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Antiviral Effects of Lactococcus lactis on Feline Calicivirus, A Human Norovirus Surrogate

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Abstract

Foodborne viruses, particularly human norovirus (NV) and hepatitis virus type A, are a cause of concern for public health making it necessary to explore novel and effective techniques for prevention of foodborne viral contamination, especially in minimally processed and ready-to-eat foods. This study aimed to determine the antiviral activity of a probiotic lactic acid bacterium (LAB) against feline calicivirus (FCV), a surrogate of human NV. Bacterial growth medium filtrate (BGMF) of Lactococcus lactis subsp. lactis LM0230 and its bacterial cell suspension (BCS) were evaluated separately for their antiviral activity against FCV grown in Crandell–Reese feline kidney (CRFK) cells. No significant antiviral effect was seen when CRFK cells were pre-treated with either BGMF (raw or pH 7-adjusted BGMF) or BCS. However, pre-treatment of FCV with BGMF and BCS resulted in a reduction in virus titers of 1.3 log10 tissue culture infectious dose (TCID)50 and 1.8 log10 TCID50, respectively. The highest reductions in FCV infectivity were obtained when CRFK cells were co-treated with FCV and pH 7-adjusted BGMF or with FCV and BCS (7.5 log10 TCID50 and 6.0 log10 TCID50, respectively). These preliminary results are encouraging and indicate the need for continued studies on the role of probiotics and LAB on inactivation of viruses in various types of foods.
1 23
Food and Environmental Virology
The Official Journal of the International
Society for Food and Environmental
Virology
ISSN 1867-0334
Volume 6
Number 4
Food Environ Virol (2014) 6:282-289
DOI 10.1007/s12560-014-9164-2
Antiviral Effects of Lactococcus lactis on
Feline Calicivirus, A Human Norovirus
Surrogate
Hamada A.Aboubakr, Amr A.El-Banna,
Mohammed M.Youssef, Sobhy A.A.Al-
Sohaimy & Sagar M.Goyal
1 23
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ORIGINAL PAPER
Antiviral Effects of Lactococcus lactis on Feline Calicivirus,
A Human Norovirus Surrogate
Hamada A. Aboubakr Amr A. El-Banna
Mohammed M. Youssef Sobhy A. A. Al-Sohaimy
Sagar M. Goyal
Received: 20 May 2014 / Accepted: 8 August 2014 / Published online: 17 August 2014
ÓSpringer Science+Business Media New York 2014
Abstract Foodborne viruses, particularly human norovi-
rus (NV) and hepatitis virus type A, are a cause of concern
for public health making it necessary to explore novel and
effective techniques for prevention of foodborne viral
contamination, especially in minimally processed and
ready-to-eat foods. This study aimed to determine the
antiviral activity of a probiotic lactic acid bacterium (LAB)
against feline calicivirus (FCV), a surrogate of human NV.
Bacterial growth medium filtrate (BGMF) of Lactococcus
lactis subsp. lactis LM0230 and its bacterial cell suspen-
sion (BCS) were evaluated separately for their antiviral
activity against FCV grown in Crandell–Reese feline kid-
ney (CRFK) cells. No significant antiviral effect was seen
when CRFK cells were pre-treated with either BGMF (raw
or pH 7-adjusted BGMF) or BCS. However, pre-treatment
of FCV with BGMF and BCS resulted in a reduction in
virus titers of 1.3 log
10
tissue culture infectious dose
(TCID)
50
and 1.8 log
10
TCID
50
, respectively. The highest
reductions in FCV infectivity were obtained when CRFK
cells were co-treated with FCV and pH 7-adjusted BGMF
or with FCV and BCS (7.5 log
10
TCID
50
and 6.0 log
10
TCID
50
, respectively). These preliminary results are
encouraging and indicate the need for continued studies on
the role of probiotics and LAB on inactivation of viruses in
various types of foods.
Keywords Norovirus Feline calicivirus Lactic acid
bacteria Probiotics Antiviral activity Lactococcus
lactis Foodborne viruses
Introduction
Foodborne illnesses associated with contaminated food
continue to plague public health as well as world econo-
mies. The economic cost of foodborne illnesses is
approximately $152 billion in the US alone (Scharff 2010).
