737640

research-article2017
IJLXXX10.1177/1534734617737640The International Journal of Lower Extremity WoundsDemetriou et al

Clinical and Translational Research

The International Journal of Lower

Antibiotic Resistance in Diabetic Foot Soft

Extremity Wounds
15
The Author(s) 2017
Tissue Infections: A Series From Greece Reprints and permissions:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/1534734617737640
https://doi.org/10.1177/1534734617737640
journals.sagepub.com/home/ijl

Maria Demetriou, MD1, Nikolaos Papanas, MD1, Periklis Panagopoulos, MD1,

Maria Panopoulou, MD1, and Efstratios Maltezos, MD1

Abstract
Diabetic foot infections are a common and serious problem for all health systems worldwide. The aim of this study was
to examine the resistance to antibiotics of microorganisms isolated from infected soft tissues of diabetic foot ulcers,
using tissue cultures. We included 113 consecutive patients (70 men, 43 women) with a mean age of 66.4 11.2 years
and a mean diabetes duration of 14.4 7.6 years presenting with diabetic foot soft tissue infections. Generally, no high
antibiotic resistance was observed. Piperacillin-tazobactam exhibited the lowest resistance in Pseudomonas, as well as in
the other Gram-negative pathogens. In methicillin-resistant Staphylococcus aureus isolates, there was no resistance to anti-
Staphylococcus agents. Of note, clindamycin, erythromycin, and amoxycillin/clavulanic acid exhibited high resistance in
Gram-positive cocci. These results suggest that antibiotic resistance in infected diabetic foot ulcers in our area is not high
and they are anticipated to prove potentially useful in the initial choice of antibiotic regimen.

Keywords
antibiotics, cultures, diabetic foot, infection, resistance

During the past years, there has been some progress in
infection control and wound healing in the diabetic foot.1,2
However, diabetic foot ulcers remain difficult to heal, easy
to deteriorate and, worse perhaps, prone to recur.3 All these
aspects call for more urgent management and/or more
urgent referral to specialist multidisciplinary teams in
patients with rapid deterioration.4 Diagnosis and aggressive
management of infection, which may include surgery,5,6 is a
vital part of treatment in such patients.6-8
The situation is far more complicated in the event of con-
comitant ischaemia.9-11 Antibiotic use is generally governed
by expert guidelines.12,13 Tissue or other specimen cultures
are helpful in guiding specific antibiotic choices.7,12
However, it is also useful to know the resistance of underly-
ing pathogens to antibiotics in the hospital where the patient
is treated or in the geographical area.12 In Greece, little is
known in this field.14 Therefore, the aim of this study was to
examine the resistance to antibiotics of microorganisms iso-
lated from soft tissue infections of diabetic foot ulcers.
Patients and Methods
The present study included 113 consecutive patients pre-
senting to our Diabetic Foot Clinic with infected diabetic
foot ulcer between January 1, 2012 and December 31, 2015.
The study was approved by the institutional ethics commit-
tee and all patients gave their informed consent.
A foot ulcer was defined as a wound located beneath the
malleoli and penetrating through all skin layers.7 Infection
was diagnosed clinically, based on the presence of 2 of the
following criteria: local swelling or induration; erythema
greater than 0.5 cm around the ulcer; local tenderness or
pain; local increase of temperature; and purulent discharge,
as described previously.7 Severity of infection was graded
according to the PEDIS classification system of the
International Working Group of the Diabetic Foot, as
already described.7 Patients with osteomyelitis (positive
probe-to-bone test and/or findings on magnetic resonance
imaging) were excluded. Osteomyelitis was an exclusion
criterion, because in such patients, bone and not tissue cul-
tures are the reference method, which would render our
results less reliable.7,15
All patients had not received antibiotics for the preceding
7 days. After debridement, tissue cultures were obtained by
4 mm biopsy punches (Kai Europe GmbH, Solingen,
Germany), as previously described.7 Specimens were placed
in sterile transport containers and sent to the Microbiology
Laboratory within 20 minutes.
1
Democritus University of Thrace, Alexandroupolis, Greece
Corresponding Author:
Maria Demetriou, G. Lambraki 138, Piraeus 18535, Greece.
Email: maria_thdemetriou@yahoo.gr
2 The International Journal of Lower Extremity Wounds 00(0)

Table 1. Antibiotic Resistance Among Gram-Negative Isolates (7 Proteus, 12 Klebsiella, 11 Pseudomonas, 8 Escherichia coli,
9 Enterobacter spp, 16 Other Gram-Negatives).

