), which may help the
replacement of empirical therapies with targeted therapies [7].
Polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), 16S rRNA
gene sequencing analysis, metagenomics (whole genome sequencing), and metatranscriptomics (RNA
profiling) have provided scientists with new insights into the bacterial community structure and the
alteration of the human microbiome during infection (Figure 4). Understanding the bacterial gene
composition and functional capabilities of pathogenic microbes using molecular-based approaches
will enable scientists to more accurately target bacterial virulence factors and enzymes associated
with infection.

4.1. Bacterial Identification of DFUs/DFIs Based on 16S rRNA Sequencing

The 16S rRNA gene has been highly conserved through bacterial evolution. This gene consists
of hypervariable regions (V1–V9), which enable the identification of different bacterial taxa [29].
A common approach to analyze 16S rRNA gene sequences is to group them into operational taxonomic
units (OTUs), based on a pair-wise sequence identity of 97%, which is commonly considered as being
equivalent to species level [30].
 J. Clin. Med. 2019, 8, 1935 6 of 18
J. Clin. Med. 2019, 8, x FOR PEER REVIEW 12 of 18

Figure 4. Overview of different approaches to evaluate bacterial species in infected cells.

Figure 4. Overview of different approaches to evaluate bacterial species in infected cells.
4.1.1. Gram-Positive Bacteria
4.1. Bacterial Identification
Comparison of DFUs/DFIs
of traditional cultureBased on 16S
and 16S rRNA
rRNA Sequencing
gene sequencing approaches to DFUs showed
thatThe
the16S
most abundant
rRNA OTU
gene has belonged
been highly to Staphylococcus
conserved throughspP. (such as
bacterial S. aureusThis
evolution. S. epidermidis),
and gene consists
which may demonstrate the critical role of Staphylococcus spp., particularly S. aureus, as
of hypervariable regions (V1–V9), which enable the identification of different bacterial taxa [29]. Aa pathogenic
common in DFUs /DFIs,
bacteriaapproach which
to analyze have
16S rRNAbeen reported
gene frequently
sequences in other
is to group themstudies that applied
into operational 16S rRNA
taxonomic
sequencing
units (OTUs),[31,32].
based onInaGardner’s study [33],
pair-wise sequence the culture-based
identity of 97%, which method underestimated
is commonly consideredtheasnumber
being
of bacterial species isolated
equivalent to species level [30].from DFUs (26 bacterial species per DFU), compared to the number of
species-level OTUs discovered by 16S rRNA sequencing analysiS. For instance, the culture-based
method
4.1.1. showed the
Gram-Positive presence of anaerobes in 27% of the samples, while 16S rRNA sequencing
Bacteria
identified anaerobic bacteria in all the samples (100%). The comparison of bacterial diversity on new
Comparison of traditional culture and 16S rRNA gene sequencing approaches to DFUs showed
and recurrent diabetic ulcers using 16S rRNA amplicon sequencing showed more reliable results
that the most abundant OTU belonged to Staphylococcus spp. (such as S. aureus and S.epidermidis),
compared to culture-based methodS. The traditional bacterial culture showed positive bacterial growth
which may demonstrate the critical role of Staphylococcus spp., particularly S. aureus, as a pathogenic
in 55% of the patients, while 16S amplicon sequencing showed positive bacterial detection in 75% of
bacteria in DFUs /DFIs, which have been reported frequently in other studies that applied 16S rRNA
the patients in new and recurrent ulcerS. Bacterial culture revealed the presence of S. aureus, anaerobes,
sequencing [31,32]. In Gardner’s study [33], the culture-based method underestimated the number of
beta-hemolytic streptococci, and Candida spP. in descending order, while 16S rRNA sequencing analysis
bacterial species isolated from DFUs (26 bacterial species per DFU), compared to the number of
showed a wider range of bacterial diversity (Peptoniphilus, Staphylococcus, Anaerococcus, Corynebacterium
species-level OTUs discovered by 16S rRNA sequencing analysis. For instance, the culture-based
Corynebacterium, Peptoniphilus, and Anaerococcus) in new and recurrent diabetic ulcers [31].
method showed the presence of anaerobes in 27% of the samples, while 16S rRNA sequencing
identified anaerobic bacteria in all the samples (100%). The comparison of bacterial diversity on new
and recurrent diabetic ulcers using 16S rRNA amplicon sequencing showed more reliable results
compared to culture-based methods. The traditional bacterial culture showed positive bacterial
growth in 55% of the patients, while 16S amplicon sequencing showed positive bacterial detection in
75% of the patients in new and recurrent ulcers. Bacterial culture revealed the presence of S. aureus,
J. Clin. Med. 2019, 8, 1935 7 of 18

