International Journal of Food Microbiology 226 (2016) 3341

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Acidied nitrite inhibits proliferation of Listeria monocytogenes

Transcriptional analysis of a preservation method
Stefanie Mller-Herbst a,b,, Stefanie Wstner a,1, Jan Kabisch c,2, Rohtraud Pichner c,3, Siegfried Scherer a,b
a
Lehrstuhl fr Mikrobielle kologie, Technische Universitt Mnchen, Weihenstephaner Berg 3, 85350 Freising, Germany
b
Zentralinstitut fr Ernhrungs- und Lebensmittelforschung (ZIEL), Technische Universitt Mnchen, Weihenstephaner Berg 3, 85350 Freising, Germany
c
Institut fr Mikrobiologie und Biotechnologie, MRI, Bundesforschungsinstitut fr Ernhrung und Lebensmittel, E.-C.-Baumann-Str. 20, 95326 Kulmbach, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Sodium nitrite (NaNO2) is added as a preservative during raw meat processing such as raw sausage production to
Received 25 June 2015 inhibit growth of pathogenic bacteria. In the present study it was shown in challenge assays that the addition of
Received in revised form 29 February 2016 sodium nitrite indeed inhibited growth and survival of Listeria monocytogenes in short-ripened spreadable raw
Accepted 5 March 2016
sausages. Furthermore, in vitro growth analyses were performed, which took into account combinations of var-
Available online 15 March 2016
ious parameters of the raw sausage ripening process like temperature, oxygen availability, pH, NaCl concentra-
Keywords:
tion, and absence or presence of NaNO2. Data based on 300 growth conditions revealed that the inhibitory
Listeria monocytogenes effect of nitrite was most prominent in combination with acidication, a combination that is also achieved during
Raw sausage short-ripened spreadable raw sausage production. At pH 6.0 and below, L. monocytogenes was unable to replicate
Nitrite in the presence of 200 mg/l NaNO2. During the adaptation of L. monocytogenes to acidied nitrite stress (pH 6.0,
Acid 200 mg/l NaNO2) in comparison to acid exposure only (pH 6.0, 0 mg/l NaNO2), a massive transcriptional adapta-
Nitrosative stress tion was observed using microarray analyses. In total, 202 genes were up-regulated and 204 genes were down-
SigB regulated. In accordance with growth inhibition, a down-regulation of genes encoding for proteins which are in-
volved in central cellular processes, like cell wall/membrane/envelope biogenesis, translation and ribosomal
structure and biogenesis, transcription, and replication, recombination and repair, was observed. Among the
up-regulated genes the most prominent group belonged to poorly characterized genes. A considerable fraction
of the up-regulated genes has been shown previously to be up-regulated intracellularly in macrophages, after ex-
posure to acid shock or to be part of the SigB regulon. These data indicate that the adaptation to acidied nitrite
partly overlaps with the adaptation to stress conditions being present during host colonization.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Listeria monocytogenes is the causative agent of human listeriosis, a
rare but serious disease, which affects mainly young, old, pregnant
and immunocompromised patients. In the European Union, 1642 con-
rmed cases were recorded in 2012 (EFSA and ECDC, 2014). The clinical
picture of listeriosis ranges from mild u-like symptoms and febrile gas-
troenteritis to severe illnesses like septicaemia and meningoencephali-
tis (Allerberger and Wagner, 2010). Due to the ability of this facultative
Corresponding author at: Lehrstuhl fr Mikrobielle kologie, Technische Universitt
Mnchen, Weihenstephaner Berg 3, 85350 Freising, Germany.
E-mail addresses: stefanie.mueller@wzw.tum.de (S. Mller-Herbst),
stefanie.wuestner@tum.de (S. Wstner), Jan.Kabisch@mri.bund.de (J. Kabisch),
rohtraud.pichner@he.hs-fulda.de (R. Pichner), siegfried.scherer@wzw.tum.de (S. Scherer).
1
Present address: Institut fr Medizinische Mikrobiologie, Immunologie und Hygiene,
Technische Universitt Mnchen, Trogerstrae 30, 81675 Mnchen, Germany.
2
Present address: Institut fr Mikrobiologie und Biotechnologie, MRI,
Bundesforschungsinstitut fr Ernhrung und Lebensmittel, Hermann-Weigmann-Strae
1, 24103 Kiel, Germany.
3
Present address: Hochschule Fulda University of Applied Sciences, Fachbereich
Oecotrophologie, Leipziger Strae 123, 36037 Fulda, Germany.
intracellular bacterium to spread directly from cell to cell (Stavru et al.,
2011), it is able to cross the intestinal epithelial cell barrier, the blood
brain barrier and the bloodplacenta barrier, which might lead to an in-
fection of the fetus in pregnant women (Mateus et al., 2013).
The ubiquitous and highly adaptable bacterium L. monocytogenes is
able to grow under harsh environmental conditions like temperatures
from 1.7 to 45.0 C, pH-values of 4.5 to 9.2 and NaCl concentrations of
up to 11.5% (Junttila et al., 1988; McClure et al., 1997; Petran and
Zottola, 1989). The ability of L. monocytogenes to proliferate at refriger-
ation temperature in food products is important for its relevance as a
food borne pathogen. The main infection sources are contaminated
raw products such as milk, cheese, chicken, sh, vegetables, fruits,
ready-to-eat products and also raw meat products.
In Germany, traditional raw sausages such as Salami or short-
ripened spreadable raw sausages are produced from raw pork and
bovine meat, according to the guidelines for meat and meat products
of the German food standard as published by the German Federal Min-
istry of Food and Agriculture (BMEL, 2014). The raw pork or bovine
meat might be a source for contamination with L. monocytogenes
(Rhoades et al., 2009; Thvenot et al., 2006). To inhibit growth of

