Probiotics & Antimicro. Prot.
DOI 10.1007/s12602-009-9017-8
Evaluation of Nisin F in the Treatment of Subcutaneous Skin
Infections, as Monitored by Using a Bioluminescent Strain
of Staphylococcus aureus
M. de Kwaadsteniet Æ C. A. van Reenen Æ
L. M. T. Dicks

Springer Science+Business Media, LLC 2009
Abstract The potential of nisin F as an antimicrobial
agent in treating subcutaneous skin infections was tested in
vivo by infecting C57BL/6 mice with a bioluminescent
strain of Staphylococcus aureus (Xen 36). Strain Xen 36
has the luxABCDE operon located on a native plasmid.
Mice were grouped into four groups: Infected with strain
Xen 36 and treated with nisin F, infected with strain Xen
36 and treated with saline (placebo), not infected and
treated with nisin (control) and not infected and not treated
(control). The immune systems of the mice were sup-
pressed with deksamethasone. Mice were treated with
either nisin F or sterile physiological saline 24 and 48 h
after infection with subcutaneously injected S. aureus Xen
36 (4 9 106 CFU). Histology and bioluminescent flux
measurements revealed no significant difference between
infected mice treated with nisin and saline, respectively.
However, infected mice treated with nisin F had an
increased number of polymorphonuclear cells when com-
pared with infected mice treated with saline. Also, not
infected mice treated with nisin F had an influx of poly-
morphonuclear cells. Nisin F is thus ineffective in com-
bating deep dermal staphylococcal infections. The apparent
immune modulation of nisin when subcutaneously injected
has to be investigated.
Keywords Nisin F
Staphylococcus aureus

Subcutaneous skin infections
M. de Kwaadsteniet
C. A. van Reenen
L. M. T. Dicks (&)
Department of Microbiology, University of Stellenbosch,
Matieland 7602, Private Bag X1, Stellenbosch 7600, South
Africa
e-mail: lmtd@sun.ac.za
Introduction
Skin and soft tissue infections (SSTIs) are an immense
health problem with symptoms ranging from mild follicu-
litis, furunculosis and atopic dermatitis to severe necro-
tizing fasciitis [15]. Prolonged and ineffectively treated
SSTIs may lead to endocartitis, osteomyelitis, meningitis,
brain abscesses and pneumonia [9]. Mild SSTIs are treated
with orally administered antimicrobial agents. Complicated
SSTIs requires hospitalization, surgical intervention and
treatment with intravenous antibiotics [14].
The SENTRY antibiotic surveillance program reported
that Staphylococcus aureus is the most frequently isolated
pathogen from SSTIs case studies in North America, Latin
America and Europe [10]. SSTIs reported in the emergency
departments in the US increased from 1.2 million in 1993
to 3.4 million in 2005. This increase coincided with the
emergence of community-associated methicillin resistant
strains of S. aureus (MRSA). Although no direct correla-
tion can be made, this theory is supported by the fact that
physicians started treating SSTIs from 2001 with antibi-
otics active against community-associated MRSA, namely
trimethoprim-sulfamethoxazole and clindamycin [12].
The occurrence of antibiotic resistance among strains of
S. aureus causing SSTIs has been reported by numerous
researchers worldwide [1] and emphasizes the need for the
development of alternative and more effective antimicro-
bial agents.
Optical imaging and bioluminescent reporter systems
have, in recent years, advanced to a point where
researchers can monitor bacterial infections in the same
animal noninvasively and in real-time over multiple time-
points. Numerous studies have shown a strong correlation
between the bioluminescence flux measurements generated
from these genetically engineered bacteria and viable cell
123

