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Combinations of maggot excretions/secretions and antibiotics are
effective against Staphylococcus aureus biofilms and the bacteria
derived therefrom
Mariena J. A. van der Plas1, 2, Cheryl Dambrot1, Heleen C. M. Dogterom-Ballering 1, Simone Kruithof 1,
Jaap T. van Dissel 1and Peter H. Nibbering1*
1
Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands;
2
Department of Surgery, Leiden
University Medical Center, Leiden, The Netherlands
*Corresponding author. Tel: þ31-71-526-2204; Fax: þ31-71-526-6758; E-mail: p.h.nibbering@lumc.nl
Received 1 October 2009; returned 20 October 2009; revised 26 January 2010; accepted 26 January 2010
Objectives: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we
reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the
bacteria. As many antibiotics are not effective against biofilms we assessed the effect of combinations of ES
and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms.
Methods: Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on
S. aureus ATCC 29213 biofilms and bacterial viability were determined using microtitre plates and in vitro
killing assays.
Results: Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin
reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown.
After a lag time the bacteria derived from biofilms became susceptible to vancomycin and clindamycin, pro-
vided that the medium was refreshed. Daptomycin dose-dependently eliminated the biofilm-derived bacteria
immediately. Furthermore, it was significantly more effective against bacteria derived from ES-exposed biofilms
than those from control biofilms. ES did not affect the activity of the antibiotics against log-phase S. aureus.
Conclusions: Combinations of maggot ES and antibiotics eliminate S. aureus biofilms and the bacteria derived
therefrom.
Keywords: Lucilia sericata, clindamycin, vancomycin, daptomycin, bacterial killing
Introduction
Chronic wounds are common in patients with vascular insuffi-
ciencies and underlying chronic conditions such as diabetes mel-
litus, as well as in patients suffering from acute, extended
trauma.
1,2
These wounds and associated amputations result in
decreased physical, emotional and social function of patients,
a reduced quality of life and major economic costs for patients,
their families and society as a whole.
3,4
A severe complication
of the healing process is bacterial colonization and subsequent
infection of the wound surface,
5–7
especially when the bacteria
are residing in biofilms.
8
These latter bacteria exhibit altered
growth characteristics and gene expression profiles as compared
with those present free in the environment, the so-called plank-
tonic bacteria.
9
Importantly, biofilm formation and the conse-
quences thereof for bacterial growth characteristics render
microorganisms resistant to the action of many antibiotics
10,11
as well as cells and effector molecules of the host’s immune
system.
7,12
Bacterial fragments/products released from biofilms
continuously attract host cells to the wound. As phagocytes
cannot ingest the biofilm-associated bacteria and therefore are
unable to eliminate the cause of infection, the subsequent
accumulation of inflammatory cells and enhanced release of
proinflammatory cytokines, proteases and reactive oxygen
species eventually leads to inactivation of growth factors and
tissue destruction,
13,14
thereby contributing to the establishment
and maintenance of chronic wounds.
Sterile larvae—maggots—of the green bottle blowfly Lucilia
sericata are used as a treatment for various types of chronic
wounds.
15 –17
Previously we reported the use of maggot
excretions/secretions (ES) to break down Staphylococcus aureus
and Pseudomonas aeruginosa biofilms.
18
However, the bacteria
released from these biofilms were not killed by ES. On the
other hand, many antibiotics cannot break down bacterial
#The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
J Antimicrob Chemother 2010; 65: 917–923
doi:10.1093/jac/dkq042 Advance publication 26 February 2010
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biofilms but effectively kill planktonic bacteria. Therefore, we
assessed the effect of combinations of maggot ES and anti-
biotics on S. aureus biofilms and on the survival of the bacteria
released from these biofilms.
Materials and methods
Maggots and maggot ES
ES of sterile second- and third-instar larvae of L. sericata (a gift from Bio-
Monde GmbH, Barsbu¨ttel, Germany) were collected as described.
19
Larvae were incubated in H
2
O(5mL/larva) for 60 min at ambient temp-
erature in the dark. Next, ES were checked for sterility and stored
at 2208C. Prior to use, ES preparations were pooled and centrifuged
at 1300 gfor 5 min at 48C to remove particulate material. ES protein
concentration was determined using the Pierce BCA Protein Assay kit
according to the manufacturer’s instructions.