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.03.006
0168-1605/ 2016 Elsevier B.V. All rights reserved.
34 S. Mller-Herbst et al. / International Journal of Food Microbiology 226 (2016) 3341
L. monocytogenes and other pathogenic bacteria, as well as growth of
spoilage bacteria, these raw sausages are traditionally produced accord-
ing to a technology referred to as hurdle technology (Leistner, 2000).
Hurdles applied during the fermentation process are the establishment
of a competitive microbiota, acidication of the product by starter cul-
tures, reduction of water activity by adding sugar/salt and drying of
the product, reduction of the redox potential by, e.g., adding ascorbate,
and addition of preservatives like the curing agent sodium nitrite.
The addition of sodium nitrite has several advantages. It contributes
to the preservation of the product, it is involved in the formation of the
typical curing color and curing avor, and it is thought to inhibit growth
of pathogenic bacteria. However, the use of sodium nitrite also has sev-
eral disadvantages. It is a food additive that requires the product to be
labeled (E 250), it is toxic in high concentrations, and might form carci-
nogenic nitrosamines upon heating or exposure to acidic conditions, e.g.
in the stomach (Cammack et al., 1999; Weitzberg and Lundberg, 2013).
Due to these downside effects, the amount of nitrite added to raw
sausage products is legally restricted to 150 ppm in raw sausages in
the European Union (European Union legislation directive 2006/52/
EC, European Parliament, 2006), and the use of the curing salt is consid-
ered critically by the health-conscious consumer.
Nevertheless, product and pathogen specic data substantiate the
importance of nitrite usage with respect to food safety. The inhibitory
effect of nitrite on growth and toxin production of Clostridium spp. in
meat products (ham, bacon, frankfurters, wiener) has been thoroughly
studied (Bowen et al., 1974; Christiansen et al., 1974; Jackson et al.,
2011; Pierson and Smoot, 1982). It was shown recently, that the addi-
tion of nitrite inhibited growth and survival of Salmonella Typhimurium
in short-ripened spreadable raw sausages (Mhlig et al., 2014b). Fur-
thermore, it has been shown previously, that the addition of nitrite
inhibited growth and survival of Listeria innocua in dry fermented sau-
sages (Hospital et al., 2012) and L. monocytogenes in vacuum packed
cooked meat (Duffy et al., 1994), in a cured cooked meat model system
(Xi et al., 2011), and in ham (Horsch et al., 2014; Myers et al., 2013).
However, it is unknown how this inhibitory effect acts at the molecular
level and how L. monocytogenes adapts to nitrite stress.
The aim of this study was to investigate the inhibitory effect of nitrite
on growth and survival of L. monocytogenes in a typical German short-
ripened spreadable raw sausage. Furthermore, the inuence of other
parameters relevant for the sausage ripening process like oxygen avail-
ability, temperature, pH-value and salt concentration on the inhibitory
effect of nitrite was investigated in vitro. Finally, global transcriptional
analyses were performed to get a better understanding of the inhibitory
effect of nitrite on L. monocytogenes at the molecular level.
2. Material and methods
2.1. Strains used in this study
The bacterial strains used in this study are listed in Table 1.
Table 1
Strains used in this study.
Strain Serotype Origin
Listeria monocytogenes EGDe 1/2a Derivative of strain NCTC 7973, which was originally isolated
from guinea pig mesenteric lymph node
Listeria monocytogenes Li 2 4b Liquor/human
Listeria monocytogenes Li 127 1/2a Liquor/human
Listeria monocytogenes Li 135 4d Sausage plant environment
SLCC: Seeligers Listeria Culture Collection.
NTCC: National Collection of Type Cultures.
Li: Listeria collection MRI Kulmbach.
2.2. Challenge experiments with short-ripened spreadable raw sausages
2.2.1. Manufacturing process
Two types of short-ripened spreadable sausages were produced
according to the amount of added NaNO2 (0 or 150 mg/kg) as de-
scribed previously (Mhlig et al., 2014b). For the production of the
sausages lard (330 g/kg) and pork (670 g/kg) were used. The day be-
fore the sausage production started, the raw meat was cut and
minced and stored over night at 4 C. At the day of production the
meat was minced to the nest granulation in a cutter, while addi-
tional ingredients were added (NaNO 2 (0 or 150 mg/kg), NaCl
(28 g/kg), glucose (2 g/kg), pepper (3 g/kg), sodium ascorbate
(0.5 g/kg) and a freeze-dried starter culture (0.8 g/kg) (BITEC-LS 1,
Gewuerzmueller, Germany, mixture of Lactobacillus sakei, Staphylococ-
cus carnosus ssp. Utilis and Kocuria varians)). The sausage meat was
then inoculated with L. monocytogenes (see below), before 200 g meat
were stuffed into cellulose hydrate casings. Four hours later, the sau-
sages were shortly immersed in a 20% potassium ascorbate solution.
For the ripening and storage process the sausages were kept in a fully