4.1.2. Gram-Negative Bacteria

Escherichia, Proteus, Klebsiella, Enterobacter, Pseudomonas, Citrobacter, Provetella, Bacteroides,
Porphyromonas, Proteobacteria, Bacteroidetes, and Fusobacteria spP. have been reported as the most
dominant Gram-negative bacterial strains by the 16S rRNA sequencing method from DFUs/DFIs,
which have also been reported using culture-based methods [31–34].

4.1.3. Polymicrobial Infection

Based on 16S rRNA analysis, deep ulcers had a more diverse bacterial community being colonized
by anaerobes, Gram-positive, and Gram-negative bacteria. Analysis of the 16S rRNA gene showed that
superficial wounds had mainly Staphylococcus spp. [33]. Similarly, assessment of 2,963 different types
of wounds (diabetic foot ulcers, venous leg ulcers, and decubitus ulcers) revealed a great bacterial
diversity in chronic wounds with a high number of Staphylococcus (63%) and Pseudomonas (25%) species,
anaerobes, and commensal bacteria [35]. Longer duration DFUs were found to be polymicrobial with
an average of 10 to 125 bacterial species [36].
Although both culture-based methods and molecular approaches have confirmed the polymicrobial
nature of DFUs/DFIs, culture-based methods may not be an appropriate method to fully evaluate this
complexity. As a result, wound care management based on inaccurate bacterial identification may lead
to unsuccessful antimicrobial outcomes.

4.1.4. Uncommon Species

16S rRNA sequencing analysis has identified a wider range of uncommon bacterial species in
DFUs/DFIs, compared to the culture-based method, which may indicate the inadequacy of culture-based
methods in the identification of anaerobes and fastidious bacteria [37]. Delftia acidovorans, Serratia
nematodiphila, Streptococcus
J. Clin. Med. salivarius,
2019, 8, x FOR PEER REVIEW Fusobacterium nucleatum, Flavobacterium succinicans, Staphylococcus
14 of 18

pettenkoferi, and many other species have been detected by the 16S rRNA sequencing method, which had
Table 1. Uncommon bacterial species isolated from diabetic ulcers using different bacterial
not commonly reported tools.
identification by culture-based methodS. Figure 5 shows uncommon bacterial species isolated
from DFUs using different identification tools.

Figure 5. Uncommon bacterial species isolated from diabetic ulcers using different bacterial
4.2. Bacterial Identification of DFUs Based on Shotgun Metagenomic Sequencing
identification tools.
In 16S ribosomal RNA (rRNA) amplicon sequencing, just a single region of the bacterial genome
(16S rRNA gene) is sequenced, but shotgun metagenomic sequencing is a whole-genome sequencing
approach. While the 16S rRNA gene is highly important in bacterial identification, it lacks
mechanistic insights into DFUs/DFIs, such as antimicrobial susceptibility pattern, functional
pathway, gene composition, biofilm, and virulence factors-related genes. Also, 16S rRNA amplicon
sequencing is less sensitive at the species level. Contamination with human mitochondrial DNA
J. Clin. Med. 2019, 8, 1935 8 of 18

4.2. Bacterial Identification of DFUs Based on Shotgun Metagenomic Sequencing

In 16S ribosomal RNA (rRNA) amplicon sequencing, just a single region of the bacterial
genome (16S rRNA gene) is sequenced, but shotgun metagenomic sequencing is a whole-genome
sequencing approach. While the 16S rRNA gene is highly important in bacterial identification, it lacks
mechanistic insights into DFUs/DFIs, such as antimicrobial susceptibility pattern, functional pathway,
gene composition, biofilm, and virulence factors-related geneS. Also, 16S rRNA amplicon sequencing
is less sensitive at the species level. Contamination with human mitochondrial DNA (mtDNA) is
another problem that can reduce microbial sequencing throughput and introduce bias in the 16S rRNA
downstream analysis [30].
Shotgun metagenomic sequencing is a genome-wide sequencing approach to assess bacterial
communities directly from infected siteS. Shotgun metagenomic sequencing can address the limitations
of culture and 16S rRNA sequencing methods and even identify unexpected microbial taxa.
This relatively young, but rapidly growing field can provide a clear insight into the potential interaction
of DFUs’ microbiota. It also provides useful data on phylogeny, new enzymes, biocatalysts, and the
function of uncultivable and unknown microbes [38]. The rapid price reduction in next-generation
sequencing and increased sequence data throughput will make shotgun metagenomic sequencing a
promising approach, in the same manner as 16S amplicon sequencing [39].

