Review Article
Submitted: 20.10.2014
Accepted: 20.12.2014
Conflict of interest
None.
Andreas Schwarzkopf 1,
Joachim Dissemond2
(1) Institut Schwarzkopf, Aura an der
Saale, Germany
(2) Department of Dermatology, Essen
University Hospital, Germany
Introduction
Bacteriologic swabs are frequently performed in patients with
chronic wounds, in order to rule out colonization with mul-
tiresistant pathogens (MRPs), such as methicillin-resistant
Staphylococcus aureus (MRSA) and multiresistant gram-ne-
gative bacteria (MRGN), as well as to identify any causati-
ve pathogens in wound infections. In a preventive manner,
swabs may also allow for calculated antibiotic therapy in
immunosuppressed wound patients. Currently, there are no
uniform standards with respect to the practical implementa-
tion of bacteriologic swabs. Among others, various sampling
techniques are being used and/or wounds are partially clean-
sed prior to swabbing [1, 2].
2015 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2015/1303
DOI: 10.1111/ddg.12611
Indications and practical
implementation of microbiologic
diagnostics in patients with
chronic wounds
Summary
Microbiology diagnostics are frequently performed in patients with chronic wounds.
However, there is currently a lack of uniformity with respect to indications as well as
the practical implementation of such workup. The fact that diagnostic results may
be significantly affected by the sampling technique used as well as the preceding
(wound) preparation underscores the need for uniform standards, which have been
missing so far.
In Germany, bacteriologic wound swabs are routinely performed, particularly with
the intent to screen for multiresistant pathogens. For this indication, prior wound cle-
ansing should be avoided, and sampling using the Essen Rotary technique provides
a quick and easy-to-use option. If there is clinical suspicion of an infection, wound
cleansing with sterile saline solution (0.9 %) and/or sterile cotton gauze should be
carried out prior to obtaining bacteriologic swabs. While routine diagnostic biopsies
are generally not required in chronic wound patients, they may be useful in case of
clinically suspected wound infections, particularly in patients with deep ulcerations,
diabetic foot syndrome, severe soft tissue infection, or fistula tissue. Moreover, biop-
sies are indispensable in the microbiology workup of specific pathogens such as my-
cobacteria, Leishmania, actinomycetes, Nocardia ssp. or molds.
The fact that test results may be significantly influenced
by the sample technique used as well as the preceding (wound)
preparation underscores the need for uniform standards for
bacteriologic swabs. The present review takes into account
current scientific knowledge, while presenting expert opinion
and recommendations based on applicable legal regulations.
Legal situation
For any given diagnostic lab test, specimen preparation is pa-
ramount. This includes selecting an appropriate container,
patient identification, obtaining the specimen using a defined
technique, filling out the lab request form as well as storage
and transport indicating the respective time and temperature.
203
 Review Article Microbiological diagnostics in chronic wounds
These components are collectively referred to as preanaly-
sis. In keeping with current guidelines on laboratory tests
(Rili-BK) issued by the German Medical Association, the
physician who orders the respective test is also responsible
for any preanalytic steps. Only after the specimen has rea-
ched the laboratory is the responsibility for the sample and its
correct processing (for example, choice and quality of culture
media, analysis and identification of bacteria, reporting with
differentiation and semi-quantitative amounts, antibiogram/
resistogram) transferred to the lab. The Rili-BK has legal
character and also describes various quality controls appli-
cable to laboratories.
There are no legally binding regulations on performing
microbiologic diagnostics. Recommendations by professio-
nal societies, such as guidelines published by the Association
of the Scientific Medical Societies in Germany (AWMF), and
quality recommendations put forth by the German Society
for Hygiene and Microbiology (DGHM), which primarily
apply to microbiology laboratories, may be used for reference
[3, 4].