automated environmental chamber (Weiss, Gieen, Germany). Tem-
perature was set to 22 C for the rst 76 h, then for 48 h to 20 C, for
24 h to 18 C and for the rest of the storage time to 17 C. The relative
humidity was set to 4050% for the rst 4 h, to 92% for 6 days and
then to 85% till the end of the storage time. There was no air velocity
within the rst 4 h of the ripening process, for the rest of the ripening
and storage period the air velocity was set to 0.050.1 m/s.
2.2.2. Inoculation of short-ripened spreadable sausages with
L. monocytogenes
First of all, an unintended contamination with L. monocytogenes of
the raw material used for short-ripened spreadable sausage fermenta-
tion was excluded by selective enrichment and plating on PALCAM
and ALOA agar plates (VWR, Ismaning, Germany) and biochemical con-
rmation according to method EN ISO 112901 (1996). Then, equal
portions of the meat batter were articially inoculated with a mixture
of three L. monocytogenes strains (Li 2, Li 127, Li 135). All strains were
lyophilized and stored in milk (10% milk powder) at 4 C. Strains were
grown separately overnight at 37 C in 9 ml of BHI broth. Cell suspen-
sions of the L. monocytogenes strains were mixed (1:1:1) and diluted
in a physiological salt solution (0.85% NaCl) to a level of 7 log10 cfu/ml.
The bacterial density was conrmed microscopically using a Thoma
cell counting chamber (Bright-Line, LO-Laboroptik, Bad Homburg,
Germany). After serial dilutions, the raw meat material was inoculated
with approximately 10 ml of the L. monocytogenes strain pool to reach
an inoculation density of about 2.7 log10 cfu/g meat mix.
2.2.3. Sampling, counting and detection of L. monocytogenes
For microbial analyses, three inoculated sausages per batch were an-
alyzed on days 0, 1, 3, 6, 13, and 28. Experiments were performed three
times independently resulting in a maximum of 9 data points per mea-
suring point. For the detection and counting of L. monocytogenes, 10 g of
Source or designations according to
other culture collections
Glaser et al. (2001)
NTCC 10527 BgVV-Berlin, Germany
University of Mannheim, Germany SLCC 6139
MRI Kulmbach, Germany
 S. Mller-Herbst et al. / International Journal of Food Microbiology 226 (2016) 3341
aseptically taken raw meat material of each sausage were added to
90 ml sterile buffered peptone water (Sin, Berlin, Germany) and ho-
mogenized in a stomacher (Stomacher 400, Seward, Worthing, UK)
for 180 s. The suspension was serially diluted in 0.85% NaCl solution
and plated on ALOA agar. The cfu/g meat matrix was determined via
counting typical L. monocytogenes colonies after aerobic incubation at
37 C for 48 h. For statistical analyses an unpaired Student's T-test was
performed (http://www.graphpad.com/quickcalcs/ttest1/).
2.2.4. Chemical analysis of short-ripened spreadable sausages
In parallel to the articially contaminated raw sausages, two types of
non-inoculated raw sausages (0 or 150 mg/kg NaNO2) were produced
for the analysis of the chemicalphysical parameters of the products.
The nitrite content was determined semi-quantitatively using the
Merckoquant nitrite test (Merck, Darmstadt, Germany) according to
the manufacturer's instructions. Determination of pH-value and water
activity (aw-value) was performed as described (Mhlig et al., 2014b).
Experiments were performed three times independently. For each ex-
periment, two non-inoculated sausages were analyzed at days 0, 1, 2,
6, 13, and 28. The mean values and standard deviations of nitrite con-
centration, the pH-value, and the aw-value were calculated.
2.3. In vitro growth analyses of L. monocytogenes
In vitro growth of L. monocytogenes EGDe exposed to a combination
of various parameters relevant for the raw sausage ripening process was
analyzed. The parameters investigated were temperature (24 C, 20 C,
17 C), oxygen availability (aerobicmicroaerophilic), pH (pH 7.0,
pH 6.5, pH 6.0, pH 5.5, pH 5.0), NaCl concentration (0%, 1%, 2%, 3%, 4%,
Carl Roth, Karlsruhe, Germany), and absence or presence of NaNO2
(0 mg/l or 200 mg/l, Sigma-Aldrich, Taufkirchen, Germany). By combin-
ing all these parameters, 300 growth conditions were investigated using
an automated turbidimetric system (Bioscreen C, Growth Curves Ab Oy
Ltd., Helsinki, Finland). Growth analyses were performed in BHI medi-
um, which was adjusted to the desired pH with concentrated lactic
acid (90%, Merck, Darmstadt, Germany) and to the respective NaCl con-
centration prior to autoclaving. A 1:5 diluted overnight culture (10 l,
overnight culture was grown in BHI broth at 37 C) were added to
190 l of the respective medium in the wells of a Honeycomb microplate
(Growth Curves Ab Oy Ltd., Helsinki, Finland), resulting in a nal 1:100
dilution of the overnight culture. For cultivation under low oxygen con-
ditions, wells were covered with 200 l parafn oil (Carl Roth, Karlsru-
he, Germany) to restrain gas exchange. Control experiments were
performed with resazurin (resazurin sodium salt, 0.0001% [w/v],
Sigma-Aldrich, Taufkirchen, Germany), a redox indicator, which
