Probiotics & Antimicro. Prot. (2013) 5:26–35
fibrous mat was directly peeled off from the grounded
milligrams of PVOH fibers was also dissolved as a nega-
collector covered with aluminum foil. Fibers were then
tive control to eliminate the possibility of PVOH inhibi-
dried under vacuum for 4 h prior to further use.
tion. An overnight culture of M. luteus in TSB (c. 109CFU/mL) was diluted 100-fold by blending with *55 °Csoft agar (TSB supplemented with 7 g/L agar). Then, 4 mL
Morphology Characterization of Antimicrobial
of the soft agar containing M. luteus was transferred onto
the surface of a TSA plate. After 30 min of solidification, asterile glass pipette (approx. 5 mm diameter) was used to
Surface images of the antimicrobial PVOH fibers with and
create wells in the agar plate. Fifty microliters of purified
without subtilosin were collected with a commercial
subtilosin, PVOH? subtilosin fibers (24 h water solution),
Nanoscope IIIa Multi-Mode AFM (Veeco Instruments,
negative control and their double dilutions were injected
Plainview, NY, USA) equipped with a J scanner, which
into the wells in duplicate. After 24 h incubation at 37 °C,
was operated in tapping mode using a silicon cantilever.
minimum inhibitory concentration (MIC) was defined as
The scanned images were obtained at the scan size of
the lowest concentration to form a visible inhibition circle
5 9 5 lm and 50 9 50 lm. The scan frequency was set at
on the M. luteus growth layer. Tests were repeated three
0.1 Hz. The section analysis embedded in the software
Nanoscope 3.0 was utilized to calculate the fiber diameterdistribution.
Skin Cytotoxicity Assays
Assessment of Subtilosin Loading Capacity
The safety of nanofiber mats with and without incorporated
and Efficiency Via Well Diffusion Assay
subtilosin for use on human skin was tested on a cultured,reconstituted human epidermal model. The EpiDerm
The evaluation of subtilosin loading capacity and effi-
(NHEK) tissue model (MatTek Corporation, Ashland, MA,
ciency was conducted by agar plate well diffusion inhibi-
USA) consists of normal, human-derived epidermal kerat-
tion assays [All nanofiber mats were sterilized by
inocytes that are cultured to form a highly differentiated
direct exposure to UV light (257 nm) in a biosafety cabinet
model of the epidermis, and allows for in vitro testing of a
(Forma Class II, A2 Biological Safety Cabinet, Thermo
compound's irritancy and cytotoxicity. All EpiDerm tis-
Fisher Scientific, Pittsburgh, PA, USA) for 10 min per side
sues were maintained at 4 °C upon receiving and prior to
prior to use. Twenty milligrams of PVOH? subtilosin
use. The standard method developed by MatTek, and later
fibers was cut with sterile tweezers, immersed in 200 lL
outlined by Frasch et al. ], was followed. Briefly, all
sterile ddH2O and kept at 4 °C for 24 h. Twenty
tissues were preincubated in room temperature culturemedium at 37 °C with 5 % CO2 for 1 h prior to use.
Nanofiber mats containing 12 % PVOH or 12 % PVOH?subtilosin were sterilized by direct exposure to UV light ina biosafety cabinet for 10 min per side, and then cut to sizeusing a sterilized size 5 cork borer (Thermo Fisher Scien-tific). These nanofiber ‘‘discs'' were applied onto the sur-face of the pre-conditioned tissues with light pressure toensure that the disc came in full contact with the tissue.
Positive (50 ll Triton-X) and negative (50 ll sterileddH2O) controls and all test compounds were assayed intriplicate. The tissues were exposed to the test compounds
Fig. 1 Schematic diagram of nanofiber formation through the process
for 24 h at 37 °C with 5 % CO2. After exposure, the tissues
of electrospinning. A PVOH and subtilosin mixed solution (final
were washed with PBS, transferred into MTT assay buffer
concentrations: 0.12 g/mL PVOH and 0.9 mg/mL subtilosin) wasloaded into a plastic syringe, which was then mounted onto a syringe
and further incubated for 3 h at 37 °C with 5 % CO2. After
pump. During the electrospinning process, a liquid polymer jet is
a second wash with PBS, the tissues were submerged in
ejected at the end of the needle when a high-voltage electric field is
isopropanol and held at room temperature overnight to
applied to the polymer solution. The liquid polymer undergoes jet
allow for extraction of the formazan dye from the tissues.
whipping and solidification, and the resultant polymer fibers are thendeposited onto the grounded aluminum collector surface. The whole
The following day, the optical density of the formazan–
process was maintained at the condition of 15 kV for voltage supply,
isopropanol mixture from each tissue was measured in
0.2 mL/h for feeding rate and 10 cm for needle-to-collector distance.
triplicate at 570 nm. Cell viability was then calculated as a
As a negative control, a 0.12-g/mL PVOH solution without subtilosin
percentage of the average negative control tissues. Tests
was electrospun by the same procedure as above. Fibers werecollected from the grounded surface after 5 h processing
were performed at least twice.