4.2.1. Gram-Positive Bacteria

Shotgun metagenomic sequencing of 100 non-infected diabetic foot ulcers was performed to
investigate the DFUs microbial community at the strain-level resolution and to identify the virulence
and pathogenicity of DFUs microbiota. S. aureus, Pseudomonas aeruginosa, and Corynebacterium striatum
were identified as the most abundant bacterial species in this study. Also, metagenomics identified the
main Staphylococcus strains, including S. aureus 7372 and S. aureus 10757 as dominant strains in DFUs
for the first time [40].

4.2.2. Gram-Negative-Bacteria
Based on shotgun metagenomic sequencing, Alcaligenes faecalis was the most abundant
Gram-negative bacteria in DFUs, which was also previously reported by the culturomic method [20,40].
The measurement of the inflammatory response in keratinocyte cells also showed that A. faecalis
could induce the production of cytokine IL-8 as a pro-inflammatory cytokine; and GM-CSF, G-CSF,
and PDFG-AB, which can lead to wound healing enhancement [40].

4.2.3. Uncommon Bacterial Species

Coagulase-negative species Staphylococcus pettenkoferi, Staphylococcus simulans, and Staphylococcus
lugdunensis were reported in less abundance from DFUs using metagenomicS. Corynebacterium striatum,
Propionibacterium spp., Porphyromonas somerae, Brevibacterium massiliense, Klebsiella oxytoca, and many
other uncommon species were also reported in DFUs using shotgun metagenomic sequencing
(Figure 5) [40]. Further investigation is needed to evaluate the importance of isolated uncommon
species in the chronicity and progression of DFIs.

4.2.4. Functional Pathways

Based on the annotation of DFUs’ microbiota pathways using SEED [41], the pathway of
carbohydrate, protein, and amino acid metabolism related genes to virulence and infection, transposable
elements, and phages were the most abundant features in DFUs’ microbiota. Deep wounds with
insufficient oxygen exchange were associated with the anaerobic glycolysis production system,
biosynthesis of saccharide, production of capsular, and extracellular polysaccharide, which all support
the presence of bacterial taxa with the property to produce a biofilm structure in deep layers of
the infection site. Whole-genome sequencing of S. aureus 7372 and S. aureus 10757 strains showed
J. Clin. Med. 2019, 8, 1935 9 of 18

the presence of the agrABCD gene, which is responsible for autoinducing peptide production and
quorum-sensing system. SA7372 was also enriched with different virulence factors related to immune
evasion, such as staphylokinase (sak) to spread the infection, an additional copy number of lukDV,
lukEv which produce neutrophil targeting leukotoxin, an additional lytN to coordinate synthesis of
the bacterial cell wall, and scn genes to inhibit opsonization and phagocytosis by the human immune
system [40].
Antimicrobial susceptibility testing of DFIs has mainly relied on culture and PCR-based methods
and is restricted to frequently isolated bacteria and commonly used antibioticS. Sample collection and
transport can also highly influence the result of antimicrobial susceptibility tests.
Identification of DFI resistome using metagenomic approaches can overcome these limitations
and provide a greater amount of information regarding all the genes involved in antibiotic resistance
(DFI resistome). Metagenomics can provide new chances for the investigation of novel resistance
mechanisms in DFUs/DFIs and accordingly the development of existing treatment approacheS. Recently
metagenomics of SA10757 strain isolated from DFUs showed the presence of different antibiotic
resistance genes, such as macrolide (ermA), tetracycline (tetA), and an aminoglycoside (ant1), sec2 and
sea, which may explain the increasing prevalence of multidrug-resistant organisms in DFUs [40].
Many aspects of pathogen detection in DFUs/DFIs have improved dramatically and are
summarized in recently published studieS. Table 1 lists commonly reported DFUs/DFIs microbiota
using different bacterial identification tools.
J. Clin. Med. 2019, 8, 1935 10 of 18