In case of a legal dispute, it is the medical experts job
to determine whether the preanalysis and the spectrum of
ordered tests were appropriate to meet the objective, namely,
detection of a given infectious pathogen. This also includes
quickly relaying test results to treating physicians. Yet, hospi-
tals continue to have deficits in this field, for example, when
initial samples are taken at the outpatient department and
the patient is then admitted to the hospital; or, when a patient
has been discharged, and results are not always sent to the
follow-up facility in a timely fashion. If the patient sustains
any damages due to delayed treatment, the responsible par-
ties may be liable.
According to 23 section 3 of the German Protection
against Infection Act, the recommendations of the Commissi-
on for Hospital Hygiene and Infection Prevention (KRINKO)
Table 1 Overview of aspects in preanalysis.
Swab
Sample container Swab with gel matrix
For PCR without gel matrix
Storage Optional: refrigerate to maintain
pathogen ratio, but loss of anaerobes
Test order Note: Screening for MRSA, MRGN, or
other pathogenic bacteria
Information given to the lab Prior antibiotic therapy
Presumed contamination during
swabbing
204 2015 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2015/1303
are considered to be of great significance. Although these re-
commendations call for wound swabs in the screening for
MRPs, they do not elaborate on sampling techniques or any
further details as to practical implementation [5]. For examp-
le, the results of MRSA-specific PCR may already be availab-
le after a few hours, however, they usually show only typical
segments of the S. aureus genome and the mecA gene.
Preanalysis in wound swabs
First, it has to be determined whether a swab or a biopsy is
best suited for bacteriologic detection. The physician must
clinically differentiate whether the microbiology workup is
done in the context of multiresistant pathogen screening, or
whether there are clinical signs of a wound infection requi-
ring systemic antibiotic therapy [1, 6]. Initial tests for mul-
tiresistant pathogens are frequently only aimed at detecting
bacterial colonization; hence, the results are generally of no
consequence for any subsequent wound therapy. If the tests
are positive for MRPs, specific hygienic and work protection
measures apply, which appear in the TRBA 250 [7] and the
June 2014 recommendations on MRSA by the Commission
for Hospital Hygiene and Infection Prevention (KRINKO) at
the Robert Koch Institute (RKI) [5].
The choice of preanalysis technique also depends on
the intended diagnostic test. For PCR testing, transporting
the swab in a gel matrix is not necessary, whereas such a gel
matrix should be used, if cultural detection is sought at an
off-site lab (Table 1) (Figure 1).
Swab preparation
There is no sufficient evidence as to the necessity for wound
cleansing prior to wound swabbing (for example, using ste-
rile 0.9 % saline solution). Taking the KRINKO concept of
Biopsy/excision
Sterile tube with a drop of sterile distilled
water or physiological saline solution
Optional: refrigerate (28 C)
Note: mycobacteria, actinomycetes,
Nocardia, other pathogenic bacteria,
Prior antibiotic therapy
Presumed contamination during biopsy
Review Article Microbiological diagnostics in chronic wounds
Figure 1 Cultural detection of Staphylococcus aureus with
ivory-colored colonies and mild hemolysis on blood agar.
strict, theoretical plausibility into account, there is some sup-
port for wound cleansing:
Possible reduction in contaminants that have migrated
from the wound margins. This especially applies to the
use of absorbent dressings.
Dilution of superficial wound substances potentially in-
fluencing bacterial growth. Also, the activity of antibac-
terial substances (for example, antiseptics) during trans-
port may be reduced or eliminated.
In putrid wound infections, dilution is desirable, as bacte-
ria may be phagocytosed, if the granulocyte count is too
high, resulting in false-negatives or inaccurate counts.
Yet, there are also drawbacks to wound cleansing prior
to sampling:
Pain caused by manipulation.
Time and effort involved.
Added cost of sterile cleansing solutions and compresses.
Certain contaminants could be eliminated, including
multiresistant pathogens, which would require further
specific measures.
2015 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2015/1303
Identification of contaminants even in the absence of
a clinically relevant infection allows for more precise
antibiotic therapy, if needed in the future.