Resistance to Antibioticsa

Other Gram-
Antibiotic Proteus Klebsiella Pseudomonas Escherichia coli Enterobacter spp Negatives
Amoxycillin + clavulanic acid 3 (42.8) 3 (25) 2 (25) 9 (100)
Cefuroxime 3 (42.8) 3 (25) 4 (50)
Piperacillin + tazobactam 1 (14.3) 0 (0) 0 (0) 0 (0) 0 (0) 1 (6.2)
Ceftazidime 1 (14.3) 1 (8.3) 1 (9.1) 0 (0) 0 (0) 2 (12.5)
Ertapenem 0 (0) 0 (0) 0 (0) 1 (11.1) 1 (6.2)
Levofloxacin 2 (28.6) 1 (8.3) 8 (72.7) 2 (25) 1 (11.1) 3 (18.7)
Moxifloxacin 2 (28.6) 1 (8.3) 8 (72.7) 2 (25) 1 (11.1) 3 (18.7)
Ciprofloxacin 0 (0) 1 (8.3) 2 (18.2) 0 (0) 0 (0) 1 (6.2)
Imipenem 2 (28.6) 0 (0) 1 (9.1) 0 (0) 0 (0) 2 (12.5)
Meropenem 1 (14.3) 0 (0) 1 (9.1) 0 (0) 0 (0) 2 (12.5)
Cotrimoxazole 5 (71.4) 2 (16.7) 10 (90.9) 2 (25) 1 (11.1) 3 (18.7)
Tigecycline 3 (42.8) 0 (0) 2 (12.5)
Colistine 1 (9.1)
a
Data are presented as absolute numbers with percentage within parentheses.

Tissue cultures were performed using standard proce-
dures, as previously described.7 After having been weighed,
homogenized, and diluted with 5 mL of thioglycolate broth,
specimens underwent serial dilutions. Appropriate media
for aerobic and anaerobic cultures were used in blood agar
and McConkey agar plates. Species identification was car-
ried by the automated system Vitek 2 and the Api 20A
(BioMerieux, Marcy lEtoile, France). Evaluation of antibi-
otic resistance was carried out by Kirby-Bauer, E-test (AB
Biodisk) and automated Vitek 2 system.16,17
Results
Overall, included were 70 men, 43 women. Patients mean
age was 66.4 11.2 years and mean diabetes duration was
14.4 7.6 years. Type 2 diabetes was diagnosed in 110
patients and type 1 diabetes in 3 patients. PEDIS infection
severity grade was 2 in 71 patients, 3 in 38 patients, and 4 in
4 patients.
Among the 113 patients, 24 had negative tissue cul-
tures. Among the positive cultures, isolates were Gram-
negatives and Gram-positives. The former included 7
Proteus, 12 Klebsiella, 11 Pseudomonas, 8 Escherichia
coli, 9 Enterobacter spp, and 16 other Gram-negatives.
The latter included 9 methicillin-resistant Staphylococcus
aureus (MRSA), 15 methicillin-sensitive Staphylococcus
aureus (MSSA), 10 other Staphylococci, 6 Streptococci,
and 8 Enterococci. Among 24 patients with Staph aureus,
this agent was isolated together with Gram-negatives in 5
patients.
Antibiotic resistance among Gram-negative isolates is
presented in Table 1. In Proteus isolates, resistance was
14.3% to piperacillin-tazobactam, 14.3% to ceftazidime,
0% to ertapenem, 14.3% to meropenem, and 28.6% to levo-
floxacin. In Klebsiella isolates, resistance was 0% to piper-
acillin-tazobactam, 0% to ertapenem, 8.3% to ceftazidime,
and 8.3% to ciprofloxacin. In Escherichia coli isolates,
resistance was 0% to piperacillin-tazobactam, ceftazidime,
ertapenem, tigecycline, and ciprofloxacin, while it was 25%
to levofloxacin. In Enterobacter spp isolates, resistance was
0% to piperacillin-tazobactam, ceftazidime, ciprofloxacin
and imipenem, while it was 11.1% to levofloxacin.
Antibiotic resistance among Gram-positive isolates is
presented in Table 2. In MRSA isolates, resistance was 0%
to vancomycin, teicoplanin, linezolide, and tigecycline,
while it was 11.1% to rifampicin and 22.2% to levofloxacin.
In MSSA isolates, resistance was 0% to vancomycin, teico-
planin, linezolide, and tigecycline, while it was 46.7% to
amoxicillinclavulanic acid and 73.3% to clindamycin. In
Enterococcus isolates, resistance was 0% to vancomycin,
teicoplanin, linezolide, and tigecycline.
In general, resistance to antibiotics was not high, and the
vast majority of patients were treated as ambulatory.
Discussion
This study has looked at antibiotic resistance of isolates
from diabetic foot infections. Generally, no high resistance
was observed. Of note, in Pseudomonas isolates, resistance
was 0% to piperacillin-tazobactam, 9.1% to ceftazidime
and 18.2% to ciprofloxacin. In contrast with the earlier
study by Shankar etal18 (which was carried out in a differ-
ent continent and was based on cultures of pus aspiration or
swabs), we found no isolates with resistance to genuine
Demetriou et al 3