Table 1. Common bacterial species isolated from diabetic ulcers using different bacterial identification tools.
Culture
Specimen
Reference Specimen Predominant Aerobe Predominant Facultative Anaerobe/Anaerobe
No.
Slater, Lazarovitch S. aureus (50%), Coagulase-negative Staphylococci (38%), Enterococcus (20%), Streptococcus (25%), Proteus (23%), Escherichia coli (17%), Klebsiella (12%), Enterobacter (10%), Pseudomonas
60 Swab, biopsy
et al., 2004 [9] Diphtheroid spP. (10%), Acinetobacter (7%) (10%), Citrobacter (8%), Anaerobic cocci (13%), Anaerobic rods (3%), Bacteroides (3%)
Streptococcus spP. (15.5%), Staphylococcus spP. (15.3%), oxacillin-susceptible S. aureus
Citron, Goldstein Curettage, (14.3%), oxacillin-resistant S. aureus (4.4%), Coagulase negative, Gram-positive cocci (45.2%), Finegoldia magna (37.4), Prevotella spP. (13.6%), Porphyromonas spP. (11.3%),
454
et al., 2007 [23] Biopsy Enterococcus spP. (13.5%), Enterobacteriaceae family (12.8%), Bacteroides fragilis group (10.2%)
Corynebacterium spP. (10.1%), P. aeruginosa (3.5%)
Al Benwan, Al Mulla
440 Curettage P. aeruginosa (17.4%), S. aureus (11.8%), methicillin-resistant S. aureus (7.7%) Enterobacteriaceae (28.5%), anaerobic Gram-negative organisms (10.8%), Enterococcus spP. (7%)
et al., 2012 [21]
Olowu, Eyaufe et al.,
150 Swab S. aureus (38%), P. aeruginosa (8%) Escherichia coli (24%), Proteus spP. (20%), Klebsiella spP. (10%)
2013 [42]
Proteus mirabilis (12.6%), Klebsiella pneumoniae (11.2%), Escherichia coli (7.2%), Enterobacter cloacae (3.2%),
Djahmi, Messad Aspiration, S. aureus (30.7%), Coagulase-negative Staphylococcus (11.2%), P. aeruginosa (8.3%),
128 Enterococcus faecalis (2.5%), Proteus vulgaris (2.5%), Streptococcus spP. (0.7%), Providencia stuartii (0.7%),
et al., 2013 [43] Biopsy, Swab Morganella morganii (5.4%), Acinetobacter baumannii (2.9%)
Klebsiella oxytoca (0.4%), Citrobacter spP. (0.4%)
Enterococcus spP. (27%), Staphylococcus: CONs (22%), Staphylococcus: COPs (7%),
Olowu, Eyaufe et al., Escherichia coli (20%), Bacillus (3%), Proteus spP. (3%), P. aeruginosa (2%), Klebsiella pneumoniae (1%),
86 Swab, Biopsy Diphtheroid spP. (2%), Acinetobacter baumannii (1%), Acinetobacter lwoffii (1%),
2013 [42] β-hemolytic streptococci (1%), Serratia liquefaciens (1%), Enterobacter gergoviae (1%)
Morganella morganii (1%), S. maltophilia (1%)
S. aureus (20.67%), Pseudomonas (13.54%), Acinetobacter baumannii (5.24%), Escherichia coli (15.72%), Klebsiella pneumoniae (13.54%), Proteus mirabilis (12.81%), Proteus species (6.11%),
Miyan, Fawwad Bone, Pus,
342 Morganella morganii (1.75%), Coagulase-negative staphylococci (0.73%), Enterococus Proteus vulgaris (4.37%), Streptococcus species (1.89%), Enterobacter Species (1.75%),
et al., 2017 [24] Biopsy
species (0.44%) Citrobacter spP. (1.46%)
Culturomics
Specimen
Reference Specimen Predominant Aerobe Predominant Facultative Anaerobe/Anaerobe
No.
Jneid, Cassir et al.,
43 Swab, debris S. aureus (52.8%), Staphylococcus lugdunensis (18.7%), S. epidermidis (11.3%) Enterococcus faecalis (45.2%), Enterobacter cloacae (22.6%), Proteus mirabilis (11.3%), Finegoldia magna (9.4%)
2018 [20]
16S rRNA Sequencing
Specimen DNA Extraction Sequencing Read per
Reference Specimen Target Region Predominant Aerobe Predominant Facultative Anaerobe/Anaerobe
No. Method Platform Sample
The most commonly found
Bead-beating combined The most commonly found species:
Ge, MacDonald et al., species: Staphylococcus spp.,
39 Biopsy with Mo Bio V1–V4 Illumina MiSeq 31,452 Streptococcus spP. Anaerococcus spp., Finegoldia spp.,
2002 [44] Corynebacterium spp.,
PowerBiofilm kit Peptoniphilus spp.
Acinetobacter spP.
Bacteroides spp.:24.2%, Peptoniphilus spp.: 13.6%, Serratia spp.: 21.4%,
Corynebacterium spp.:14.4%,
Qiagen TissueLyser Finegoldia spp.: 6.7%, Anaerococcus spp.: 7.7%, Prevotella spp.: 7.4%,
Dowd, Wolcott et al., Streptococcus spp.: 36.5%,
40 Debridement combined with - bTEFAP - Peptostreptococcus spp.: 8.7%, Porphyromonas spp.:7%,
2008 Pseudomonas spp.: 14.5%,
QIAamp DNA Mini Kit Actinomyces spp.: 5.7%, Varibaculum spp.: 9%,
Staphylococcus spp.: 8.3%
Fusobacterium spp.:5.6%, Citrobacter spp.: 9.5%, Rothia spp.: 5.8%
Bead-beating combined
Gardner, Hillis et al., Roche 454 FLX S. aureus: 96.5%, S. epidermidis:
52 Swab with a QIAampDNA V1–V3 5634 Proteobacteria (9.8%), Bacteroidetes (7.3%), and Fusobacteria (1.4%)
2013 [33] Titanium 0.4%), Actinobacteria (14%)
Mini Kit
J. Clin. Med. 2019, 8, 1935 11 of 18