Enteric viruses, particularly human norovirus (NV) and
hepatitis virus type A, are the leading causes of viral
foodborne illnesses (Anonymous 2012; Koopmans and
Duizer 2004). Human NV, one of the top five highest-
ranking pathogens with respect to the total cost of food-
borne illness in the US, belongs to family Caliciviridae and
is a well-known cause of ‘‘winter-vomiting disease’’ or
‘stomach-flu’’ (ECDC 2013; Scharff 2012). The U.S.
Centers for Disease Control and Prevention (2013) reported
that NV causes 19–21 million cases of acute gastroenteritis
annually in the US and leads to 1.7–1.9 million outpatient
visits, 400,000 emergency room visits, 56,000–71,000
hospitalizations, and 570–800 deaths, mostly among young
children. More than half of all foodborne disease outbreaks
H. A. Aboubakr S. M. Goyal (&)
Department of Veterinary Population Medicine, College of
Veterinary Medicine, University of Minnesota, 1333 Gortner
Ave, St. Paul, MN 55108, USA
e-mail: goyal001@umn.edu
H. A. Aboubakr S. M. Goyal
Veterinary Diagnostic Laboratory, College of Veterinary
Medicine, University of Minnesota, 1333 Gortner Ave, St. Paul,
MN 55108, USA
H. A. Aboubakr A. A. El-Banna M. M. Youssef
Food Science and Technology Department, Faculty of
Agriculture, Alexandria University, Aflaton St., El-Shatby,
P.O. Box 21545, Alexandria, Egypt
S. A. A. Al-Sohaimy
Department of Food Biotechnology, Arid Land Cultivation and
Development Institute, City of Scientific Research and
Technology Applications, New Borg El Aarab,
Alexandria 21934, Egypt
123
Food Environ Virol (2014) 6:282–289
DOI 10.1007/s12560-014-9164-2
Author's personal copy
due to a known cause reported to CDC from 2006 to 2010
was attributed to NV. In the European Union, caliciviruses
(primarily NV) were responsible for 507 of 675 foodborne
viral outbreaks (European Food Safety Authority 2009).
The minimal effect of most food processing methods on
the inactivation of foodborne viruses has been reviewed
(Baert et al. 2009; FAO/WHO 2008; Hirneisen et al. 2010).
In addition, recent experiments with NV in a variety of
foods revealed that freezing, cooling, and mild heat treat-
ment (minimal food processing) were not effective in sig-
nificantly reducing virus titers (Mormann et al. 2010).
Thus, development of novel, efficient and safe strategies
for controlling viral contamination of foods is of great
interest to food scientists and food producers. In this
regard, biopreservation (control of one organism by
another) has received much attention in the last decade
(Dalie
´et al. 2010).
Among natural biological antagonists, lactic acid bac-
teria (LAB), a part of the intestinal microflora, have been
widely used for the production of fermented foods. These
bacteria have a long history of use in foods and are known
to have beneficial health effects in humans. Many com-
pounds are produced during LAB fermentation some of
which have an antimicrobial activity. These compounds
include: hydrogen peroxide, organic acids, diacetyl,
hydroxyl fatty acids, proteinaceous compounds, and bac-
teriocins (Dalie
´et al. 2010). The antagonistic effects of
LAB against pathogenic bacteria e.g., Listeria monocyt-
ogenes,Staphylococcus aureus,Staphylococcus epidermi-
dis,Streptococcus sanguins,Proteus mirabilis, and
Yersinia spp. have been reported (Al Askari et al. 2012;
Cizeikiene et al. 2013; Dalie
´et al. 2010; Koo et al. 2012;
Schwenninger et al. 2011).