numbers in tissue homogenates. Bioluminescent imaging
(BLI) has been used for studying skin infections and the
treatment of such infections with antibiotics [4, 11].
Nisin F is a natural nisin variant with in vitro antimi-
crobial activity towards clinical strains of S. aureus [6]. We
have shown that nisin F plays a protective role against
S. aureus infections in the respiratory tract of Wistar rats
[7]. The aim of this study was to investigate the antimi-
crobial effect of nisin F against S. aureus when injected
subcutaneously in C57BL/6 mice.
Materials and Methods
Bacterial Strains and Culture Conditions
Staphylococcus aureus Xen 36 (Caliper Life Sciences,
Hopkinton, MA) has a stable copy of the modified
Photorhabdus luminescence luxABCDE operon at a single
integration site on a native plasmid. The parental strain,
S. aureus ATCC 49525, has been isolated from a patient
with bacteremia. S. aureus Xen 36 was cultured in brain
heart infusion (BHI, Biolab, Biolab Diagnostics, Midrand,
South Africa) at 37C. Lactococcus lactis subsp. lactis F10
was cultured in De Man Rogosa Sharpe (MRS) broth
(Biolab) at 30C.
In Vitro Studies
Nisin F, produced by L. lactis subsp. lactis F10 [6], was
partially purified by ammonium sulphate precipitation and
dialysed, according to the method described by Sambrook
et al. [13]. The peptide was concentrated by freeze-drying
in ampules. Antimicrobial activity was determined by
using the agar-spot test method and expressed as arbitrary
units (AU) per ml. One AU is the reciprocal of the highest
serial two-fold dilution showing a clear zone of inhibition
of the indicator strain. An 18-h-old culture of S. aureus
Xen 36 (106 CFU per ml), embedded in BHI soft agar (1%,
w/v), was used as indicator.
The stability of nisin F in blood was tested by spotting
samples onto sheep blood agar seeded with S. aureus Xen
36 (106 CFU per ml). The activity of nisin F (AU ml-1)
was determined as described before. The control experi-
ment was performed in a similar way, but with S. aureus
Xen 36 inoculated into BHI agar.
Animal Model
Approval for the experiments was obtained from the Ani-
mal Ethics Committee of Stellenbosch University (ethics
reference number 200801024). C57BL/6 mice weighing
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Probiotics & Antimicro. Prot.
between 20 and 25 g were divided into four groups (six per
group). The mice were housed in plastic cages in animal
rooms with constant environmental conditions and were
fed a standard rodent diet. The immune system of the
animals was suppressed by adding deksamethasone
(2.5 mg l-1) to their drinking water. The hair on the back
of each mouse was removed with Veet Lotion Hair
Remover (Reckitt Benckiser, Elandsfontein, South Africa)
and an area marked for treatment before the trial started.
Mice in groups 1 and 2 were subcutaneously infected with
10 ll S. aureus Xen 36 (4 9 106 CFU). Strain Xen 36
contains a plasmid with the luxABCDE operon and
becomes bioluminescent when exposed to 490 nm. Mice in
groups 3 and 4 were not infected with S. aureus Xen 36.
After 24 and 48 h of infection, mice in group 1 were
treated by injecting 10 ll nisin F (256 AU) subcutaneously
into the area of infection. With in vitro experiments, 10 ll
of nisin F (25,600 AU ml-1) spotted onto plates seeded
with 106 CFU ml-1 yielded a clear inhibition zone. Based
on these results, we have decided to use the same dosage
for in vivo experiments. Mice in group 3 (not infected)
received the same treatment. Mice in group 2 were injected
with 10 ll sterile physiological saline. Mice in group 4
were not infected and not treated.
Five days after the first infection all mice were eutha-
nized by an overdose with pentobarbitone sodium (Centaur
Labs, Bayer Animal Health Isando, South Africa) admin-
istered intraperitoneally. The chest cavity was opened and
blood samples collected by cardiac puncture of the right
ventricle.
In Vivo Imaging
Subcutaneous infections by S. aureus were monitored by
scanning the mice in the IVIS 100 Imaging System
(Caliper Life Sciences). The animals were anaesthetised
with 2% isoflurane and imaged each day for a maximum of
5 min using a CCD camera (IVIS 100 Imaging System).
Living Image software (Caliper Life Sciences, Hopkinton,
MA, USA) was used to detect and quantify total photon
emission (number of photons/s/cm2) from defined regions
of interest (ROI) within each image.
Histology
Skin sections of three rats from each group were aseptically
removed, fixed in 4% (v/v) formaldehyde (PBS) for 24 h at
25C, embedded in paraffin, sectioned and stained with
hematoxylin and eosin (H&E). The samples were pro-
cessed and analysed at Pathcare Veterinary Pathologists
(Pathcare, Dietrich, Voigt, Mia and Partners, Goodwood,
South Africa).
Probiotics & Antimicro. Prot.

Results and Discussion be degraded by enzymes in blood when injected

Research on nisin in the treatment of subcutaneous infec-
tions is limited. To the best of our knowledge, only one
paper has been published on the control of subcutaneous
infections with a bacteriocin. Ghiselli et al. [8] infected
subcutaneously implanted grafts with Staphylococcus epi-
dermis. Grafts soaked in nisin before implantation had
lower infection counts (6.2 9 103 ± 1.3 9 103 CFU) than
grafts not treated (4.8 9 107 ± 2.0 9 106 CFU) [8]. In
this study, we investigated whether nisin F, injected sub-
cutaneously, were antimicrobial against a pathogenic strain
of S. aureus (Xen 36) in vivo. High levels of nisin F
(2 9 256 AU) have been used to compensate for the pos-
sible inactivation of the lantibiotic.
Identical levels of antimicrobial activity (6,400 AU ml-1)
were recorded when samples were spotted onto blood
agar and BHI agar. This suggested that nisin F would not
subcutaneously.
Photon emissions from mice that have been infected
with S. aureus Xen 36, and then treated with nisin F (group 1)
and saline (group 2) increased by 0.5 log after the first
24 h. Abscesses underneath the skin were visible after 24 h
in mice from both groups. At 24 and 48 h either nisin F
(group 1) or sterile physiological saline (group 2) was
administered subcutaneously. No significant difference
could be detected between group 1 and group 2 throughout
the duration of the trial (Figs. 1, 2). Histology detected
multifocal areas of necrosis with complete loss of cellular
detail were in the dermal stromas of the mice in both
groups. Small hair fragments were also visible. Subacute
active necrotic dermatitis and cellulitis were hence recor-
ded in the dermal stromas and deeper subcuticular tissues
of skin from mice in both groups, confirming that the
infection has not been cured. Nisin F, administered at