showed that addition of parafn oil indeed resulted in the generation
of a microaerophilic atmosphere. NaNO2 was either omitted or added.
Plates were steadily shaken at medium intensity and the required con-
stant temperature in the analyzer. The experiment was conducted for
27 h. The absorbance of each microwell was recorded automatically
every 30 min at an optical density of 600 nm (OD600). The gain in pop-
ulation density was calculated from the difference of the highest OD600
and the starting OD600 (OD600max OD600start) for each single growth
curve. The mean of the gain in population density (GPD) and the stan-
dard deviation was calculated for each growth condition from six mea-
suring points derived from three biologically independent experiments
performed in duplicate.
2.4. Transcriptomic analyses
2.4.1. Exposure of L. monocytogenes EGDe to acidied nitrite stress
A L. monocytogenes EGDe overnight culture (2.53.0 ml) was used to
inoculate 150 ml BHI medium (500 ml conical ask). Cells were grown
at 24 C under constant shaking (150 rpm) to an OD600 of 0.790.86.
Then, two 50 ml aliquots were centrifuged (4186 g, 8 min, RT). The pel-
lets were resuspended either in BHI pH 6.0 (pH adjusted with lactic acid
35
[90%, Merck, Darmstadt, Germany] prior to autoclaving) without NaNO2
(0 mg/l) or in BHI pH 6.0 containing 200 mg/l NaNO2. Cells were further
incubated for 1 h at 24 C before they were again collected by centrifu-
gation (4186 g, 10 min, RT). Finally, the cell pellet was snap frozen in liq-
uid nitrogen and stored at 80 C until further processing.
2.4.2. RNA isolation
RNA isolation was performed as described (Mller-Herbst et al.,
2014).
2.4.3. Microarray analyses
Microarray analyses and data evaluation were performed according
to (Mller-Herbst et al., 2014). In total, 6 biological replicates of RNA
sets were analyzed in technical duplicates. In cDNA synthesis reactions
of three RNA sets, cDNA derived from L. monocytogenes EGDe cells incu-
bated in BHI pH 6.0 at 24 C with 200 mg/l NaNO2 was labeled with Cy3-
dCTP and cDNA of cells incubated in BHI pH 6.0 without NaNO2 was la-
beled with Cy5-dCTP (GE Healthcare, Freiburg, Germany). Dye swap
was performed for the three other RNA sets.
Genes were considered to be up- or down-regulated if (i) at least for
nine valid spots the log2 ratio of medians (200 mg/l NaNO2/0 mg/l
NaNO2) was 1.0 or 1.0 and (ii) the arithmetic mean of the log2
ratio of medians of valid spots was 1.0 or 1.0. Log2 RTL (relative
transcription level) indicates the arithmetic mean of the log2 ratio of
medians of all valid spots. The data discussed in this publication have
been deposited in NCBI's Gene Expression Omnibus (Edgar et al.,
2002) and are accessible through GEO Series accession number
GSE66472.
2.4.4. qPCR
Reverse transcription of 1 g total RNA was performed with an all-
in-one reaction mix (qScript cDNA Super-Mix, Quanta BioSciences, Gai-
thersburg, USA) according to the manufacturer's protocol. For qPCR in
the SmartCycler (Cepheid, Sunnyvale, USA) or iCycler (Bio-Rad Labo-
ratories GmbH, Mnchen, Germany), a ready-to-use reaction mix (Per-
feCta SYBR Green FastMix, Quanta BioSciences, Gaithersburg, USA) was
used. Each analysis was performed in duplicate for at least three biolog-
ically independent experiments. Data were analyzed by the Relative Ex-
pression Software Tool Multiple Condition Solver REST-MCS
version 2, a Calculation Software for the Relative Expression in real-
time PCR using Pair Wise Fixed Reallocation Randomisation Test
(Pfaf et al., 2002). Transcription of the 16S rRNA gene was used for nor-
malization. Oligonucleotides used for qPCR analyses are summarized in
Table S1 in the supplementary material.
3. Results and discussion
3.1. Nitrite inhibited growth and survival of L. monocytogenes in situ
To address the question whether the addition of nitrite to short-
ripened spreadable raw sausages indeed contributes to an increased mi-
crobial safety of these products, challenge assays were performed
(Fig. 1). At the day of production and ripening day 1, there was no
statistically signicant difference for the bacterial load in sausages
produced without nitrite compared to those produced with nitrite.
From day 2 on there was a higher amount of L. monocytogenes detect-
able in sausages produced without nitrite. This difference was statisti-
cally signicant for days 2 and 13. At days 6 and 28 the number of
L. monocytogenes was also lower in the sausages produces without ni-
trite, although the difference is not quite statistically signicant
(p 0.1). In the sausages without nitrite, L. monocytogenes was able to
proliferate within the rst days of the ripening process, leading to a sig-
nicant increase of the L. monocytogenes number on ripening day 2
compared to day 0 or day 1. In the further ripening process, the number
of L. monocytogenes decreased. A signicant reduction compared to the
starting number, however, could only be observed on day 28. In the
36 S. Mller-Herbst et al. / International Journal of Food Microbiology 226 (2016) 3341