Antibiotics
Stock solutions of vancomycin (Pharmachemie B.V., Haarlem, The
Netherlands), daptomycin (Cubicin, Chiron Corporation Limited, Uxbridge,
UK) and clindamycin (Upjohn GmbH, Heppenheim, Germany) were dis-
solved in distilled water to a final concentration of 10 g/L.
S. aureus cultures
S. aureus ATCC 29213 (Manassas, VA, USA) were grown in tryptone soya
broth (TSB) at 378C under vigorous shaking. The MIC values for this
strain are 0.5– 2 mg/L for vancomycin, 0.25– 1 mg/L for daptomycin
and 0.06 –0.25 mg/L for clindamycin.
20
Biofilm assay
Biofilm formation of S. aureus in 96-well polyvinyl chloride (PVC) plates
was conducted as described.
18
In short, bacteria from overnight cultures
were diluted 1:1000 and 5 mL of these bacterial suspensions were added
to each well containing 100 mL of ‘biofilm medium’ consisting of 0.5TSB
supplemented with 0.2% (w/v) glucose. After 24 h, planktonic cells were
removed and 100 mL of biofilm medium with or without antibiotics (1 –
400 mg/L) and/or ES (20– 200 mg/L) were added to the biofilms. At the
indicated time intervals, planktonic cells were harvested from these
wells and the numbers of viable bacteria were determined microbiologi-
cally using serial dilutions of these suspensions plated six times each
onto agar plates. The lower detection limit of this method is 100 cfu/
well. In addition, after washing the wells with tap water, biofilms were
exposed to a 1% (w/v) Crystal Violet solution for 15 min, washed and
then incubated in absolute ethanol for 15 min to extract the Crystal
Violet retained by the cells. Next, this solution was used to quantify the
amount of biofilm by measuring the absorbance at 590 nm.
Furthermore, we investigated the effect of antibiotics on bacteria
derived from the biofilms and subsequently transferred to fresh biofilm
medium. For this purpose, the planktonic cells were removed from
24-h-old biofilms, and fresh biofilm medium was added to the wells con-
taining ES (20 –200 mg/L) or H
2
O as a control. After an additional 24 h,
the bacteria released from the biofilms were harvested and 25 mLof
these bacterial suspensions were transferred to wells of a PVC plate con-
taining 75 mL of TSB medium supplemented with antibiotics; the final
concentration of the medium was 0.5TSB and 0.2% glucose. After
3 and 24 h, the numbers of surviving bacteria were determined microbio-
logically as described above.
Concentration– effect relationship for antibiotics
on exponentially growing S. aureus
To further determine the concentration– effect relationship for antibiotics
on planktonic S. aureus in the presence or absence of ES, in vitro killing
assays were conducted as described
21
with minor modifications. Bacteria
in mid-log phase were centrifuged at 2000 gfor 10 min, washed twice
with PBS and resuspended to a concentration of 110
7
bacteria/mL of
biofilm medium supplemented with antibiotics (0.005– 500 mg/L) and/
or ES (20 –200 mg/L). Subsequently, 100 mL aliquots of these bacterial
suspensions were transferred to wells of a 96-well PVC plate and incu-
bated at 378C. After 1, 2 and 3 h, the numbers of surviving bacteria
were determined microbiologically as described above.
Next, the differences between the logarithms (base 10) of the
numbers of cfu in the absence and presence of antibiotics and/or ES
were calculated for each timepoint.
22
For further calculations, the
highest value of the net killing rate during the 3 h of exposure was
used (E
R
). The concentration– effect relationship was established by
using the Hill equation:
ER¼ER;max C=ðEC50 þCÞ
where E
R
,max is the estimated maximal killing rate, Cthe antibiotic con-
centration (mg/L) and EC
50
the estimated antibiotic concentration at
which 50% of the maximal killing is reached. The parameters of this
pharmacodynamic model were calculated in SPSS using non-linear
regression analysis.
Statistical analysis
Statistical analyses were performed using Graphpad Prism version 4.02.
Statistical differences between the values for ES-incubated and
control-incubated bacteria were analysed using a paired t-test. The
level of significance was set at Pvalues ,0.05.