Table 2. Antibiotic Resistance Among Gram-Positive Isolates (9 MRSA, 15 MSSA, 10 Other Staphylococci, 6 Streptococci, and
8 Enterococci).

Resistance to Antibioticsa

Other
Antibiotic MRSA MSSA Staphylococci Streptococci Enterococci
Amoxycillin + clavulanic acid 7 (77.8) 7 (46.7) 2 (20) 8 (100)
Vancomycin 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Levofloxacin 2 (22.2) 0 (0) 1 (10) 0 (0) 6 (75)
Moxifloxacin 2 (22.2) 0 (0) 1 (10) 0 (0) 6 (75)
Ciprofloxacin 6 (66.6) 3 (20) 4 (40) 2 (33.3) 4 (50)
Teicoplanin 0 (0) 0 (0) 0 (0) 0 (0)
Rifampicin 1 (11.1) 3 (20) 0 (0)
Cotrimoxazole 2 (22.2) 1 (6.7) 4 (40) 1 (16.7)
Linezolide 0 (0) 0 (0) 0 (0) 0 (0)
Tigecycline 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Clindamycin 8 (88.9) 11 (73.3) 3 (30) 5 (83.3) 8 (100)
Erythromycin 8 (88.9) 11 (73.3) 3 (50)

Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus.

a
Data are presented as absolute numbers with percentage within parentheses.