Fig. 1 A representative mouse

from each group depicts a time
series of photon detection
(photons per second per square
centimetre) from metabolically
active S. aureus Xen 36.
Group1: Infected and treated
with nisin F, group 2: infected
and treated with saline, group 3:
not infected and treated with
nisin F and group 4: not infected
and not treated

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 Probiotics & Antimicro. Prot.

6.5 Infected areas were also associated with a high infil-

6.0 tration of polymorphonuclear cells, lymphocytes and
Log10 values

5.5 fibroblasts (Fig. 3a). However, infected areas that have

5.0
4.5
4.0
3.5
3.0
1 2 3 4 5 6
Days
Fig. 2 Photon emissions (photons/s/cm2/sr) from defined regions of
interest (ROI) within selected images of mice in each group. Group1
(-•-): Infected and treated with nisin F, group 2 (-j-): infected and
treated with saline, group 3 (-m-): not infected and treated with nisin
F and group 4 (-h-): not infected and not treated
2 9 256 AU, 24 h apart, could thus not control the growth
of S. aureus Xen 36 in subcutaneous infections. The reason
for this is not clear. It may be that the level of infection
(number of S. aureus Xen 36 cells) was too high for the
nisin F dosage applied. However, if this was the case, one
would have expected slight variations in the photon read-
ings. The high standard deviations recorded for biolumi-
nescent flux measurements are normal. The level of
cutaneous infections caused by S. aureus may vary and are
not the same for each animal. The onset of the lesions may
also differ between mice. Similar observations have been
documented for cutaneous staphylococcal infections in
humans [3].
been treated with sterile saline showed less polymorpho-
nuclear cells (Fig. 3b). This suggests that the innate
immune system of mice treated with nisin F have been
more successful in actively combating the infection or that
the administration of nisin F could have modulated the
innate immune system.
The total photon emissions from mice not infected and
treated with nisin F (group 3) and mice not infected and not
treated (group 4) were significantly lower than the total
photon emissions from the infected groups (groups 1 and 2),
as expected. However, the total photon emissions recorded
for group 3 were slightly higher than the total photon
emissions of group 4 (Figs. 1, 2). This is an interesting
phenomenon since the only variable between the two
groups was the administration of nisin which does not
bioluminescence.
The skin pathology of the mice in group 3 (not infected
and treated with nisin F) ranged from no histological
changes to focal acute dermatitis. Focal areas of oedema
with mild infiltration of polymorphonuclear cells, and a
few macrophages, were detected in the dermal stroma of
mice with focal acute dermatitis and mice with mild focal
dermal oedema. The cytoplasma of the macrophages were
discoloured in the latter mice, probably due to pigment in
the protein product (Fig. 3c). No histological changes were

Fig. 3 Hematoxylin and Eosin

stains of skin tissue from a
mouse infected with S. aureus
Xen 36 and treated with nisin F,
b mouse infected with S. aureus
Xen 36 and treated with saline,
c mouse not infected and treated
with nisin F and d mouse not
infected and not treated
(control)