Recently, there has been an increased interest in using
LAB and other probiotic bacteria as viral inhibitors against
coronavirus (Maragkoudakis et al. 2010), herpes simplex
virus (Khani et al. 2012), human immunodeficiency virus
(Martı
´n et al. 2010), influenza virus (Kobayashi et al. 2011;
Lee et al. 2013; Youn et al. 2012), rotavirus (RV; Mara-
gkoudakis et al. 2010), and vesicular stomatitis virus (VSV;
Botic
´et al. 2007). Lactococcus lactis (formerly, Strepto-
coccus lactis) is one of the most important LABs. It is a
Gram-positive bacterium used extensively in the produc-
tion of butter milk and cheese (Madigan et al. 2012). Other
uses include the production of pickled vegetables, beer or
wine, bread, and other fermented foodstuff, such as soy-
milk kefir. This organism has a homofermentative metab-
olism and produces L-(?)-lactic acid (Samarz
ˇija et al.
2001). It can also produce D-(-)-lactic acid when cultured
at low pH (A
˚kerberg et al. 1998). The capability to produce
lactic acid is one of the reasons why L. lactis is one of the
most important microorganisms in the dairy and food
industries and has achieved the GRAS (generally regarded
as safe) status (FDA 2012).The present study was under-
taken to determine the antiviral activity of L. lactis subsp.
lactis LM0230 against feline calicivirus (FCV), a surrogate
of NV.
Materials and Methods
Bacterial Strain
Lactococcus lactis subsp. lactis LM0230 was kindly pro-
vided by Dr. Dan O’Sullivan, Professor of Food Microbi-
ology, Department of Food Science and Nutrition,
University of Minnesota. The strain was maintained at
-20 °C in De Man, Ragosa, and Sharp (MRS) broth
(Oxoid, Basingstoke, England) supplemented with 20 %
(v/v) glycerol as a cryoprotective agent.
Preparation of Bacterial Growth Medium Cell-Free
Filtrate (BGMF) and Bacterial Cell Suspension (BCS)
The bacterium was grown in 30 mL MRS broth for 24 h at
30 ±02 °C, under anaerobic conditions. The culture was
centrifuged at 2,0009gfor 15 min. The supernatant was
collected and divided into two portions. One portion (its
measured pH was 3.7) was filter-sterilized using 0.22 lm
PVDF membrane filters (Millex
Ò
.GV, Millipore, Bedford,
MA) and was labeled as ‘raw BGMF’. The second portion
was adjusted to pH 7.0 ±0.05 using 1 M sodium
hydroxide solution, filter-sterilized, and labeled as ‘pH-7
adjusted BGMF’. The BCS was prepared by washing the
pellet of bacteria obtained above twice with sterile peptone
phosphate water broth (PPWB; Fluka, Switzerland) to
remove excess MRS followed by centrifugation at
2,0009gfor 15 min. The washed pellet was re-suspended
in 10 mL of PPWB. The viable bacterial cell count was
determined spectrophotometrically by measuring the opti-
cal density (OD) at 620 nm against cell-free PPWB as a
blank. A standard curve was created by plotting ODs of
10-fold serial dilutions of a standard BCS versus mathe-
matically calculated colony forming units (CFUs)/mL of
each dilution. The CFU/mL of the standard BCS was
measured initially using the plate count technique on MRS
agar plates.
Cell Line and Growth
A Crandell–Reese feline kidney (CRFK) cell line was
obtained from Veterinary Diagnostic Laboratory, Univer-
sity of Minnesota, USA. Cells were grown in Corning
cellgro minimum essential medium (MEM) with Earle’s
salts and L-glutamine (Mediatech, Inc., USA) supple-
mented with 8 % fetal bovine serum (FBS) and standard
Food Environ Virol (2014) 6:282–289 283
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antibiotics at 37 °Cin5%CO
2
in tissue culture flasks until
confluent monolayer of cells is formed. The cell culture
was regularly passaged. To perform biological assays, the
cells were seeded in 96 well plates (5 910
4
cells/well) and
incubated for 48 h at 37 °C under 5 % CO
2
to reach the
monolayer.