anti-Pseudomonas agents. Other studies19-23 concur with
our own finding of low resistance to anti-Pseudomonas
agents, but again the majority of cultures in those studies
was pus aspiration or swab. Resistance was not high in
Proteus, E coli, Enterobacter spp, MRSA, MSSA,
Enterococcus, non-aureus Staphylococci, Streptococci, and
other Gram-negative isolates, either.
These results, which, to the best of our knowledge,
appear to be the first systematic presentation based on tissue
culture in Greece, suggest that piperacillin-tazobactam
exhibits the lowest resistance in Pseudomonas as well as in
the other Gram-negative pathogens. Ceftazidime, cipro-
floxacin and carbapenems also exhibited low resistance in
Gram-negative isolates. The low resistance in Gram-
negative isolates from diabetic foot infections agrees with
other studies as well.18-22
In MRSA isolates, there was no resistance to anti-Staph-
ylococcus agents, in line with Shankar etal,18 Abdulrazak
etal,20 Bravo-Molina etal,21 and Hatipoglou etal.22,23 This
is important, given the concern about the increasing preva-
lence of MRSA in the community and in diabetic foot infec-
tions, with the potential to turn into a therapeutic challenge.8
In MSSA and Streptococci, rifampicin and cotrimoxazole
exhibited low resistance, whereas clindamycin and erythro-
mycin exhibited very high resistance. The high resistance
rates of clindamycin, erythromycin and amoxicillin/clavu-
lanic acid is probably attributable to the hitherto very wide-
spread prescription of these agents in Greece. Naturally,
resistance rates vary between studies. Bravo-Molina etal21
have reported excellent efficacy of rifampicin in MRSA and
MSSA. Hatipoglou etal22,23 have reported adequate effi-
cacy of cotrimoxazole in Staph aureus and Streptococcus
isolates. At variance with our findings, Bravo-Molina etal21
and Hatipoglou etal22 found no high resistance to clindamy-
cin in Gram-positive cocci. Similarly, Hatipoglou etal22
found lower then ourselves resistance to erythromycin in
MSSA and Streptococci.
In our study, levofoxacin was very efficacious in MSSA
and Streptococci, while there was moderate resistance to
this agent in MRSA, E coli, and Enterobacter spp isolates
and very low resistance in Klebsiella isolates. Citron etal19
have reported 27% resistance to levofloxacin in Gram-
positive isolates and 6% in Enterobacteriacae.
It is anticipated that the present findings may prove clini-
cally useful in terms of the initial choice of antibiotic regi-
men in infected diabetic foot ulcers in our area. Certainly, it
should not be underestimated that clinical experience and
individual patient evaluation are needed when choosing the
initial antibiotics, given that knowledge on the underlying
pathogens cannot be based on the clinical presentation
per se.10-13,24 In this context, an additional evidence of com-
plexity is that some patients with clinically infected diabetic
foot ulcer exhibited negative tissue cultures: this has already
been highlighted as a reason for the clinician to be prudent,
continue efficacious treatment and, perhaps, even obtain
further tissue specimens.7 At the end of the day, further
experience in the susceptibility and resistance to antibiotics
in Greece is highly welcome, inasmuch as the diabetic foot
remains in 2017 a common3 and highly expensive25 compli-
cation of diabetes.
The strength of this study is the use of tissue and not
swab culture (which is less reliable).7 Moreover, the num-
ber of patients was not small. A limitation is the exclusion
of patients with osteomyelitis, which is relevant for clinical
4 The International Journal of Lower Extremity Wounds 00(0)
practice, but bone culture would have been properly
required.7,15 Moreover, determination of antibiotic resis-
tance required subgroup analysis based on types of isolates,
and this led to small subgroups. A final limitation relates to
the role of ulcer duration. This merits further study, but a
larger prospective study beyond the scope of this work
would have been required.
In conclusion, no high antibiotic resistance was, gener-
ally, observed. Piperacillin-tazobactam exhibited the low-
est resistance in Gram-negative pathogens. In MRSA
isolates, there was no resistance to anti-Staphylococcus
agents. Importantly, however, clindamycin, erythromycin,
and amoxycillinclavulanic acid exhibited high resistance
in Gram-positive cocci. It is anticipated that these results
may prove useful in the initial choice of antibiotic regimen
in our area.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of inter-
est with respect to the research, authorship, and/or publication of
this article: Dr N. Papanas has been an advisory board member
of TrigoCare International; has participated in sponsored studies
by Novo Nordisk and Novartis; has received honoraria as a
speaker for Astra-Zeneca, Eli-Lilly, Novo Nordisk, and Pfizer;
and has attended conferences sponsored by TrigoCare
International, Novo Nordisk, Sanofi-Aventis, and Pfizer. Dr P.
Panagopoulos has been an advisory board member of GS and
MSD, has received honoraria by ABBVIE, GS, and attended