Probiotics & Antimicro. Prot. (2013) 5:26–35
and because we found an unusually high inhibition at200 lg/mL, a virucidal assay was performed to determine
Antiviral Activity: Subtilosin Inhibits HSV-1
whether subtilosin at this concentration produces a direct
effect on the viral particle. To that purpose, viral suspen-sions were incubated with subtilosin 200 lg/mL for 15, 45
To assess the antiviral activity of subtilosin, we first
and 90 min. Then, aliquots were taken, diluted in MM and
determined the subtilosin's concentration that reduced cell
used to infect Vero cell monolayers to test virus survival
viability to 50 % as compared to the control (CC50) by
using a plaque assay. The incubation of viral suspension
using the MTT method. A virus yield inhibition assay was
with subtilosin for 15 min at 37 °C resulted in an inhibition
then performed to determine cell-free infectivity and the
of the viral titer of 99 %. After 45 min of treatment, the
selectivity index (SI), that is, the relationship between CC50
reduction in viral infectivity was higher than 99.99 %,
and EC50 values. For comparative purposes, the inhibitory
indicating that subtilosin is a potent virucidal agent active
effect of ACV and FOS was also assayed. Between 12 and
against tk-positive and tk-deficient (data not shown) HSV-1
100 lg/mL, subtilosin inhibited HSV-1 replication in a
dose-dependent manner (Fig. ) and a remarkably strong
To establish if subtilosin's inhibitory activity at low
concentration was due to its direct effect on the viral par-
200 lg/mL subtilosin. At a concentration of 100 lg/mL,
ticle, we performed a similar experiment at concentrations
subtilosin inhibited replication in a 99 % and at 12 lg/mL,
below 200 lg/mL. Treatment of viral suspensions for 1 h
a concentration 26-fold lower than the CC50, subtilosin still
at 37 °C with 6 to 100 lg/mL of subtilosin did not reduce
inhibited approximately 90 % of HSV-1. A 50 % of inhi-
viral infectivity (Fig. ), indicating that the reduction in the
bition was found with subtilosin at a concentration of
viral titers at these concentrations (as seen in Fig. ) were
9.6 lg/mL. Lower concentrations of the compound did not
only due to antiviral activity.
display considerable antiviral activity. The SI value for
In order to determine whether subtilosin interferes with
subtilosin against HSV-1 tk? was 33, while the values for
the initial steps of the viral multiplication cycle at a non-
ACV and FOS were 957 and 14, respectively. On the other
virucidal concentration, a time of addition experiment was
hand, subtilosin rendered a SI value of 31 against tk-defi-
performed. For that purpose, 50 lg/mL of subtilosin was
cient HSV-1, while the SI value for ACV was only 10.3
added to HSV-1 infected Vero cells at different times after
infection and at 24 h p.i., cell-free infectivity was deter-
Since it has been reported that microbicidal peptides
mined. As it is depicted in Fig. , when subtilosin was
such as defensins may inactivate HSV on direct contact
present starting from 1, 3, 5 or 8 h p.i., virus yields werereduced by approximately 2 log, suggesting that subtilo-sin's antiviral activity is not due to an inhibition of earlystages of viral multiplication.
To further characterize the inhibitory action of subtilo-
sin, Vero cells were infected with HSV-1 and treated withdifferent concentrations of the peptide ranging from 25 to100 lg/mL. Afterward, a polyacrylamide gel electropho-resis followed by a Western blot analysis was performed todetermine whether subtilosin reduces viral protein synthe-sis. As shown in Fig. viral protein gD synthesis was notaffected by the different concentrations used in thisexperiment, suggesting that the antiviral mechanism ofsubtilosin does not interfere with this or previous steps inthe viral multiplication cycle.