Table 1. Cont.
16S rRNA Sequencing
Specimen DNA Extraction Sequencing Read per
Reference Specimen Target Region Predominant Aerobe Predominant Facultative Anaerobe/Anaerobe
No. Method Platform Sample
New ulcers: Staphylococcus
Bead-beating combined
Smith, Collier et al., Illumina Hiseq (31.25%), Corynebacterium (25%) New ulcers: Peptoniphilus (37.5 %), Anaerococcus (31.25%)
16 Swab with a QIAamp DNA V4 110,447
2016 [31] 2500 Recurrent ulcers: Recurrent ulcers: Peptoniphilus (12%), Anaerococcus (25%)
Mini Kit
Corynebacterium (31.25%)
S. epidermidis (38%), S. aureus
(33%), S. haemolyticus (21%),
S. lugdunensis (18%),
Stenotrophomonas maltophilia Finegoldia magna (25%), Enterococcus faecalis (17%), Anaerococcus
Bead-beating combined
(16%), P. aeruginosa (14%), vaginalis (13%), Streptococcus agalactiae (10%), Enterobacter hormaechei
Wolcott, Hanson with High PurePCR
910 Biopsy V1–V3 Roche 454 - Corynebacterium (13%), (9%), Prevotella bivia (9%), Delftia acidovorans (5%), Serratia
et al., 2016 [35] Template Preparation
Corynebacterium striatum (12%), nematodiphila (5%), Proteus mirabilis (4%), Streptococcus salivarius,
Kit
Staphylococcus pettenkoferi (9%), Fusobacterium nucleatum, Bacteroides fragilis, Flavobacterium succinicans
Acinetobacter baumannii (5%),
Corynebacterium jeikeium (5%)
Ralstonia pickettii
Bead-beating combined
Gardiner, Vicaretti The most commonly found species: Staphylococcus, followed by Acinetobacter, Corynebacterium,
257 Swab, Biopsy with BioStic DNA V4 Illumina Miseq -
et al., 2017 [45] unclassified Enterobacteriacea.
extraction kit
Staphylococcus (22.77%,
Bead-beating combined S. aureus (13.3%), Staphylococcus
Loesche, Gardner
100 Swab with a QIAamp DNA V1–V3 Illumina Miseq 22,070 pettenkoferi (5.3%)), Anaerococcus (7%)
et al., 2017 [46]
Mini Kit Streptococcus (11.98%),
Corynebacterium (11.46%)
Metagenomicss
Specimen DNA Extraction Bacterial DNA Sequencing Read per
Reference Specimen Predominant Aerobe Predominant Facultative Anaerobe/Anaerobe
No. Method Enrichment Platform Sample
Staphylococcus (18.95%:
S. aureus, S. aureus 7372,
S. pettenkoferi, S. epidermidis,
S. simulans, S. lugdunensis),
Corynebacterium (14.64%,
NEBNext Corynebacterium striatum,
Bead-beating combined
Kalan, Meisel et al., Microbiome C. jeikeium, C. amycolatum,
100 Swab with a QIAamp DNA HiSeq 4000 144,416,914 Anaeroccocus, Porphyromonas, Prevotella, Veillonella spp.
2018 [40] DNA C. pseudogenitalium,
Mini Kit
Enrichment kit C. tuberculostearicum,
C. resistens), Pseudomonas
(9.37%, P. aeruginosa,
P. alcaliphila), Streptococcus
(7.32%, S. agalactiae,
S. dysgalactiae, S. anginosus)
J. Clin. Med. 2019, 8, 1935 12 of 18