conferences sponsored by Actelion, Janssen, BMS, and MSD.
Professor E. Maltezos has participated in sponsored studies by
Novo Nordisk and Novartis and attended conferences sponsored
by Wyeth, Pfizer, and Bayer.
Funding
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
References
1. Papanas N, Mani R. Advances in infections and wound heal-
ing for the diabetic foot: the die is cast. Int J Low Extrem
Wounds. 2013;12:83-86.
2. Papanas N, Demetzos C, Pippa N, Maltezos E, Tentolouris
N. Efficacy of a new heparan sulfate mimetic dressing in
the healing of foot and lower extremity ulcerations in type 2
diabetes: a case series. Int J Low Extrem Wounds. 2016;15:
63-67.
3. Armstrong DG, Boulton AJM, Bus SA. Diabetic foot ulcers
and their recurrence. N Engl J Med. 2017;376:2367-2375.
4. Papanas N, Mani R. How to cope with the increasing burden
of the diabetic foot: better three hours too soon than a minute
too late. Int J Low Extrem Wounds. 2014;13:171-172.
5. Panagopoulos P, Drosos G, Maltezos E, Papanas N. Local
antibiotic delivery systems in diabetic foot osteomyelitis:
time for one step beyond? Int J Low Extrem Wounds. 2015;14:
87-91.
6. Aragn-Snchez J. Seminar review: a review of the basis
of surgical treatment of diabetic foot infections. Int J Low
Extrem Wounds. 2011;10:33-65.
7. Demetriou M, Papanas N, Panopoulou M, Papatheodorou K,
Bounovas A, Maltezos E. Tissue and swab culture in diabetic
foot infections: neuropathic versus neuroischemic ulcers. Int
J Low Extrem Wounds. 2013;12:87-93.
8. Eleftheriadou I, Tentolouris N, Argiana V, Jude E, Boulton
AJ. Methicillin-resistant Staphylococcus aureus in diabetic
foot infections. Drugs. 2010;70:1785-1797.
9. Edmonds M. Double trouble: infection and ischemia in the
diabetic foot. Int J Low Extrem Wounds. 2009;8:62-63.
10. Manu CA, Mustafa OG, Bates M, etal. Transformation of the
multidisciplinary diabetic foot clinic into a multidisciplinary
diabetic foot day unit: results from a service evaluation. Int J
Low Extrem Wounds. 2014;13:173-179.
11. Aragn-Snchez J, Lzaro-Martnez JL, Hernndez-Herrero
C, etal. Surgical treatment of limb- and life-threatening
infections in the feet of patients with diabetes and at least one
palpable pedal pulse: successes and lessons learnt. Int J Low
Extrem Wounds. 2011;10:207-213.
12. Lipsky BA, Berendt AR, Cornia PB, etal. 2012 Infectious
Diseases Society of America clinical practice guideline for
the diagnosis and treatment of diabetic foot infections. Clin
Infect Dis. 2012;54:e132-e173.
13. Dumville JC, Lipsky BA, Hoey C, Cruciani M, Fiscon M,
Xia J. Topical antimicrobial agents for treating foot ulcers
in people with diabetes. Cochrane Database Syst Rev.
2017;6:CD011038.
14. Papanas N, Lazarides MK. Diabetic foot amputations in
Greece: where do we go from here? Int J Low Extrem Wounds.
2011;10:4-5.
15. Markanday A. Diagnosing diabetic foot osteomyelitis:
narrative review and a suggested 2-step score-based diag-
nostic pathway for clinicians. Open Forum Infect Dis.
2014;1:ofu060.
16. Miller JM, Holmes HT, Krisher K. General principles of
specimen collection and handling. In: Murray PR, Baron
EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. Manual of
Clinical Microbiology. 8th ed. Washington, DC: ASM Press;
2003:55-66.
17. OHara CM, Weinstein MP, Miller JM. Manual and auto-
mated systems for detection and identification of microorgan-
isms. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA,
Yolken RH, eds. Manual of Clinical Microbiology. 8th ed.
Washington, DC: ASM Press; 2003:185-207.
18. Shankar EM, Mohan V, Premalatha G, Srinivasan RS, Usha
AR. Bacterial etiology of diabetic foot infections in South
India. Eur J Intern Med. 2005;16:567-570.
19. Citron DM, Goldstein EJ, Merriam CV, Lipsky BA,
Abramson MA. Bacteriology of moderate-to-severe diabetic
foot infections and in vitro activity of antimicrobial agents. J
Clin Microbiol. 2007;45:2819-2828.
20. Abdulrazak A, Bitar ZI, Al-Shamali AA, Mobasher LA.
Bacteriological study of diabetic foot infections. J Diabetes
Complications. 2005;19:138-141.
21. Bravo-Molina A, Linares-Palomino JP, Lozano-Alonso S,
Asensio-Garca R, Ros-De E, Hernndez-Quero J. Influence
Demetriou et al 5
of wound scores and microbiology on the outcome of the
diabetic foot syndrome. J Diabetes Complications. 2016;30:
329-334.
22. Hatipoglu M, Mutluoglu M, Uzun G, Karabacak E, Turhan V,
Lipsky BA. The microbiologic profile of diabetic foot infections
in Turkey: a 20-year systematic review: diabetic foot infections
in Turkey. Eur J Clin Microbiol Infect Dis. 2014;33:871-878.
23. Hatipoglu M, Mutluoglu M, Turhan V, etal. Causative patho-
gens and antibiotic resistance in diabetic foot infections: a
prospective multi-center study. J Diabetes Complications.
2016;30:910-916.
24. Grigoropoulou P, Eleftheriadou I, Jude EB, Tentolouris N.
Diabetic foot infections: an update in diagnosis and manage-
ment. Curr Diab Rep. 2017;17:3.
25. Petrakis I, Kyriopoulos IJ, Ginis A, Athanasakis K. Losing a
foot versus losing a dollar; a systematic review of cost stud-
ies in diabetic foot complications. Expert Rev Pharmacoecon
Outcomes Res. 2017;17:165-180.