Virus Propagation and Titration
FCV, strain 255, was used in the experiments. The virus
was propagated in CRFK monolayers. Flasks containing
CRFK cell monolayers were infected with FCV. When
cytopathic effect (CPE) was observed by inverted micro-
scope (24–48 h after infection and incubation at 37 °C) the
supernatant containing the virus was collected after freez-
ing and thawing three times followed by centrifugation at
3,0009gfor 15 min. Virus was stored at -80 °C until
used. For virus titration, the 50 % tissue culture infectious
dose (TCID
50
) method was used. In which, serial 10-fold
dilutions of samples were prepared in MEM containing
4 % FBS and inoculated in confluent CRFK monolayers
prepared in 96-well microtiter plates using three wells per
dilution. The cells were examined for the development of
CPE daily up to 5 days. The endpoint was taken as the
highest dilution of the virus which produced CPE in 50 %
of the inoculated cells. Viral titers were calculated by the
Karber formula (Karber1931) and were expressed as
TCID
50
/0.1 mL.
Cytotoxicity Assay
The minimum non-toxic dilutions (MNTDs) of each type
of BGMF were determined based on cellular morphologi-
cal alteration method described by Orhan et al. (2010).
Briefly, several dilutions of each BGMF prepared in MEM
were inoculated in monolayers of CRFK cells contained in
96-well microplates at 100 lL/well followed by incubation
for 48 h at 37 °C under 5 % CO
2
. Dilutions that were not
toxic to viable cells were labeled as non-toxic and were
also compared with non-treated cells (negative control) for
confirmation. The lowest non-toxic dilutions were chosen
as (MNTDs).
Antiviral Assays
The anti-FCV activity of L. lactis LM0230 and its
metabolites was assayed by three different methods. In
which, FCV titers of treated and non-treated virus or cells
(control) were calculated.
(i) Pre-treatment of cells with BGMF after discarding
its growth medium, the CRFK cell monolayers
were covered with 100 lL of non-toxic dilutions
(MNTDs) of the two different types of BGMF
(1:10 diluted and undiluted) from raw and pH
7-adjusted BGMF, respectively. After incubation
at 37 °Cin5%CO
2
incubator for various
incubation times (30 min, 90 min, and 24 h), the
monolayers were washed with MEM. Immedi-
ately, the washed monolayers were infected with
100 lL of FCV 10-fold serial dilutions.
(ii) Pre-treatment of cells with BCS, the CRFK
monolayers were incubated with 20, 50, and
100 lL of BCS (5.1 910
8
CFU/mL) for 30, 60,
and 90 min at 37 °Cina5%CO
2
incubator.
After incubation the non-bound bacteria were
removed by washing two times with MEM
100 lL each. The monolayers were then infected
with FCV dilutions.
(iii) Pre-treatment of virus with BGMF, aliquots
(250 lL) of FCV suspension were mixed sepa-
rately with equal volumes of raw BGMF and pH
7-adjusted BGMF (both undiluted) in 1.5 mL
sterile Eppendorf tubes. After incubation at 37 °C
in 5 % CO
2
incubator for different times (30 min,
90 min, and 24 h), 10-fold serial dilutions were
prepared from each mixture followed by infection
of CRFK monolayers.
(iv) Pre-treatment of virus with BCS (virus adsorption
to bacterial cells), aliquots (250 lL) of FCV
suspension were separately mixed with equal
volumes of BCS containing different bacterial
cell counts (1 910
8
,2910
8
,3910
8
CFU/
250 lL) in 1.5 mL sterile Eppendorf tubes. After
incubation at 37 °Cin5%CO
2
incubator for
different times (30 min, 90 min, and 24 h), the
mixtures were centrifuged at 12,0009gfor 3 min.
10-Fold serial dilutions of the supernatant were
prepared in MEM and 100 lL of each dilution
was used to infect the CRFK monolayers for
titration.
(v) Co-treatment of cells and virus with BGMF,
10-fold serial dilutions of FCV were prepared in
different solutions of raw BGMF and pH
7-adjusted BGMF as diluents followed by infec-
tion of CRFK monolayers. Three different dilu-
tions of raw BGMF and pH 7-adjusted BGMF
(1:10, 1:20, 1:30, and undiluted, 1:5, 1:10 v/v in
MEM medium) were used, respectively.