5. DFUs/DFIs Enter the Metatranscriptomic Era

While bacterial pathogens destroy human cells by cellular pathway manipulation for multiplication
and survival, human cells respond to these pathogens through cascade changes in the innate and
humoral immune system. Inconstancy of wound microbiota and bacterial transition patterns over
time is thought to play an important role in the healing process of DFUs/DFIs [47].
RNA sequencing has emerged as an accurate technology to study metabolically active microbiota
and the host-microbes transcriptome, simultaneously avoiding many drawbacks of microarrays, such
as cross-hybridization, background noise, and restriction of gene identification [48].
Different types of RNA such as coding (mRNA) and non-coding RNA (microRNAs, siRNA,
snoRNAs) also can be analyzed by metatranscriptomic approacheS. Transcriptional responses of wounds
to P. aeruginosa infections showed that 2 h after bacterial infection, wounds had down-regulation of
ncRNAs (snoRNA, miRNA, and RNU6 pseudogenes), while 6 and 24 h after infection, wounds
had down-regulation of protein-coding genes with an overrepresentation of ncRNAs prior to
down-regulation of skin-enriched coding gene expression in the host. These findings suggest
that regulation of different classes of ncRNAs follow a consecutive and harmonized pattern during
transition state from inflammation to the healing process [48].
Furthermore, possible correlations between the expression of different virulence genes in S. aureus
isolated from DFUs have also been identified [49]. In this regard, scientists have used oligonucleotide
microarrays designed for specific encoding genes in the S. aureus genome to identify the bacterial
genomic profile in uninfected and infected diabetic foot ulcerS. A comparison of S. aureus expressed
virulence genes in infected and uninfected ulcers showed that toxic shock syndrome toxin (tsst),
leukocidins (lukF, lukS, lukS-PV, and lukF-PV), exfoliatins (etA, etB), and enterotoxins (sea, seb, sec, sek,
seq) were not identified in uninfected ulcers (grade 1), but are mainly expressed in infected ulcers
(grade 2–4). Additionally, a Panton-Valentine leukocidine gene (encoding β-pore-forming toxin) was
present in deep ulcers (Grade 4). In a similar study, the expression profile of uninfected ulcers (grade 1)
and infected ulcers (grade 2–4) was examined using conventional PCR of cap8, sea, sei, lukE, and hlgv
geneS. Isolated strains from infected ulcers were found to remarkably be more virulent than strains
from uninfected ulcerS. A combination of these five virulence genes may help differentiate infected
ulcers from non-infected ulcers and to anticipate wound status at the follow-up [50].
This information indicates the importance of gene expression profiling during infection progression,
which can improve our understanding of disease development, as well as prognosis and development
of therapeutical approacheS. Shotgun metagenomic sequencing and metatranscriptomics can predict
the relative abundance of DFUs/DFIs microbiota, which can express a particular virulence gene based
on infection severity. As this topic is still in its infancy, further investigations are warranted to evaluate
if screening of wound microbial activity of specific wound bacterial species and metabolic gene
expression can determine if a DFU will become infected. By using molecular analysis, more bacterial
communities are predicted to be isolated from DFUs/DFIs, particularly from deep infection ulcers,
which are an optimal place for the growth of anaerobeS. Due to the excellent performance of molecular
methods in recent years, it is likely that phenotypical methods will be replaced with DNA-based
methods as frontline diagnostic tests in the future. New findings based on molecular technologies can
improve traditional therapies and replace them with more evidence-based therapy.