Fig. 2 Subtilosin inhibits HSV-1 replication in a dose-dependentmanner. Vero cells infected with HSV-1 tk? (m.o.i. = 1) were
Nanofiber Morphology
incubated with different concentrations of subtilosin for 24 h. Then,supernatants were harvested and cell-free virus yields were deter-mined by a plaque assay. Values are mean ± SD of the log reduction
Figure shows the tapping mode-atomic force microscopy
in HSV-1 titers for each concentration compared to no drug. Data are
(TP-AFM) images of antimicrobial PVOH fibers. Rela-
from three independent experiments, where each titration was carried
tively straight fibers were formed from the pure PVOH
out in duplicate. Percentages of inhibition of the different treatmentsare expressed in each column
solution (Fig. a), and an individual PVOH fiber's
Probiotics & Antimicro. Prot. (2013) 5:26–35
Table 1 Cytotoxicity and antiviral activity of subtilosin, aciclovir (ACV) and foscarnet (FOS) against HSV-1
Structural formula
Phosphonoformic acid trisodium salt
CC50: compound concentration required to reduce cell viability by 50 %, as determined by the MTT method
EC50: compound concentration required to reduce virus yield by 50 %
SI (selectivity index): ratio CC50/EC50
* Structural formula of subtilosin reproduced from Marx et al. []
Fig. 4 Low concentrations of subtilosin do not produce a virucidaleffect against HSV-1. HSV-1 tk? was incubated with subtilosin atconcentrations ranging from 6 to 100 lg/mL or MM for 60 min at37 °C to assay virucidal activity of subtilosin at concentrations thatdisplay antiviral activity. Then, aliquots were taken and Vero cells
Fig. 3 Subtilosin inactivates HSV-1 particles at a concentration of
were infected to test virus survival using a plaque assay. Values are
200 lg/mL. HSV-1 tk? and tk deficient (data not shown) were
mean ± SD from three independent experiments, where each titration
incubated with subtilosin at a concentration of 200 lg/mL or with
was carried out in duplicate
MM for 0, 15, 45 and 90 min at 37 °C. Then, aliquots were taken andVero cells were infected to test virus survival using a plaque assay.
Values are means of HSV-1 tk? infectivity ± SD from three
An individual PVOH? subtilosin fiber diameter was
independent experiments, where each titration was carried out in
determined to be 278 nm (Fig. d). This phenomenon is
likely due to subtilosin's interaction with PVOH, resulting
diameter was 567 nm (Fig. as calculated by the section
in weakened entanglement between the PVOH polymer
analysis embedded in the Nanoscope software. Addition of
chains, shifting the critical entanglement concentration of
subtilosin affected the fiber's morphology, resulting in
PVOH solution toward a higher value and subsequently
thinner fibers from the electrospinning process (Fig. c).
reducing fiber diameter.
Probiotics & Antimicro. Prot. (2013) 5:26–35
equal to 60 lg/mL. Thus, the loading capacity was calcu-lated to be 2.4 mg subtilosin per gram of fiber.
Since the water acting as a solvent in the subtilo-
sin ? PVOH solution was evaporated and dried during theelectrospinning and vacuum processes, 1 g of nanofiberwas assumed to be primarily composed of PVOH. Since8.3 mL of 0.12 g/mL PVOH solution was required to form1 g of fiber, the amount of subtilosin used in the blendedsolution was 7.6 mg. Thus, loading efficiency was calcu-lated to be 31.6 %. The loss of subtilosin may be the resultof the high voltage applied ] or the difficulty of sub-tilosin release from encapsulation.
Nanofibers Cytotoxicity
Fig. 5 Effect of time of subtilosin addition on HSV-1 production.
Vero cells infected with HSV-1 (m.o.i. = 1) were incubated withMM (VC) or MM containing subtilosin 50 lg/mL added either at 1, 3,
The safety of subtilosin-based nanofibers for use on human
5 or 8 h p.i. Cultures were further incubated at 37 °C until 24 h p.i
skin tissues was tested using the in vitro EpiDerm model.
Then, cell-free virus yields were determined by plaque assay. Values
After 24 h of exposure, the tissues exposed to the negative
are mean ± SD from three independent experiments, where each
titration was carried out in duplicate
2O) and PVOH fibers retained 100 % viability.
Tissues exposed to the PVOH? subtilosin fibers retainedan average of 98.5 % viability in comparison to the neg-ative control. There was zero viability for the tissuesexposed to the positive control, Triton-X.