(vi) Co-treatment of CRFK cells with BCS and virus,
the CRFK monolayers were inoculated with 20,
50, and 100 lL of BCS (5.1 910
8
CFU/mL).
Immediately, the monolayers were infected with
serial 10-fold dilutions of FCV prepared in MEM.
After the fifth day of incubation, the wells were
washed two times with MEM 100 lL each, to
284 Food Environ Virol (2014) 6:282–289
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remove the bacterial cells overlaying layers which
prevent observation of CPEs under microscope.
Cell control and bacterial-treated cell control
wells were done for discrimination between CPE
versus intact cells, and normal CPE versus bac-
terial-contaminated cells, respectively.
Statistical Analysis
Each titration was carried out in triplicate and each
experiment was triplicated. The results are the
mean ±standard deviation. The analysis of variance
(ANOVA) was generated by Ftest. The statistical analysis
was carried out using STATISTICA software, v. 10
(Statsoft, Inc., USA).
Results
Cytotoxicity of BGMF–CRFK Cells
Raw BGMF exhibited toxicity to CRFK cells at 0 and 1:5
dilutions while higher dilutions exhibited no toxicity. On
the other hand, pH 7-adjusted BGMF did not show any
toxicity in diluted or undiluted forms.
Antiviral Activity of L. lactis LM0230
(i) Pre-treatment of cells with BGMF, the CRFK
cells were pre-treated with raw and pH 7-adjusted
BGMFs at their MNTDs (1:10 and 0 dilution,
respectively) for 30 min, 90 min or 24 h. As
shown in Fig. 1, there is no significant decrease
(PC0.01) in FCV titer after pre-treatment of
CRFK cells either with raw or pH 7-adjusted
BGMF. The time of pre-treatment also had no
significant effect (PC0.01).
(ii) Pre-treatment of cells with BCS, the CRFK cells
were pre-treated with various BCS volumes (20, 50,
and 100 lL) to examine the effect of number of
bacterial cells on the capability of CRFK cells to
support FCV replication. The cells were pre-treated
for 30, 60, or 90 min for each BCS volume. Except
for a little decrease in FCV titer (0.5 log
10
TCID
50
/
0.1 mL) with CRFK treated with 100 lL of BCS for
90 min, neither bacterial cell count nor the treat-
ment time had any significant effect on FCV titer
(P\0.01; data not shown).
(iii) Pre-treatment of FCV with BGMF, the pre-treat-
ment of FCV with raw BGMF for 30 min, 90 min,
and 24 h resulted in significant reductions
(P\0.01) in FCV titer by approximately 0.7,
1.0, and 1.3 log
10
TCID
50
/0.1 mL, respectively,
whereas pre-treatment with pH 7-adjusted BGMF
led to non-significant decreases (PC0.01) with
all pre-treatment times (Fig. 2).
(iv) Pre-treatment of FCV with BCS (virus adsorption
to bacterial cells), the pre-treatment of FCV with
BCS containing 1 910
8
,2910
8
and
3910
8
CFU of L. lactis LM0230 resulted in
non-significant decreases (PC0.01) in FCV titers
at either 30 or 90 min (Fig. 3). However, virus
titers were significantly reduced (P\0.01) when
the virus was treated for 24 h with BCS containing
1910
8
,2910
8
, and 3 910
8
CFU (approxi-
mately 1.2, 1.3, and 1.8 log
10
TCID
50
/0.1 mL
reductions, respectively).
Fig. 1 Effect of pre-treatment of CRFK cells with raw and
pH 7.0-adjusted BGMF on FCV titer. Pre-treatment times used were
30 min, 90 min, and 24 h. Data shown are an average of triplicate
experiments. Error bars represent standard deviations
Fig. 2 Effect of pre-treatment with raw and pH 7.0-adjusted BGMF
on FCV titer. Treatment times used were 30 min, 90 min, and 24 h.