Results
Effect of antibiotics and ES on S. aureus biofilms
The results showed a dose-dependent increase in biofilm size by
vancomycin and daptomycin already within 3 h (Table 1). This
effect persisted over the next 21 h. In contrast, clindamycin
dose-dependently decreased the amount of biofilm; after 3 h
of incubation the biofilm diminished by 28% (Table 1). As
reported previously,
18
within 3 h ES degraded the S. aureus bio-
films completely and this effect was not counteracted by the
antibiotics (data not shown).
Effect of combining ES and antibiotics on the viability
of S. aureus released from biofilms
To find out if the biofilm-derived bacteria are susceptible to anti-
biotics, we incubated the biofilms with various concentrations of
antibiotics and/or ES and determined the number of viable bac-
teria at different intervals.
Preliminary experiments revealed no reduction in the number
of viable bacteria when using 10 mg/L of vancomycin and dap-
tomycin. Vancomycin at concentrations of 50 mg/L slightly but
significantly reduced the number of viable bacteria at 24 h
(Figure 1a), but not after 3 h (data not shown). Daptomycin
dose-dependently reduced the number of biofilm-derived bac-
teria within 3 h; a 3 log reduction was seen for 400 mg/L (data
not shown). This reduction in bacterial numbers continued for
van der Plas et al.
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the next 21 h (Figure 1b). After 3 h of incubation, the number of
viable bacteria was 1 log lower in the presence of clindamycin
compared with control incubations of bacteria derived from
either ES-treated or control biofilms (data not shown). Over the
following 21 h, no increase in bacterial numbers was observed
in the presence of clindamycin (Figure 1c). Furthermore, a dose-
dependent effect of clindamycin was observed at the lowest
concentrations used in the experiments (i.e. 1, 5 and 10 mg/L,
resulting in a reduction in the number of viable bacteria by
53%+9%, 78%+4% and 80%+14%, respectively), whereas
maximal inhibition was reached with clindamycin concentrations
.10 mg/L. Of note, ES (200 mg/L) did not affect the antibacterial
activity of the antibiotics (Figure 1a – c); 20 mg/L ES yielded
similar results (data not shown).
Effect of ES and antibiotics on biofilm-derived bacteria
transferred to fresh biofilm medium
As large numbers of bacteria derived from the biofilms remained
viable in the presence of the antibiotics, we considered the possi-
bility that these biofilm-derived bacteria were in a dormant state
making them resistant to these antibiotics. Therefore, bacteria
derived from ES-incubated or control-incubated biofilms were
transferred to fresh biofilm medium supplemented with
antibiotics.
Vancomycin failed to affect the number of viable bacteria at
3 h but induced a 2 log reduction in bacterial counts at 24 h.
This effect was independent of the dose of antibiotics or
whether the biofilms had been exposed to ES (Figure 2a). Dapto-
mycin dose-dependently reduced the number of bacteria within
3 h. Moreover, the bactericidal effect of daptomycin against bac-
teria derived from biofilms exposed to 200 mg/L ES (Figure 2b),
but not to 20 mg/L ES (data not shown), was higher than that
against bacteria from control biofilms. After 24 h, all bacteria
were killed by the various concentrations of daptomycin (data
not shown). Clindamycin prevented an increase in the number
of bacteria at 3 h of incubation (data not shown) and the
number of bacteria remained constant during the following
21 h (Figure 2c); the activity of clindamycin against bacteria
derived from ES-exposed biofilms was similar to that from
control biofilms.
Effect of ES on the concentration–effect relationship
of antibiotics on exponentially growing S. aureus
To investigate the effect of ES on the activity of the antibiotics, we
determined the killing curves for the various antibiotics using log-
phase bacteria. The results showed a dose-dependent reduction in
the number of viable S. aureus by all three antibiotics; the
maximum effect of daptomycin was higher than that of clindamy-
cin and vancomycin, which were equally effective against the bac-
teria (Figure 3a– c). The estimated EC
50
and E
R
,max values are
given in Table 2. The activity of the antibiotics was not affected
by 20 or 200 mg/L ES. Of note, 500 mg/L daptomycin was suffi-
cient to kill all bacteria within 1 h in four out of five experiments
under all conditions. Furthermore, the maximum effect of clinda-
mycin was observed at 1 mg/L, the maximum effect of vancomy-
cin was observed at 10 mg/L and 500 mg/L daptomycin was
required to reach a maximal effect.