Technique for wound swabbing
Current literature offers only little information on wound
swabbing. Although the RKI does not provide detailed inst-
ructions, either, the use of tap water as cleansing solution has
unequivocally been dismissed. Neither do standard micro-
biology textbooks contain any detailed suggestions [8]. Most
publications recommend the following procedures:
Levine technique: applying slight pressure, a sample is
taken from an area measuring about 1 cm 2 at the center
of the lesion, or from an area of the wound that appears
clinically infected.
Z technique: avoiding the wound margins, swabs are
passed over the wound surface in a zigzag fashion.
Essen Rotary: moving from the periphery toward the
center of the wound (covering the entire wound area),
a sample is taken using a spiraling motion and applying
slight pressure.
The Levine technique was recommended in a consensus
statement by the World Union of Wound Healing Societies
(WUWHS) in 2008 [9]. A prospective clinical study compa-
red the bacteriologic results of the Levine technique with the
Z technique. In 83 wounds, the Levine technique was supe-
rior to the Z technique for both acute and chronic wounds.
The authors suggested an increased release and uptake of
wound fluid as potential reason [10]. They also found that
the Levine technique led to an increase in the number of bac-
teria detected. Another prospective clinical study showed
similar results in 50 chronic wound patients [11], which is
why the Levine technique has internationally frequently been
propagated as gold standard in the microbiology workup of
infected chronic wounds [12]. In a recent study on 50 pati-
ents with noninfected chronic leg ulcers, various swabbing
methods were compared to the Essen Rotary method com-
monly used in Germany (Figure 2). Six bacteriologic swabs
were taken per patient. The bacterial spectrum on the wound
surface was found to be very diverse, sometimes leading to
vast differences among samples from various areas of the
wound. MRSA-positive patients presented a special problem:
in two out of five Levine technique patients, MRSA would
have gone undetected. The study also showed that, compared
to the Levine technique, the Essen Rotary method detected
significantly more bacterial species [13]. Several compara-
tive clinical studies have shown that biopsies of superficial,
noninfected chronic wounds are only required in specific
situations [1417].
205
 Review Article Microbiological diagnostics in chronic wounds
Figure 2 Bacteriologic swab using the Essen Rotary in a wound
clinically not infected. Here, the swab was specifically aimed at
detecting multiresistant pathogens (MRPs) at initial presentation.
Detection of specific pathogens
Certain pathogens require a biopsy for identification (due to
the necessity for special culture media and particularly due to
their slow growth), as they would otherwise go undetected in
routine wound swab testing. There is no specification in the
literature on how large biopsies need to be for further workup.
For practical reasons, however, they should not be smaller
than 4 mm in diameter. Clinical indications for a biopsy inclu-
de cutaneous tuberculosis and the more common non-tuber-
culous mycobacteria that cause Buruli ulcer, which is particu-
larly prevalent in tropical regions. Given that the microscopic
detection of these pathogens requires special stains, laborato-
ries must be specifically informed about the clinical suspicion
of mycobacteria [8]. Molecular biology tests represent another
alternative [18]. Actinomycetes are usually found in fistulas
(pus) or biopsies. Leishmania species can also only be detected
by special staining of biopsy material [8].
Current AWMF guidelines
While taking wound biopsies on a routine basis has been pro-
pagated in North America in recent years, in Europe, various
206 2015 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2015/1303
swabbing techniques have been recommended. The following
is a summary of current information and recommendations,
as found in the AWMF guidelines:
S3 guidelines of the German Society of Phlebology
(DGP): Diagnosis and therapy of venous leg ulcers [19]:
wound infections identification of the causative pa-
thogens by microbial culture, and analysis of their sen-
sitivity to antibiotics by resistogram should be perfor-
med in case of primary suspicion of a mixed infection
or poor response to initial therapy. Analysis of the bac-
terial spectrum by microbial culture of biopsy material
offers no added advantage over a wound swab.