Fig. 1. Impact of NaNO2 on growth and survival of L. monocytogenes in short-ripened spreadable raw sausages. The cfu of a pool of three L. monocytogenes strains (Li 2, Li 127, Li 135) per
gram raw sausage was determined on ripening days 0, 1, 2, 6, 13 and 28 for sausages produced without nitrite (0 mg/kg NaNO2, dark gray bars) or for sausages cured with nitrite
(150 mg/kg NaNO2, light gray bars). The cfu of L. monocytogenes/g sausage was determined as the mean of nine data points derived from three biologically independent experiments.
Error bars indicate the standard deviations. A Student's-T-test was performed to determine the signicance level for the deviations of the cfu L. monocytogenes/g sausage in sausages
with 0 mg/kg NaNO2 compared to sausages produced with 150 mg/kg NaNO2, for the deviations at successive time points, and for the deviations on days 1, 2, 6, 13 and 28 compared
to day 0 (** very signicant: p 0.01, *** extremely signicant: p 0.001, for results that were not quite statistically signicant: 0.1 p N 0.05 the p value is indicated, no marking: p N 0.1).

presence of NaNO2, no increase in the number of L. monocytogenes could
be observed in the rst days of the ripening process in the traditionally
produced short-ripened spreadable raw sausages. The bacterial load of
L. monocytogenes was the same on ripening days 0, 1, and 2. Already
on day 6 a slight reduction compared to the starting number was ob-
served. This reduction was signicant on days 13 and 28. Addition of
150 mg/kg NaNO2 inhibited proliferation of L. monocytogenes during
the rst days of the ripening process, which resulted in a stronger de-
crease of the bacterial load of L. monocytogenes on ripening/storage
days 6, 13, and 28 compared to sausages produced without nitrite.
The inhibitory effect of nitrite on the proliferation of
L. monocytogenes during the rst days of the ripening process could be
a direct effect of the presence of nitrite itself or an indirect effect. The
presence of nitrite could have inuenced other parameters like pH or
aw, which in turn might have inuenced growth and survival of
L. monocytogenes. To address this question, the physicalchemical
parameters were determined in control sausages which were produced
either with or without nitrite. First of all, the concentration of nitrite
was determined in the two types of raw sausages. As expected, no ni-
trite was detectable in control sausages produced without nitrite. For
the raw sausages produced with nitrite, a decrease of nitrite was
observed (Table 2). The detectable amount of nitrite dropped to zero
on day 6. This is in line with the inhibition of the proliferation of
L. monocytogenes observed during the rst two days of the ripening pro-
cess in the sausages produced with nitrite compared to those produced
without nitrite. pH- and aw-value development in the L. monocytogenes
free control sausages was typical for short-ripened spreadable raw sau-
sage (Table 2), both dropped steadily from ripening day 0 to day 28.