Discussion
The main conclusion from the present study is that combinations
of maggot ES and antibiotics can break down S. aureus biofilms
and subsequently eliminate the bacteria derived therefrom. This
conclusion is based on the following observations. First, ES broke
down established biofilms within 3 h and this effect was not nega-
tively or positively affected by the antibiotics. In the absence of ES,
samples containing vancomycin or daptomycin, antibiotics whose
activity depends on their action on the bacterial cell envelope,
lacked activity against biofilms; similar findings were observed
for the b-lactam antibiotic flucloxacillin (M. J. A. van der Plas,
C. Dambret and P. H. Nibbering, unpublished observations).
In contrast, clindamycin and linezolid (M. J. A. van der Plas,
C. Dambret and P. H. Nibbering, unpublished observations)
decreased the amount of biofilm, albeit that they were unable
to completely eradicate it in the 24 h incubations applied here.
Secondly, biofilm-derived bacteria became more susceptible to
the action of vancomycin and clindamycin after being
Table 1. Effect of antibiotics on established biofilms of S. aureus
Vancomycin Daptomycin Clindamycin
mg/L 3 h 24 h 3 h 24 h 3 h 24 h
0 0.18+0.02 0.30+0.02 0.21+0.01 0.30+0.02 0.21+0.01 0.29+0.02
1 0.19+0.03 0.26+0.04 0.20+0.03 0.27+0.03 0.20+0.03 0.24+0.04
5 0.21+0.03 0.29+0.05 0.20+0.03 0.26+0.05 0.17+0.03 0.24+0.04
10 0.27+0.04* 0.38+0.06* 0.23+0.04 0.35+0.07 0.17+0.04 0.24+0.04
50 0.26+0.04* 0.36+0.05* 0.26+0.04* 0.45+0.10* 0.16+0.01* 0.24+0.01*
100 0.26+0.04* 0.40+0.03* 0.27+0.01* 0.39+0.03* 0.16+0.01* 0.22+0.01*
200 0.26+0.04* 0.35+0.02* 0.28+0.02* 0.36+0.03* 0.16+0.01* 0.21+0.02*
400 0.22+0.01* 0.43+0.03* 0.33+0.02* 0.38+0.04* 0.15+0.01* 0.21+0.02*
Results (OD
590
) are means+SEM of 4 experiments.
For all samples, the addition of ES resulted in total breakdown of the biofilms (OD ,0.10).
*Values are significantly different from those for control biofilms (P,0.05).
Effect of maggot excretions/secretions and antibiotics on S. aureus
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transferred to fresh medium than when the bacteria remained
in the biofilm wells. An explanation for this result could be that
the bacteria derived from biofilms are in a static/dormant state
and therefore less susceptible to antibiotics that solely target
multiplying bacteria.
9
In contrast, daptomycin showed direct
activity against biofilm-derived bacteria whether or not they
were transferred to fresh medium. Moreover, ES (200 mg/L)
enhanced the antibacterial activity of daptomycin, but not of
vancomycin and clindamycin, against biofilm-derived S. aureus
transferred to fresh medium. Although we cannot explain the
latter results, they probably depend on the specific pharmacody-
namic mechanisms of daptomycin. Thirdly, ES did not alter the
activity of the antibiotics against exponentially multiplying
bacteria.
In the interpretation ofthe current findings, the following points
need be considered. First, we performed all experiments with a
single ATCC reference strain of S. aureus. In addition, our preliminary
results show that maggot ES can prevent methicillin-resistant
(a)
cfu/well
108
107
106
105
104
103
102
101
108
107
106
105
104
103
102
101
108
107
106
105
104
103
102
101
0
–+–+–+–+–+
0 50 50 100 100 200 200 400 400
Vancomycin (mg/L)
Daptomycin (mg/L)
(b)
cfu/well
0
–+–+–+–+–+
0 50 50 100 100 200 200 400 400
Clindamycin (mg/L)
(c)
cfu/well
0
–+–+–+–+–+
0 50 50 100 100 200 200 400 400
ES
ES
ES
Figure 1. Effect of antibiotics, ES and combinations thereof on
biofilm-derived S. aureus. Biofilms were washed and then incubated
with increasing concentrations of vancomycin (a), daptomycin (b) or
clindamycin (c) in the absence or presence of 200 mg/L ES for 24 h.