S3 guidelines of the German Society for Wound Healing
and Wound Treatment (DGfW): local therapy of chronic
wounds in patients with risk factors such as peripheral ar-
terial occlusive disease, diabetes mellitus, or chronic venous
insufficiency [20]. Wound cleansing with medicinal lar-
vae may only be done after ruling out colonization with
Pseudomonas aeruginosa immediately prior to their
use, a wound swab should be taken to exclude infection.
S1 guidelines put forth by the task force on hygiene
in hospitals and private doctors offices of the AWMF
working group: obtaining, storing, and transporting spe-
cimens for microbiology diagnostics [3]. Material from
wounds and infectious lesions. Indications: superficial
and deep infections of the skin, mucous membranes and
soft tissues, abscesses, osteomyelitis, and fistulas. Materi-
al: sterile swabs, sterile sharp spoon or syringe with can-
nula (for puncturing), transport medium (should also be
suitable for anaerobes), and disposable gloves. Procedu-
re: put on gloves, swabbing (without prior skin disinfec-
tion); after removing any coating, collect material from
the depth of the wound; or with a sharp spoon from the
skin (suspected fungal infection); or from the borders in
chronic wounds; or use puncturing methods (after disin-
fecting the skin) for abscesses or deep wound infections
to collect pus or exudate. Place the swab or aspirated
material in a suitable transport tube or container with
transport medium. Send immediately to the laboratory;
if transport is delayed, store in a refrigerator (46 C).
S1 guidelines of the German Society for Pediatric Sur-
gery: wounds and wound treatment [21]. Wound infec-
tion therapy: wound swab.
National treatment guidelines (NVL) for type 2 diabetes:
prevention and therapeutic strategies for foot complica-
tions [22]. Deep tissue samples are superior to super-
ficial wound swabs; a sample should be taken from any
ulcer persisting for more than 30 days. The tissue sample
should be taken after mechanical wound cleansing.
The following guidelines that deal with chronic wounds
do not contain any specific information on bacteriologic tests:
Review Article Microbiological diagnostics in chronic wounds
S1 guidelines of the task force on hygiene in hospitals
and private doctors offices of the AWMF working
group: chronic wounds and those healing by secondary
intention hygiene requirements [4].
S3 guidelines of the German Society for Angiology So-
ciety for Vascular Medicine: guidelines on the diagnosis
and treatment of peripheral arterial occlusive disease
(PAOD) [23].
Discussion
In the context of modern, guideline-based treatment of chro-
nic wound patients, microbiologic aspects also have to be ta-
ken into account [24, 25]. However, a review of the current
literature shows it is still impossible to issue unequivocal,
evidence-based recommendations on the practical implemen-
tation of bacteriologic diagnostics in these patients. The fol-
lowing is therefore aimed at critically discussing various re-
levant aspects and summarizing their clinical consequences.
The section on wound infections In the DGP guidelines
recommends bacteriologic swabs whenever a mixed infection is
suspected or response to initial therapy has been poor. Yet, this
particular procedure seems to be geared towards the clinical
suspicion of erysipelas. For venous leg ulcers, it is clearly stated
that culturing biopsy tissue offers no advantage over taking a
swab in regard to microbiologic results [19]. The DGfW guide-
lines, which primarily focus on wound treatment aspects, con-
tain a statement on bacteriologic swabs only in the section on
wound cleansing using medicinal larvae [20]. In the guidelines
of the task force hygiene in hospitals and private doctors of-
fices, indications for bacterial workup include superficial and
2015 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2015/1303
deep infections of the skin, mucous membranes and soft tissu-
es, abscesses, osteomyelitis, and fistulas. Here, various utensils,
such as sterile swabs or a sterile sharp spoon, are listed without
elaborating on their diagnostic value. As to the procedure, it
is recommended to take a swab from the depth of the wound
after removing any coating. In chronic wounds, material may
also be obtained from the wound margins using a sharp spoon
[3]. In the section on wound infections in the guidelines of
the German Society for Pediatric Surgery, the point on thera-
py is followed simply by the word wound swab without any
further details on implementation or preparations. Given that
this is followed by the removal of necrotic areas plus , one
may assume that, in case of clinical suspicion of a wound infec-
tion, a bacteriologic swab is to be taken prior to the removal of
necrotic areas and additional measures,. Later, wound smears
are again discussed in a section on special types: primarily in-
fected wounds (bite/gun shot wounds) [21]. In the NVL on pre-
ventive and therapeutic strategies for foot complications, deep
tissue samples are advised (at least) in type 2 diabetes patients
with such lesions. Sampling should be performed after mecha-
nical wound cleansing. As this information is listed under an-
tibiotic therapy, it is safe to assume that this recommendation
applies to cases with clinically suspected wound infections, and
the procedure should be carried out prior to administering sys-
temic antibiotics [22].