Antimicrobial peptides have shown to act against viruses in
Fig. 6 Viral protein gD synthesis is not affected by subtilosin
many different ways. Most of them exert their activity by
treatment. Vero cells infected (Lanes 2–5) or not (Lane 1) with HSV-
directly inactivating the viral particle or inhibiting early
1 tk? at an m.o.i. of 1 were incubated at 37 °C for 1 h to allow
events in virus multiplication cycle For example, the
internalization. Afterward, the inoculum was discarded and themonolayers were covered with MM or MM containing subtilosin at
peptides MCP-1 and MCP-2, the human neutrophil peptide
different concentrations (Lane 2: virus control; lane 3: 25 lg/mL;
(HNP)-1 and brevinin-1 inactivate viral particles [,
lane 4: 50 lg/mL; lane 5: 100 lg/mL) and incubated at 37 °C. At
. On the other hand, dermaseptins, lactoferricin and the
24 h p.i. cell lysates were collected and subjected to Western blotting
polyphemusin analog T22 seem to interfere at the level of
and glycoprotein gD was analyzed with an antibody against gD. Toverify equal loading, Western blotting was performed with an
virus–cell interface, thus blocking viral entry [,
antibody to ERK1. Data are from one of two different experiments
However, other antimicrobial peptides exert their antiviralactivity by preventing virus multiplication, as melittin andcecropin which were shown to inhibit the HIV replication by
Loading Capacity and Loading Efficiency
suppressing viral gene expression Other bacteriocinssuch as CRL35 and ST4 V, isolated from Enterococcus
Loading capacity was defined as the amount of subtilosin
faecium and Enterococcus mundii respectively, inhibited the
released from a gram of subtilosin ? PVOH nanofiber.
HSV-1 and HSV-2 replication in a dose-dependent manner
Loading efficiency was defined as the ratio of loading
, ]. In this work, we studied subtilosin's activity
capacity to the amount of subtilosin used in the blended
against HSV-1. We found that subtilosin at a non-cytotoxic
solution to produce 1 g of nanofiber.
concentration of 200 lg/mL inactivates HSV-1 viral parti-
First, the MIC of subtilosin against M. luteus was
cles. The incubation of viral suspension with 200 lg/mL of
determined by well diffusion assay as 60 mg/L. The four-
subtilosin for 15 min was enough to reduce infectivity by
fold dilution of a 20-mg nanofiber per 200 lL ddH2O
99 % and after 45 min of treatment the reduction in viral
solution, equivalent to 25 mg fiber/mL ddH2O, was the
infectivity was higher than 99.99 %. Interestingly, at lower
lowest dilution that retained antimicrobial activity, estab-
non-virucidal doses, subtilosin inhibits HSV-1 multiplica-
lishing it as the MIC. The concentration of subtilosin
tion cycle in a dose-dependent manner. At subtilosin's
released from 25 mg of fiber in 1 mL of sterile ddH2O was
concentration of 100 lg/mL, 99 % inhibition of HSV-1
Probiotics & Antimicro. Prot. (2013) 5:26–35
Fig. 7 Surface morphology ofpolyvinyl alcohol-basednanofibers with and withoutincorporated subtilosin. Tappingmode-atomic force microscopy(AFM) images of PVOHelectrospun fibers includinga 50 lm 9 50 lm height imageof PVOH electrospun fibers;b 5 lm 9 5 lm height imageof PVOH electrospun fibersc 50 lm 9 50 lm height imageof PVOH ? subtilosinelectrospun fibers;d 5 lm 9 5 lm height imageof PVOH ? subtilosinelectrospun fiber
multiplication was achieved. Remarkably, 12 lg/mL sub-
reduce HSV and HIV transmission, because they do not
tilosin, a concentration 26-fold lower than the CC50, caused
prevent subclinical reactivations of the virus novel
more than 90 % inhibition of HSV-1. In addition, subtilosin
treatment methods are of critical importance. One possible
was equally active against HSV-1 tk? and tk-deficient
alternative is the use of subtilosin in a nanofiber-based for-
strains and rendered higher SI values than FOS, raising the
mulation. As we demonstrated, subtilosin is readily encap-
possibility for using subtilosin against ACV-resistant
sulated into PVOH fiber mats, which may be directly applied
mutants. It was previously mentioned that antimicrobial
onto areas of infection. Moreover, nanofibers both with and
peptides generally display virucidal activity or interfere with
without incorporated subtilosin were demonstrated to be fully
early events in viral multiplication cycle. Interestingly,
safe for human use. Heunis and Dicks have demonstrated
subtilosin is able to inactivate viral particles and to inhibit
the feasibility of incorporating a variety of different types of
viral multiplication, although in different concentrations.