Thereafter, the number of viable bacteria in the medium was
determined microbiologically. Results of 4– 6 experiments are shown,
with lines representing the medians. The values from the samples
containing antibiotics are significantly different from those of the
control wells. Filled circles, no ES; open circles, 200 mg/L ES.
(a)
cfu/well
109
108
107
106
105
104
103
102
101
109
108
107
106
105
104
103
102
101
109
108
107
106
105
104
103
102
101
0
–+–+
*****
–+–+–+
0 50 50 100 100 200 200 400 400
Daptomycin (mg/L)
Vancomycin (mg/L)
(b)
cfu/well
0
–+–+–+–+–+
0 50 50 100 100 200 200 400 400
Clindamycin (mg/L)
(c)
cfu/well
0
–+–+–+–+–+
0 50 50 100 100 200 200 400 400
**
ES
ES
ES
Figure 2. Effect of antibiotics on the numbers of viable S. aureus derived
from ES-exposed and control-incubated biofilms transferred to new
wells. Biofilms were washed and then incubated with ES (200 mg/L) or
not for 24 h and thereafter transferred to wells containing fresh medium
supplemented with various concentrations of vancomycin (a),
daptomycin (b) or clindamycin (c). For vancomycin and clindamycin the
bacteria were recovered after a 24 h incubation and for daptomycin after
3 h. Finally, the number of viable bacteria in the medium was
determined microbiologically. Results of 6– 7 experiments are shown,
with the lines representing the medians. Values are significantly different
from those for bacteria derived from control-incubated biofilms
(*P,0.05 and **P,0.005). Filled circles, no ES; open circles, 200 mg/L ES.
van der Plas et al.
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S. aureus biofilm formation and break down established biofilms
(M. J. A. van der Plas and P. H.Nibbering, unpublished observations).
Nevertheless, we cannot conclude that our findings are generaliz-
able to all S. aureus strains and/or other bacterial species.
However, in agreement with our results, several reports have
described daptomycin as being one of the most effective antibiotics
in the control of biofilm-related S. aureus infections whereas clinda-
mycin and vancomycin were less effective.
23,24
Secondly, the con-
centrations of antibiotics used in the in vitro biofilm assay are
relatively high compared with the free, active antibiotic concen-
trations generally achieved in patients (10–40 mg/L vancomycin,
1–15/20 mg/L daptomycin, 1–20 mg/L clindamycin). However,
concentrations of antibiotics similar to those used in the current
study can be attained in wounds after topical application. Thirdly,
at their MIC values vancomycin or daptomycin did not affect the
biofilm size, whereas at higher concentrations biofilm formation
was enhanced. It should be realized that MIC concentrations of
antibiotics did not reduce the numberof viable biofilm-derived bac-
teria whereas the higher, biofilm-enhancing concentrations did. In
agreement, supra-MIC concentrations of antibiotics are reported to
be effective against killing of bacteria released from biofilms,
whereas sub-MIC and MIC levels were not.
25
Fourthly, in contrast
to the above-mentioned reports, we did not observe a reduction
in biofilm size when using low levels of antibiotics. The explanation
for these contradictory results could be the method of quantifi-
cation. We used Crystal Violet staining to quantify the amount of
biomass whereas many reports describe the use of redox indicators
to measure the metabolic activity of the bacteria. However,
reduced metabolic activity does not exclude similar or even
increased biomass. In agreement with this, it is reported that
several antibiotics, including vancomycin, reduce the redox poten-
tial of bacteria without reducing the matrix.
26
This may lead to bac-
terial multiplication from the remaining matrix and may even
contribute to the development of resistance against the antibiotics.
Clearly, more research should be done into the effect of antibiotics
on biofilms and the bacteria derived from these structures. Fifthly,
previously we reported that the active molecule in ES is heat
labile.