Based on the current literature, superficial bacteriologic
swabs are a sufficient screening method in patients with chro-
nic wounds, and should be done without prior cleansing. In
most cases, swabs are taken to rule out MRPs. While detecti-
on usually does not alter treatment, it does necessitate specific
hygiene measures when treating these patients [5, 6, 26, 27].
Figure 3 Patient with a clinically
non-infected venous leg ulcer. Micro-
biology workup may be considered
at initial presentation to rule out
colonization with MRPs. In this case,
a bacteriologic swab may be carried
out, for example using the Essen
Rotary without prior wound clean-
sing (a). Patient with a mixed venous
and arterial leg ulcer and suspected
wound infection. Prior to initiation of
any systemic antibiotic therapy, a bac-
teriologic swab should be performed
with prior wound cleansing (b).
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 Review Article Microbiological diagnostics in chronic wounds
Table 2 Indications for microbiologic diagnostics.
Bacteriologic swab without prior wound cleansing
Detection/exclusion of multiresistant pathogens (screening)
Bacteriologic swab with prior wound cleansing
Detection of causal pathogens in wound infections
Colonization/infection with yeasts
Biopsy/excision
Wound infection in patient with deep ulcerations, for
example, diabetic foot syndrome
Fistula tissue, if fistula contents cannot be obtained
Suspected pathogens: mycobacteria, Leishmania,
actinomycetes, Nocardia, molds
Wound infections with negative swabs (no pathogens
detected)
The Essen Rotary method is a quick and easy-to-use modifi-
cation of conventional swabbing methods, such as the Levine
technique and the Z technique; it involves only slightly more
effort and offers significantly higher sensitivity in detecting su-
perficial bacterial colonization in chronic wound patients [13].
If, on the other hand, the objective is to identify pathogens
causing wound infections in chronic wounds or to potentially
detect yeasts, the wound should be cleansed prior to any bacte-
riologic swab [28]. Wound cleansing may be done using sterile
physiologic saline and/or mechanically with sterile compresses
[8] (Figure 3a, b). Routine biopsies for pathogen detection are
not necessary in patients with chronic wounds. Yet, diagnostic
biopsies may be advisable, if there is clinical suspicion of a
wound infection, especially in patients with deep ulcerations,
diabetic foot syndrome, severe soft tissue infections, or fistu-
la tissue. Moreover, if certain pathogens are suspected, such
as mycobacteria, Leishmania, actinomycetes, Nocardia, or
molds, biopsy material should be used for microbiologic dia-
gnostics. Finally, a biopsy may also be required, if swabs have
failed to show any pathogens [8] (Table 2).
Correspondence to
Prof. Dr. med. Joachim Dissemond
Klinik und Poliklinik fr Dermatologie, Venerologie
und Allergologie
Universittsklinikum Essen
Hufelandstrae 55
45122 Essen
Germany
E-mail: joachim.dissemond@uk-essen.de
208 2015 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2015/1303
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