6. Treatment of DFIs
While antibiotic treatment for DFIs is initially prescribed empirically, accurate bacterial
identification of DFIs can improve therapeutic approacheS. The selection of the most effective antibiotic
is a vital step to reduce the treatment period, prevent the expansion of resistant bacterial strains,
and limit health costs [51]. Methicillin-resistant S. aureus (MRSA) is a common bacterial pathogen
in DFIs and is a difficult-to-treat infection. Compared to non-MRSA infections, diabetic people with
MRSA infections have a greater than fivefold increase in mortality [52]. The susceptibility pattern of
J. Clin. Med. 2019, 8, 1935 13 of 18

Staphylococcus spP. and Gram-negative bacteria should regularly be monitored in DFIs, while other
organisms may be analyzed selectively [53].
In the following section, wounds have been classified based on the Infectious Disease Society of
America system (IDSA).

6.1. Non-Infected Wounds

To prevent possible infection and improve the wound-healing process, different classes of
antibiotics are being prescribed by clinicians, even for uninfected woundS. There is, however,
no consensus to support this practice, which may also increase treatment-related adverse events
(TRAEs) and the expansion of resistant bacteria. The European Wound Management Association has
prohibited antibiotic prescription for uninfected wounds [54].

6.2. Mild Infections

Mild infections are characterized by inflamed subcutaneous tissue, which may vary in size but
are found less than 2 cm from the surrounding of the ulcer. Gram-positive cocci, S. aureus, and
group B streptococci have been frequently isolated in the mild stage. Hence, antibiotics that target
Gram-positive bacteria may be a good choice for this stage of infection. Mild infections can be healed
using oral antibiotics (quinolones, trimethoprim-sulfamethoxazole, and clindamycin). Based on the
IDSA guideline, mild infections require a shorter course of antibiotic treatment (up to two weeks),
compared to moderate and severe infections [55].

6.3. Moderate to Severe Infections

A heterogeneous mixture of aerobic Gram-positive cocci, aerobic Gram-negative Bacilli, and
anaerobic pathogens can be isolated from moderate to severe stages of infection [56]. Based on the
IDSA guideline, moderate to severe infections need up to four weeks of antibiotic therapy [55]. Narrow
spectrum oral antibiotics and broad-spectrum parenteral antibiotics are administered for mild and
severe infections, respectively. In either case, the presence of Staphylococcus and Streptococcus species
should always be considered [51]. Cotrimoxazole, tetracyclines, rifampin, linezolid, fluoroquinolones,
and clindamycin have a proper absorption rate and are effective for systemic circulation when
administered orally for moderate to severe infections [57].

6.4. Natural Antimicrobial Peptides

Due to the high incidence of multidrug-resistant bacterial strains, traditional antibiotics have
become less effective against a broad range of bacteria. Natural antimicrobial peptides (AMPs) or host
defense peptides (HDPs), which are derived from host innate immune responses, have broad-spectrum
activity against Gram-positive and Gram-negative bacteria. Based on previous reports, antimicrobial
proteins isolated from the small intestine of rabbits were found to display antimicrobial activity against
S. aureus, P. aeruginosa, E. coli, and C. albicans [58].

6.5. Bacteriophages
Conventional antibiotic therapies are less useful for MRSA infectionS. Bacteriophage therapy is a
novel and potential alternate therapy to overcome MRSA infectionS. It is recommended to combine
the bacteriophage and antibiotic therapies to increase the efficiency of MRSA treatment [59]. Due to
the complexity of bacterial community colonized in DFIs, a mixture of phages may require to target a
wider range of the bacterial population. Anti-Staphylococcus phages have a broad range of activity;
hence, two or three different phages are required to target different strains of S. aureus. In contrast,
Gram-negative bacteria require a higher range of phages (>10) [60].
J. Clin. Med. 2019, 8, 1935 14 of 18

6.6. Negative-Pressure Wound Therapy

Negative-pressure wound therapy (NPWT) is a vacuum sealing method to control wound swelling
and stimulate the formation of granulation tissue to speed up the wound healing process [61]. Based on
a study conducted on 342 patients, wounds treated with NPWT healed in a shorter period of time,
compared to traditional dressings [62]. NPWT can also be combined with the installation of effective
antibacterial agentS. It has been reported that patients with NPTW combined with antiseptic installation
experienced shorter hospitalization, compared to NPTW without antiseptic installation [63]. However,
due to insufficient published data in this regard, the productivity of this method has not been well
documented yet.