18
Our recent studies into the effects of various substances
affecting structural features of proteins and inhibiting enzymic
activities indicated that the molecule in ES responsible for
S. aureus biofilm breakdown may be a serine protease (M. J. A.
van der Plas, unpublished observations). Currently, we are purifying
the active molecules from maggot ES by activity-guided chromato-
graphy. However, more research is required before the identity of
the active component of maggot ES is clarified. Obviously, appli-
cation of purified maggot-derived compounds instead of live
maggots will definitely increase the acceptance and use of this
very effective therapy for chronic—non-healing—wounds.
What is the clinical relevance of our findings? The failure of
antibiotics to affect biofilms and the bacteria derived therefrom
parallels their overall lack of activity against bacterial coloniza-
tion and infection of chronic wounds where biofilm formation
may be prominent.
7,8,10,27
Therefore, biofilm matrices and the
associated bacteria have to be targeted simultaneously to eradi-
cate chronic infections. Previously we found that maggot ES
break down biofilms of S. aureus
18
without killing the released
bacteria. Here we report that the released bacteria become sus-
ceptible to the actions of antibiotics that fail to affect
biofilm-associated microorganisms when they start multiplying.
Therefore, combinations of maggot ES and antibiotics would
ensure complete breakdown of the biofilms, thereby preventing
bacterial re-growth from the remaining matrix, and prompt anti-
biotic action against the bacteria released from the biofilms.
Additionally, these bacteria will be subjected to the effector
7
(a)
X/ = no ES
= 20 mg/L ES
= 200 mg/L ES
5
6
4
ER
3
2
1
0
0.001 0.01 0.1 1
Vancomycin (mg/L)
10 100 1000
0.0001 0.001 0.01 0.1 1 10 100
/
/
8
7
(b)
X/ = no ES
= 20 mg/L ES
= 200 mg/L ES
5
6
4
ER
3
2
1
0
0.1 1 10
Daptomycin (mg/L)
100 1000 10000
/
/
7
(c)
X/ = no ES
= 20 mg/L ES
= 200 mg/L ES
5
6
4
ER
3
2
1
0
Clindamycin (mg/L)
/
/
Figure 3. Effect of ES on the concentration–effect relationships of the
antibiotics against log-phase S. aureus. Bacteria were incubated with
increasing concentrations of vancomycin (a), daptomycin (b) or
clindamycin (c) in the presence or absence of ES (20–200 mg/L).
Results of 6– 8 experiments are shown. The dose –effect relationships
were calculated from these data using the Hill equation.
Table 2. Pharmacodynamic parameters of the antibiotics and ES
Vancomycin Daptomycin Clindamycin
ES
(mg/L) E
R
,max/h
EC
50
(mg/L) E
R
,max/h
EC
50
(mg/L) E
R
,max/h
EC
50
(mg/L)
0 1.65 0.167 5.41 46.24 1.64 0.036
20 1.77 0.080 7.25 57.99 1.57 0.025
200 1.98 0.110 5.77 39.30 2.30 0.024
Effect of maggot excretions/secretions and antibiotics on S. aureus
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mechanisms of the immune system and ingestion by
maggots.
28,29
Thus, addition of maggots or maggot ES to anti-
biotics may become a promising approach for the treatment of
chronically colonized/infected surfaces of unresponsive wounds.
In this respect, it should be realized that some current treatment
modalities, where maggots apparently are used as a replace-
ment for instead of as an adjunct to antibiotics, often overesti-
mate bacterial killing by ES when applied in therapeutically
relevant amounts.
18
Of note, antibiotics including vancomycin
and clindamycin have no detrimental effects on maggot
growth and survival.
30
Based on our results and other
reports,
23,24
daptomycin and ES combined appear particularly
promising for the treatment of biofilm-related S. aureus wound
infections. Daptomycin, in contrast to vancomycin and cationic
antimicrobial peptides, kills bacteria without inducing bacterial
lysis.
31 –33
As chronic wounds often are characterized by pro-
longed and dysregulated inflammatory responses,
13,34 –36
decreased bacterial lysis may reduce proinflammatory responses
to bacterial products by immune cells, thereby contributing to
the healing process.
32
Acknowledgements
We thank Emile F. Schippers for technical assistance.
Funding
This study was supported by internal funding.
Transparency declarations
None to declare.
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