Table 2
Physicalchemical parameters during short-ripened spreadable raw sausage production.

Raw sausage type Ripening day/storage day

(0 or 150 mg/kg NaNO2)
0 1 2 6 13 28

Nitrite concentration in mg/kg

0 mg/kg NaNO2 00 00 00 00 00 00
150 mg/kg NaNO2 123 5 88 3 37 10 00 00 00

pH-Value
0 mg/kg NaNO2 5.68 0.05 5.61 0.08 5.48 0.06 5.28 0.05 5.15 0.04 5.14 0.05
150 mg/kg NaNO2 5.70 0.13 5.76 0.09 5.60 0.18 5.44 0.14 5.28 0.16 5.21 0.03

aw-Value
0 mg/kg NaNO2 0.9593 0.0020 0.90.5566 0.0029 0.9560 0.0040 0.9525 0.0024 0.9515 0.0024 0.9366 0.0220
150 mg/kg NaNO2 0.9580 0.0038 0.9590 0.0016 0.9578 0.0023 0.9572 0.0035 0.9553 0.0016 0.9524 0.0021
 S. Mller-Herbst et al. / International Journal of Food Microbiology 226 (2016) 3341 37

Comparing pH- and aw-value in the control sausages, marginally higher
pH- and aw-values could be observed in the sausages produced with ni-
trite. This might, however, be due to non-specic uctuations. There-
fore, these data did not indicate a massive impact of nitrite on other
parameters that might contribute to the inhibition of proliferation dur-
ing the rst ripening days.
From this data it can be concluded that it is nitrite or its deriva-
tives which were responsible for growth inhibition in short-
ripened spreadable raw sausages, especially during the rst days of
the ripening process, when nitrite can be measured in the product.
Such an observation was already described for other types of raw
sausages such as long ripened sausage-type Salami (Kabisch et al.,
2012 [German]).
3.2. Nitrite inhibits growth of L. monocytogenes in vitro
To further investigate if parameters of the ripening process might in-
uence the inhibitory effect of nitrite on growth of L. monocytogenes,
in vitro growth analyses were performed (Fig. 2).
Lowering the pH by adding lactic acid caused a reduced growth
of L. monocytogenes at all temperatures both under aerobic and
microaerophilic conditions. An increase in the NaCl concentration led
to a reduced gain in population density under aerobic conditions.
Under microaerophilic conditions, an increase in the NaCl concentration
sometimes even promoted growth. Such a protective effect of NaCl
under stressful conditions was already reported previously for
Escherichia coli O157:H7 (Jordan and Davies, 2001). In all cases, the ad-
dition of NaNO2 had an adverse effect on growth of L. monocytogenes
EGDe. This inhibitory effect of NaNO2 was most prominent in combi-
nation with acidication of the medium. At a pH of 6.0 and below,
the presence of nitrite almost completely inhibited growth of
L. monocytogenes EGDe, irrespective of the temperature, the oxygen
supply or the applied NaCl concentration.
This inhibitory effect of acidied nitrite on growth and proliferation
of L. monocytogenes is in accordance with a previous study (McClure
et al., 1991). However, in the respective study, the pH was adjusted
with the inorganic acid HCl in contrast to this study, in which the pH
was adjusted with lactic acid, which is also formed by starter cultures
during food fermentation. Such an inhibitory effect of nitrite at an acidic
pH, which was adjusted with lactic acid, was reported previously for
in vitro growth and survival of Listeria ivanovii (Gnzle et al., 1997).
A combination of acidic pH and the presence of nitrite is also applied
during the raw sausage ripening process, especially during the rst days
of ripening. It has been proposed that under these conditions it is not ni-
trite itself, which is acting as inhibitory agent. Rather, reactive nitrogen
intermediates (RNIs) like nitric oxide (NO) and other nitrogen oxides
(N2O3, NO2), nitrous acid (HNO2), and, in the presence of oxygen,
peroxynitrite (ONOO), which derive from nitrite upon acidication,
are thought to mediate the effect (reviewed in (Cammack et al., 1999;
Lundberg et al., 2004)). It is not known, which of these RNIs are actually
produced in which quantities in the raw sausage product.
3.3. Acidied nitrite induced massive transcriptional changes in
L. monocytogenes
To better understand the profound inhibitory effect of acidied ni-
trite on growth and survival of L. monocytogenes, global transcriptional
analyses were performed by microarrays. The array analyses revealed
extensive transcriptional changes in response to addition of nitrite at
an acidic pH. In total, 406 genes showed a nitrite dependent change in
transcription, with 202 genes being up-regulated and 204 genes being
down-regulated by addition of 200 mg/l NaNO2 (Table S2).

Fig. 2. Nitrite dependent growth of L. monocytogenes EGDe in combination with different temperatures, oxygen availability, pH-values and NaCl concentrations. L. monocytogenes was
grown at 17 C (a, d), 20 C (b, e), and 24 C (c, f) under aerobic (ac) and microaerophilic (df) conditions. Growth was investigated for different pH-values (pH 7.0, pH 6.5, pH 6.0,
pH 5.5, pH 5.0) and different NaCl concentrations (0%, 1%, 2%, 3%, 4%) in the absence (0 mg/l NaNO2, blue) or presence (200 mg/l NaNO2, green) of NaNO2. The gain in population
density (GPD) was calculated as the mean from three biologically independent experiments performed in duplicates. Error bars indicate standard deviation.
38 S. Mller-Herbst et al. / International Journal of Food Microbiology 226 (2016) 3341