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Probiotics & Antimicro. Prot.
observed in the skin of mice from group 4, i.e. not infected
and not treated (Fig. 3d).
Atopic dermatitis, although not yet defined as an auto-
immune disease, is initiated and maintained by the actions
of inflammatory cells such as cytokines, chemokines and
T-cells [5]. Mice treated with nisin (both the infected and
non-infected groups) had a higher number of polymor-
phonuclear cells in the dermal stromas of the skin tissue.
Great care must therefore be taken since subcutaneously
injected nisin F, if acting as an immune modulator, could
worsen atopic dermatitis. The fact that some of the mice in
group 3 (not infected but treated with nisin F) developed
symptoms of dermatitis further supports this theory.
This study emphasizes the importance of investigating
the effect of lantibiotics on the immune system and to
determine immune and disease conditions where their
application may be detrimental to the host. A study per-
formed by Besselink et al. [2] illustrates that a pharma-
ceutical product that is considered safe for human
consumption, and widely used, may under specific cir-
cumstances be dangerous.
In this study, we have shown that subcutaneous
administration of nisin F has no effect on deep dermal
staphylococcal infections. Furthermore, the safety of
administrating nisin F subcutaneously is questionable since
it may modulate the innate immune system. Further studies
are in progress to determine if nisin F may be used as a
topical agent in the curing of superficial skin infections.
Acknowledgements Pathcare Veterinary Pathologists (Pathcare,
Dietrich, Voigt, Mia and Partners, Goodwood, South Africa) for
histology and blood cell counts and Dr. R. Smith, Department of
Physiology, University of Stellenbosch, for advice on animal care.
The research was supported by a grant from the National Research
Foundation (NRF), South Africa.
References
1. Awad SS, Elhabash SI, Lee L, Farrow B, Berger DH (2007)
Increasing incidence of methicillin-resistant Staphylococcus
aureus skin and soft-tissue infections: reconsideration of empiric
antimicrobial therapy. Am J Surg 194:606–610
2. Besselink MG, van Santvoort HC, Buskens E, Boermeester MA,
van Goor H, Timmerman HM, Nieuwenhuijs VB, Bollen TL, van
Ramshorst B, Witteman BJ, Rosman C, Ploeg RJ, Brink MA,
Schaapherder AF, Dejong CH, Wahab PJ, van Laarhoven CJ, van
der Harst E, van Eijck CH, Cuesta MA, Akkerman LM, Gooszen
HG, Dutch Acute Pancreatitis Study Group (2008) Probiotic
prophylaxis in predicted severe acute pancreatitis: a randomized,
double-blind, placebo-controlled trial. Lancet 371:651–659
3. Bunce C, Wheeler L, Reed G, Musser J, Barg N (1992) Murine
model of cutaneous infection with gram-positive cocci. Infect
Immun 60:2636–2640
4. Burkatovskaya M, Tegos GP, Swietlik E, Demidova TN, Castano
A, Hamblin MR (2006) Use of chitosan bandage to prevent fatal
infections developing from highly contaminated wounds in mice.
Biomaterials 27:4157–4164
5. Chan LS (2008) Atopic dermatitis in 2008. Curr Dir Autoimmun
10:76–118
6. De Kwaadsteniet M, Ten Doeschate K, Dicks LMT (2008)
Characterization of the structural gene encoding nisin F, a new
lantibiotic produced by a Lactococcus lactis subsp. lactis isolate
from freshwater fish (Clarius gariepinus). Appl Environ Micro-
biol 74:547–549
7. De Kwaadsteniet M, Ten Doeschate K, Dicks LMT (2009) Nisin
F in the treatment of respiratory tract infections caused by
Staphylococcus aureus. Lett Appl Microbiol 48:65–70
8. Ghiselli R, Giacometti A, Cirioni O, Dell’Acqua G, Mocchegiani
F, Orlando F, D’Amato G, Rocchi M, Scalise G, Saba V (2004)
RNAIII-inhibiting peptide and/or nisin inhibit experimental vas-
cular graft infection with methicillin-susceptible and methicillin-
resistant Staphylococcus epidermis. Eur J Vasc Endovasc Surg
27:603–607
9. Lo BM, Erwin EA (2008) Missed epidural brain abscess after
furunculosis. Am J Emerg Med 26:522
10. Moet GJ, Jones RN, Biedenbach DJ, Stilwell MG, Fritsche TR
(2007) Contemporary causes of skin and soft tissue infections in
North America, Latin America, and Europe: report from the
SENTRY Antimicrobial Surveillance Program (1998–2004).
Diagn Microbiol Infect Dis 57:7–13
11. Mortin LI, Li T, van Praagh AD, Zhang S, Zhang XX, Alder JD
(2007) Rapid bactericidal activity of daptomycin against methi-
cillin-resistant and methicillin-susceptible Staphylococcus aureus
peritonitis in mice as measured with bioluminescent bacteria.
Antimicrob Agents Chemother 51:1787–1794
12. Pallin DJ, Egan DJ, Pelletier AJ, Espinola JA, Hooper DC,
Camarqo CA (2008) Increased US emergency department visits
for skin and soft tissue infections, and changes in antibiotic
choices, during the emergence of community-associated methi-
cillin-resistant Staphylocococcus aureus. Ann Emerg Med
51:291–298
13. Sambrook JE, Fritsch F, Maniatis J (1989) Molecular cloning: a
laboratory manual, 2nd edn. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY
14. Wilson SE (2001) Clinical trial results with linezolid, an oxazo-
lidinone, in the treatment of soft tissue and postoperative Gram-
positive infections. Surg Infect (Larchmt) 2:25–35
15. Young LM, Price CS (2008) Community-acquired methicillin-
resistant Staphylococcus aureus emerging as an important cause
of necrotizing fasciitis. Surg Infect (Larchmt) 9:469–474
123