Data are average of triplicate experiments. Error bars represent
standard deviations
Food Environ Virol (2014) 6:282–289 285
123
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(v) Co-treatment of cells and virus with BGMF, co-
treatment of CRFK simultaneously with BGMFs
and FCV as virus infection led to significant
decreases in FCV titers (P\0.01; Fig. 4). The
highest decrease in virus titer (7.5 log
10
TCID
50
/
0.1 mL) was obtained by co-treatment with pH
7-adjusted BGMF at MNTD (0 dilution). There
were lower decreases in FCV titers with higher
dilutions of pH 7-adjusted BGMF (1.3 and 1.0
log
10
TCID
50
/0.1 mL with 1:5 and 1:10 dilutions).
Similar trend was seen with co-treatment with raw
BGMF. The highest decrease in FCV (P\0.01)
was attained with MNTD (1:10 dilution) of raw
BGMF followed by 1:20 dilutions to be 1.5, 1.3
log
10
TCID
50
/0.1 mL reduction, respectively. The
highest dilution (1:30) showed non-significant
decrease in the virus titer (0.3 log10 TCID
50
/
0.1 mL).
(vi) Co-treatment of cells with BCS and virus, the co-
treatment of CRFK monolayers with different
volumes (20, 50, and 100 lL) of BCS
(5.1 910
8
CFU/mL) during simultaneous FCV
infection resulted in significant decreases
(P\0.01) in FCV titer versus its titer with
control monolayers (without BCS). The highest
decrease in FCV titer (6.0 log
10
TCID
50
/0.1 mL)
was attained by treatment with 100 lL followed
by approximately 5.7 and 5.0 log
10
TCID
50
/
0.1 mL when CRFK monolayer was treated by
50 and 20 lL of BCS, respectively (Fig. 5). There
were no statistically significant differences
(PC0.01) between the decreasing values attrib-
uted to the three BCS volumes used.
Discussion
To study the antiviral activity of LAB and probiotics, L.
lactis ssp. lactis LM0230 was chosen as a model because it
is a common LAB with probiotic properties (Heoa et al.
2013). The FCV was chosen as a surrogate of NV because
the former does not grow in vitro although several attempts
have been made to accomplish this task (Guix et al. 2007;
Malik et al. 2005; Straub et al. 2007). In addition, the FCV
has been used as a surrogate to evaluate the efficacy of
Fig. 3 Effect of pre-treatment with BCS on FCV titer. Different
bacterial cell counts (1 910
8
,2910
8
, and 3 910
8
CFU) and three
treatment times for each were used. Data are average from triplicate
experiments. Error bars represent standard deviations
Fig. 4 Effect of co-treatment of CRFK with FCV and raw or
pH 7.0-adjusted BGMF (three different dilutions of each). Data are
average from triplicate experiments. Error bars represent standard
deviations
Fig. 5 Effect of co-treatment of CRFK with FCV and different
volumes of BCS (20, 50, and 100 lL). Data are average from
triplicate experiments. Error bars represent standard deviations
286 Food Environ Virol (2014) 6:282–289
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common preservation processes used in the food industry
(Baert et al. 2009; Butot et al. 2008). The FCV belongs to
the same Caliciviridae family as does human NV (Bidawid
et al. 2000; D’Souza et al. 2006).
To avoid interference of BGMF toxicity with viral
CPEs, preliminary cytotoxicity assays were done to
determine the MNTD of each type of BGMF. The cyto-
toxicity of raw BGMF at 0 and 1:5 dilutions may have been
due to their low pH (pH 3.7 and 5.0, respectively) since 0
dilution of pH 7-adjusted BGMF did not show any toxicity.
Pre-treatment of CRFK cells with BGMF or BCS had
non-significant decreases in FCV titer ranged from 0 to
68 % (less than one log
10
TCID
50
/0.1 mL). This result is in
agreement with an earlier report in which 68 and 60 % of
VSV infectivity was diminished when IPEC-J2 cells were
pre-treated with BGMF or BCS of certain LAB strains
(Botic
´et al. 2007).
Significant time-dependent decrease in FCV titer was
obtained by pre-treatment of FCV with raw BGMF but not
with pH 7-adjusted BGMF. The main difference between
the two types of BGMF is the status of lactic acid excreted
in the growth medium by L. lactis. Lactic acid was neu-
tralized by sodium hydroxide in pH 7-adjusted BGMF.