6.7. Hyperbaric Oxygen Therapy

Hyperbaric oxygen therapy (HBOT) is the exposure of diabetic ulcers to a high concentration of
oxygen in an adjustable atmospheric pressure. Based on previous findings, HBOT could decrease lower
extremity amputation rate in patients with diabetic foot ulcers [64]. There is, however, no published
data available to evaluate the effect of hyperbaric oxygen therapy on bone or tissue infections.

6.8. Stem Cell Therapy

Embryonic stem cells can transform into known differentiated cell types in the human body.
Stem cell therapy can decrease the chance of amputation in diabetic people with ischaemic diabetic
foot ulcers [65,66].

6.9. Off-Loading
Off-loading is primarily to keep the pressure off the affected area by using special boots, casts,
or shoes to help the foot ulcers heal as quickly as possible. It also can reduce the risk of severe
complications [57].

7. New Insights into DFIs Treatment Based on Molecular Findings

Despite many advances in the microbiology field, bacterial antibiotic resistance is still a major
challenge, and little is known about the region, distribution, and diversity of resistance genes
particularly for uncultivable bacteria, which constitute a large portion of the bacterial population
and may play significant roles in infection progression. Several novel antibiotic resistance genes,
including bleomycin, aminoglycosides, tetracycline, and β-lactam, have been identified using DNA
sequence-based approaches [67]. However, DNA-based techniques only suggest the presence of a
gene that may encode enzymes involved in resistant bacteria, but it does not necessarily confirm
that the gene is functionally expressed or not. Metatranscriptomics can help us to reach this level
of understanding and more important to study the host-pathogen co-transcriptome profile [47].
The study of slow-growing, fastidious, anaerobic, and unknown pathogens, which normally have
been underestimated by culture-based methods will provide an early warning system necessary for
modification and alteration of antibiotic therapy. The useful information from shotgun metagenomic
sequencing and metatranscriptomic analysis can help researchers to anticipate the bacterial resistance
profile before it emerges clinically. So far, our knowledge is limited to culturable bacteria and few
studies conducted on 16S rRNA sequencing analysis on DFUs/DFIS. We need to apply new molecular
approaches to track the transcriptional program involved in bacterial survival and how pathogens can
subvert antimicrobial strategies, not only in culturable bacteria, but also in uncultivable microorganisms
in DFUs/DFIs.

8. Conclusions
Continuous evaluation of the feet, proper use of antibiotics, surgical procedures, and multifaceted
approaches emphasizing better diagnostic methods can prevent infection progression, and, more
J. Clin. Med. 2019, 8, 1935 15 of 18

importantly, the risk of lower extremity amputation. Researchers and clinicians should be up-to-date
and have an understanding of new methods of prevention, diagnosis, and treatment of DFIs.
There have been many studies on the bacteriology of DFUs/DFIs over the past decades with
varying, and sometimes inconsistent resultS. These discrepancies might be due to demographical and
geographical differences, various processes of sampling, human errors, sample size, and different
bacterial identification methods used.
Even though significant advances have been made to manage DFIs, many unanswered questions
about DFUs/DFIs microbiota exist. These questions will require help from new and advanced molecular
technologieS. A diverse range of studies has successfully evaluated transcriptional pathways involved
in intramacrophage survival [68] and alteration of the bacterial transcriptomic profile in adaptation to
human cells [69]. However, the host inflammatory responses and major bacterial metabolisms involved
in DFIs have not been profiled yet. The output of dual meta-transcriptomic analysis or profiling of
dynamic host-pathogen interactions offer strong prospects for further research on DFUs/DFIs.
It may be concluded that molecular approaches are more reliable than traditional methods in the
study of DFUs/DFIs microbiota and can provide greater insights into DFI microbiology. However, due
to the paucity of information, more investigation is needed to decide which method should be chosen
as the primary identification tool.

Author Contributions: F.S.H. designed and wrote the first draft of the study. M.Z., K.V., D.G.A., H.H. reviewed
and commented on the manuscript. All authors have read and approved the final manuscript.
Funding: The authors have not received any funding or benefits from the industry to conduct this study.
Conflicts of Interest: The authors declare no conflict of interest.

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