For a better overview of these massive transcriptional changes,

Fig. 3. Validation of microarray analysis by qPCR. The nitrite dependent transcription of
the genes lmo0133, lmo0200, lmo0202, lmo0433, lmo0995, lmo0997, lmo2067, lmo2158,
lmo2200, lmo2230, lmo2695, and lmo2748 (up-regulated in BHI, pH 6.0 in the presence
of 200 mg/l NaNO2 according to microarray analyses) as well as that of the genes
lmo1015, lmo1755, lmo1808, lmo2063, and lmo2630 (down-regulated in BHI, pH 6.0 in
the presence of 200 mg/l NaNO2) was investigated in L. monocytogenes EGDe by qPCR.
The log2 RTLs of the respective genes in nitrite treated (BHI pH 6.0, 200 mg/l NaNO2) vs.
untreated (BHI pH 6.0, 0 mg/l NaNO2) cultures of the microarray analysis were plotted
against the mean of the log2 RTLs of the qPCR data. The coefcient of determination
R2 = 0.88 was calculated with Microsoft Excel.
The accuracy of the microarray analysis was conrmed by qPCR
analyses, validating the NaNO2 dependent transcriptional change for
12 up-regulated and 5 down-regulated genes (Fig. 3). The results from
microarray analysis and qPCR analysis correlated well (R2 = 0.88),
which conrmed the reliability of the transcriptional data.
regulated genes were summarized according to functional groups
based on their clusters of orthologous genes (COGs) (ftp://ftp.ncbi.nlm.
nih.gov/genomes/Bacteria/Listeria_monocytogenes_EGD_e_uid61583/
NC_003210.ptt) (Fig. 4). Down-regulated genes encoded, among others,
proteins involved in cell wall/membrane/envelope biogenesis (26
genes), information storage and processing (translation, ribosomal
structure and biogenesis, 37 genes), transcription (10 genes), and repli-
cation, recombination and repair (12 genes). The down-regulation of
genes involved in central cellular processes and the synthesis of central
cellular structures reected the observed growth inhibition of
L. monocytogenes by acidied nitrite. Genes encoding for proteins in-
volved in metabolism were equally up- or down-regulated by acidied
nitrite, indicating an adaptation of the metabolism to the respective en-
vironmental condition. The most prominent group of up-regulated
genes belonged to poorly characterized genes (not assigned to COGs
[32 genes], general function based on prediction only [33 genes], and
unknown function [41 genes]).
3.3.1. Genes induced by acidied nitrite were up-regulated in vitro in
macrophages
Interestingly, among 202 up-regulated genes, 94 (= 46.5%)
have been found to be up-regulated intracellularly in macrophages
(Chatterjee et al., 2006) (summarized in Table S2), where
L. monocytogenes is exposed to acidic conditions and RNIs, at least in
the phagolysosome. NO is one of the RNIs, which is generated from ni-
trite after acidication (reviewed in Lundberg et al., 2004) and is also
produced by the inducible nitric oxide synthase in activated macro-
phages (Beckerman et al., 1993; Higginbotham et al., 1992). The present
study is, to our knowledge, the rst one that describes the adaptation of