Therefore, its effect was eliminated by transforming lactic
acid into its sodium salt (sodium lactate) at pH 7.0. This
explanation is supported by a similar study in which pre-
treatment of FCV with 0.3 % D,L-lactic acid solution (pH
3.4–3.5) at 20 °C led to 1.3 log
10
reduction in FCV titer
(Straube et al. 2011). The pH of undiluted raw BGMF in
our study was 3.7. We hypothesize that the viral capsid
proteins are denaturated due to the effects of acid pH on
non-enveloped viruses (Rodger et al. 1977; Straube et al.
2011), thus preventing viral attachment to its host cells.
Pre-treatment of FCV with BCS resulted in a decreased
virus titer after 24 h but not after 30 or 90 min. Similarly,
Botic
´et al. (2007) reported 70 % reduction in infectivity of
VSV after 24 h incubation with different LAB strains.
They attributed this reduction to the adsorption or binding
of the virus on the surface of LAB strains probably because
peptidoglycans in the cell walls of LAB trapped the virus
(Botic
´et al. 2007). The cell wall of L. lactis is also known
to have a peptidoglycan structure consisting of A4a-type
peptidoglycan, with a monomer primary structure (Glc-
NAc-MurNAc-L-Ala-a-D-Glu-L-Lys-D-Ala) and a D-Asp in
the interpeptide bridge, attached to the a-amino group of
Lys (Courtin et al. 2006). Some Lactobacillus strains have
been shown to trap HIV virions by binding the mannose
sugar rich ‘‘dome’’ of their attachment glycoprotein gp 120
(Carlson et al. 2004; Chang et al. 2009). A similar mech-
anism may also have worked in the bacterium–virus
interaction system of the present study.
In co-treatment experiments, the FCV titers were
reduced by both types of BGMFs, but complete inhibition
of FCV infectivity was only attained when undiluted pH
7-adjusted BGMF was used (Fig. 4). We hypothesize that
the extracellular metabolites of L. lactis excreted in BGMF
might prevent the attachment of FCV to the cells affecting
its entrance into the cells. The observed antiviral activities
of pH 7-adjusted BGMF indicate that lactic acid may not
be the key factor in this action where it was transformed to
sodium lactate during pH adjustment of BGMF. It has been
reported previously that metabolites of L. lactis such as
bacteriocins (Akkoc¸ et al. 2011; Choi et al. 2000; Samar-
z
ˇija et al. 2001) and hydrogen peroxide (Grufferty and
Condon 1983; Samarz
ˇija et al. 2001; Van Niel et al. 2002)
may be responsible for such action. Antiviral activity of
bacteriocins and bacteriocin-like substances produced by
LAB, probiotics, and certain other bacteria has been
reported (Ermolenko et al. 2010; Saeed et al. 2007;
Todorov et al. 2005; Torres et al. 2013; Wachsman et al.
2003). Hydrogen peroxide is also a well-known antiviral
substance (Roberts and Antonoplos 1998). Antiviral
activity of probiotic bacteria against VSV has been attrib-
uted to their metabolites (Botic
´et al. 2007).
Co-infection of CRFK with FCV and BCS showed about
7.5 log
10
TCID
50
/0.1 mL (*100 %) reduction in FCV
infectivity (Fig. 4). In similar work, the infectivity of VSV
was decreased by 60 % when IPEC-J2 cells co-infected
with VSV and different LAB strains (Lactobacilli and
Bifidobacteria) and VSV (Botic
´et al. 2007). Our results are
also in agreement with Maragkoudakis et al. (2010) who
observed significant decreases in infectivity of transmissi-
ble gastroenteritis coronavirus (TGEV) and RV when
hosting cells co-infected with the viruses in presence of
Lactobacillus sp. It was hypothesized that LAB cells
induced release of reactive oxygen species such as NO
-
and H
2
O
2
, which may be responsible for killing the studied
viruses (TGEV and RV; Maragkoudakis et al. 2010).