antimicrobials into nanofibers, and proven that the fibers
Furthermore, it does not seem to affect any viral multipli-
retain antimicrobial activity against bacterial pathogens such
cation step up to viral protein synthesis as shown by the time
as methicillin-resistant Staphylococcus aureus, Escherichia
of addition of subtilosin experiment and Western blot
coli, Pseudomonas aeruginosa and Klebsiella pneumoniae.
analysis, suggesting that it may interfere with late stagessuch as viral formation and release. Taking into account thatsome studies suggest that subtilosin binds to lipid mem-
branes, that high concentrations of the compound may resultin membrane permeabilization [] and that viral formation
In this report, the natural, safe antimicrobial peptide sub-
and release are viral steps particularly sensible to membrane
tilosin was shown to have potent virucidal and antiviral
structure; future analysis will be conducted to elucidate the
activity against HSV-1. Preliminary research on the safety
precise mechanism underlying the antiviral activity of
of subtilosin in a novel nanofiber formulation indicates that
the formulation has potential for use in human applications.
Considering the incidence of HSV infections and the
Further research will be conducted to elucidate the
issues regarding the clinical approved antiherpetic drugs,
molecular mechanism underlying the antiviral activity of
such as the reported emergence of ACV, PCV and FOS
subtilosin, to optimize the nanofiber formulation and to
resistant mutants and that it has been recently shown that the
assay the activity of subtilosin nanofibers against this and
available antiviral treatment of herpetic infections fail to
other Herpesviridae family members.
Probiotics & Antimicro. Prot. (2013) 5:26–35
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acyclovir-resistant herpes simplex virus type 1 in trigeminal
tides. Eur J Clin Microbiol Dis 19:187–194
Probiotics & Antimicro. Prot. (2013) 5:36
Erratum to: Safety, Formulation and In Vitro Antiviral Activityof the Antimicrobial Peptide Subtilosin Against Herpes SimplexVirus Type 1
Nicola´s I. Torres • Katia Sutyak Noll • Shiqi Xu •Ji Li • Qingrong Huang • Patrick J. Sinko •Mo´nica B. Wachsman • Michael L. Chikindas
Published online: 16 February 2013Ó Springer Science+Business Media New York 2013
Erratum to: Probiotics & Antimicro. Prot.
Transparency declaration: Nicola´s I. Torres, Katia Sut-
yak Noll and Shiqi Xu equally contributed to the researchand to this manuscript. Mo´nica B. Wachsman, Michael L.
In the original version of this paper, the article note
Chikindas and Qingrong Huang are the manuscript's senior
(Transparency declaration) was missed out unfortunately,
authors in the areas of virology, bacteriology and formu-
which should have come in the front page of the article.
lation chemistry, correspondingly.
The missed article note is as follows.
The online version of the original article can be found under doi:
N. I. Torres M. B. WachsmanLaboratorio de Virologı´a, Departamento de Quı´mica Biolo´gica,Facultad de Ciencias Exactas y Naturales, Universidad deBuenos Aires, Ciudad Universitaria, Pabello´n 2, Piso 4, 1428Buenos Aires, Argentina
K. Sutyak Noll S. Xu J. Li Q. Huang M. L. Chikindas (&)School of Environmental and Biological Sciences,Rutgers, The State University of New Jersey,65 Dudley Road, New Brunswick, NJ 08901, USAe-mail: [email protected];[email protected]
K. Sutyak NollKraft Foods, Inc., 555 South Broadway, Tarrytown, NY 10591,USA
P. J. SinkoDepartment of Pharmaceutics, Ernest Mario School ofPharmacy, Rutgers, The State University of New Jersey,160 Frelinghuysen Road, Piscataway, NJ 08854, USA
Source: http://foodsci.rutgers.edu/chikindas/MChikindas%20Manuscripts/PAAP-2013-5-26.pdf
Elementalwatson "la" revista ………………. Revista cuatrimestral de divulgación "En el conocimiento y la cultura no Año 4, número 11 sólo hay esfuerzo sino también placer. Llega un punto donde estudiar, o investigar, o Universidad de Buenos Aires Ciclo Básico Común (CBC) aprender, ya no es un esfuerzo y es puro
The diabetic foot infection can lead to tissue necrosis and amputation. Diabetes is the leading non-traumatic cause of major amputation of the lower limbs. Miles J levyJonathan ValabhjiQ2 NeuropathyNerve damage due to disease of the vasa nervorum results in a ‘glove and stocking' sensorimotor peripheral neuropathy that may progress proximally. The motor component results in dener-vation of the small muscles of the foot, leading to: • hyperextension at the metatarsophalangeal joints