Competition between bacterial cells and FCV for attaching
to the functional receptors on the cells may also help elu-
cidate these results. It is also possible that LAB may
establish a ‘‘cross talk’’ (some sort of signaling) or alter the
state of the epithelial cells and macrophages, which leads
to an antiviral response as suggested by Botic
´et al. (2007).
Finally, four possible mechanisms of the anti-FCV
effect of L. lactis subsp. lactis LM0230 can be proposed.
First, the lower pH related to the excretion of lactic acid by
LAB may be responsible for denaturation of capsid pro-
teins of the virus preventing its attachment to host cells.
Second, the peptidoglycan structure of LAB may trap viral
particles. Third, production of different metabolites (such
as bacteriocins and hydrogen peroxide) can prevent the
entrance of the virus into host cells thereby inhibiting its
replication. Finally, the competition between the bacterial
cells and the virus for attachment on host cells may be
occurred. In addition, the induction effect of the bacterium
Food Environ Virol (2014) 6:282–289 287
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for the host cells to produce reactive oxygen substances
might kill the virus.
In conclusion, this study reported for the first time, an
antiviral effect of L. lactis subsp. lactis LM0230 (as a dual
model of LAB and probiotics) against FCV as a human NV
surrogate. This indicates that LAB and probiotics-based
fermented food may hold a promise in preventing food-
borne viruses and that these bacteria hold promise as bio-
preservative agents in controlling the contamination of
foods with viruses. Although preliminary, the results pre-
sented here are of particular importance and merit further
investigation to understand deeply the mechanisms of LAB
and probiotics antiviral effect and to study its activity in
food models.
Acknowledgments Funding provided by the Cultural Affairs and
Mission Sector, Ministry of Higher Education and Scientific
Research, Egypt is gratefully acknowledged.
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The objective of this study was enumeration, identification of lactic acid bacteria LAB from dried fruits and testing their antibacterial activity against different types of bacteria. Dilution method and cultivation in selective media was used for enumeration LAB, the isolates were identified by their physiological and biochemical characteristics and their antibacterial activity was performed by the agar well diffusion method. The results showed that thirty-seven isolates of LAB were isolated from tested samples, The isolates belonged to Lactococcus lactis subsp lactis (8), Lactococcus raffinolactis (6), Streptococcus thermophilus (6), Pediococcus acidilactici (3), Lactobacillus delbrueckii subsp. bulgaricus (2), Lactobacillus helveticus (3), Lactobacillus plantarum(4), Lactobacillus alimentarius (1), Lactobacillus brevis (2) and Lactobacillus fermentum(2).The results of antibacterial activity showed that seven CFSs of LAB had antibacterial activity against at least four strains tested. Lactobacillus fermentum had the best activity, they inhibited eight strains from sixteen tested strains, such as Streptococcus spp, Streptococcus sanguins, Staphylococcus epidermis, Staphylococcus aures, Proteus mirabilis, Hafnia alveie and Yersinia spp. In general, CFSs wewre active against the Gram positive more than Gram negative strains. MICs values were between 25 -100 AU/ml.
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We investigated the effects of a potential probiotic strain Lactococcus lactis subsp. lactis I2 on the immune response and growth of olive flounder (Paralichthys olivaceus), and their capacity to prevent streptococcosis after Streptococcus iniae challenge. The L. lactis subsp. lactis I2 strain, isolated from olive flounder intestine, was supplemented orally as a feed additive (~ 108 CFU g− 1) to fish for 5 weeks. Compared with the untreated group, the rate of growth was increased in the I2-diet group. The administration of I2 to olive flounder enhanced non-specific immune parameters, such as lysozyme, antiprotease, serum peroxidase and blood respiratory burst activities. At 9 days after challenge with S. iniae (108 CFU), the untreated control group experienced a 90% mortality rate, whereas all of the I2-cell-supplemented fish survived. These results show that L. lactis subsp. lactis I2 exerted beneficial effects as a probiotic and has potential as an alternative to antibiotics for the prevention of streptococcosis in aquaculture.