Fig. 4. Transcriptional adaptation of L. monocytogenes EGDe to acidied nitrite. The transcription prole of cells grown in BHI pH 6.0 was compared to that of cells cultivated in BHI pH 6.0
with 200 mg/l NaNO2. Up- or down-regulated genes were grouped according to the NCBI COGs (clusters of orthologous groups). Each bar represents the number of genes, which were
down-regulated (gray bars) or up-regulated (black bars) by acidied nitrite. Since one gene can be assigned to more than one COG class, the total number of COG assignments is
greater than the number of differentially expressed genes.
 S. Mller-Herbst et al. / International Journal of Food Microbiology 226 (2016) 3341
L. monocytogenes to acidied nitrite. However, several studies have been
performed on how other Gram-positive and Gram-negative bacteria
adapt to nitric oxide stress. The transcriptional adaptation of Bacillus
subtilis, another low GC Gram-positive bacterium which belongs to the
order Bacillales, to NO was reported (Moore et al., 2004). The gene
showing the highest induction by NO was hmp, encoding for the NO de-
toxication avohemoglobin Hmp. Hmp contributes to survival of NO
stress in several bacteria, such as B. subtilis (Rogstam et al., 2007), Staph-
ylococcus aureus (Richardson et al., 2006), Salmonella enterica serovar
Typhimurium (S. Typhimurium) (Stevanin et al., 2002) and E. coli
(Membrillo-Hernndez et al., 1999). Furthermore, it has been described
previously that hmp contributes to withstand acidied nitrite stress in S.
Typhimurium in vitro (Mhlig et al., 2014b). However, this well studied
NO detoxication system, which oxidizes NO to NO 3 aerobically and re-
duces NO to N2O anaerobically (reviewed in (Forrester and Foster,
2012)), is not present in L. monocytogenes. Neither are alternative NO
detoxication systems encoded on the L. monocytogenes chromosome
(Glaser et al., 2001), like the avorubredoxin NorV and the cytochrome
c nitrite reductase NrfA, for which a NO detoxication function has been
proposed in S. Typhimurium and E. coli (Gardner et al., 2002; Mills et al.,
2008; Poock et al., 2002). This suggests that L. monocytogenes does use
alternative systems to cope with NO stress, which might be found
among the poorly described genes that are up-regulated both intracel-
lularly and after exposure to acidied nitrite.
3.3.2. Genes differentially regulated after exposure to acidied nitrite were
differentially regulated after acid shock
Besides NO other reactive nitrogen species like nitrogen oxides, ni-
trous acid, and, in the presence of oxygen, peroxynitrite, might be
formed from nitrite upon acidication (reviewed in (Cammack et al.,
1999; Lundberg et al., 2004)).
It was shown recently, that exposure to acidied nitrite leads to in-
tracellular acidication of the cytoplasm in S. Typhimurium (Mhlig
et al., 2014a). It was speculated that this might be due to formation of
nitrous acid from nitrite under acidic conditions. Nitrous acid, an un-
charged molecule, which can easily diffuse across membranes, is
deprotonated in the near neutral cytoplasm resulting in intracellular
acidication. The transcriptomic data from the present study indicate
that intracellular acidication may also take place in L. monocytogenes.
Many of the genes found to be differently regulated by acidied nitrite
have recently been shown to be differently regulated after exposure to
acid stress at 25 C (Neuhaus et al., 2013). Of the 202 genes which
were up-regulated after acidied nitrite stress, 138 (68.3%) have been
shown to be induced after acid shock, whereas of the 204 genes which
were down-regulated by acidied nitrite stress 162 (79.4%) have been
shown to be less transcribed after exposure to acidic conditions (sum-
marized in Table S2). These data indicate that L. monocytogenes was ex-
posed to a more severe acid stress when exposed to acidied nitrite
than to acidication alone, an observation that can be explained by
the hypothesis of intracellular acidication.
3.3.3. Genes induced by acidied nitrite were regulated positively by the
sigma factor B (SigB)
It was observed that 122 genes which were up-regulated by acidied
nitrite have either been shown to be positively regulated by SigB or to
contain a putative SigB binding site in their promotor region (Abram
et al., 2008b; Hain et al., 2008; Oliver et al., 2010; Raengpradub et al.,
2008) (summarized in Table S2). SigB is needed for the initiation of
RNA synthesis of multiple stress related genes. Thus, SigB has been
shown to contribute to survival of various stresses, such as acid stress
(Abram et al., 2008a; Chaturongakul et al., 2011; Oliver et al., 2010;
Raengpradub et al., 2008), which again might reect an increased intra-
cellular acidication after exposure to nitrite at acidic conditions. How-
ever, the adaptation of B. subtilis to NO stress also includes a differential
expression of SigB regulated genes (Moore et al., 2004). This suggests
that a differential expression of SigB regulated genes does not
39
necessarily indicate an increased intracellular acidication, but might
reect the adaptation of L. monocytogenes to RNIs by inducing a general
stress response.
3.3.4. Genes induced by acidied nitrite were direct target genes of the
transcriptional repressor CtsR
Besides SigB, the repressor CtsR has been postulated to be negatively
involved in resistance to a variety of stresses in L. monocytogenes, like
high hydrostatic pressure, high temperature, H2O2 or acid stress
(Karatzas and Bennik, 2002; Karatzas et al., 2003; Van Boeijen et al.,
2010). A direct regulation of CtsR has been shown for the class III heat
shock proteins encoded by clpB (lmo2206) (Chastanet et al., 2004),
clpP (lmo2468), clpE (lmo0997), clpB (lmo2206) and the clpC operon
(ctsR[lmo0229]-lmo0230-lmo0231-clpC[lmo0232]), which contains ctsR
and is therefore autoregulated (Nair et al., 2000). All these direct target
genes with the exception of clpC have been shown to be up-regulated
after exposure to acidied nitrite stress in L. monocytogenes. ATP depen-
dent Clp proteases mediate ATP-dependent proteolysis or might func-
tion as molecular chaperons involved in protein folding and assembly.
They consist of an ATPase subunit, which belongs to the 100 kDa heat
shock protein (HSP100) Clp family (clpB, clpC, clpE) and a proteolytic
subunit (clpP). The ATP-dependent Clp proteases share structural and
functional similarity with the eukaryotic proteasome (reviewed in
(Porankiewicz et al., 1999)). Indeed, in Mycobacterium tuberculosis, a
bacterium expressing a proteasome homolog, this proteasome is
thought to protect the cell from acidied nitrite stress by degrading pro-
teins that are irreversibly oxidized, nitrated or nitrosated (Darwin et al.,
2003). A similar function can be anticipated for the ATP dependent Clp
proteases in L. monocytogenes after exposure to acidied nitrite stress.
4. Conclusion
Data from this study clearly demonstrate that combining acidica-
tion and presence of nitrite is a successful hurdle strategy to inhibit pro-
liferation of the human pathogen L. monocytogenes in vitro and in the
short-ripened spreadable raw sausage product. This observation is
reected by the transcriptional data that indicate a shut-down of central
cellular processes. L. monocytogenes induces a general (acid) stress re-
sponse upon exposure to acidied nitrite, which is in accordance with
the hypothesis that nitrous acid is generated from nitrite under acidic
conditions. As the production of nitrous acid (an equilibrium reaction
depending on the nitrite concentration and the pH) might indeed con-
tribute to the inhibition of the proliferation of L. monocytogenes in the
raw sausage product, it is tempting to speculate that a similar inhibitory
effect may be achieved in a sausage product in the presence of lower ni-
trite concentrations when the pH of the product is lowered by the use of
alternative starter cultures, a hypothesis that is currently under
investigation.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijfoodmicro.2016.03.006.
Acknowledgment
This research project was supported by the German Ministry of
Economics and Technology (via AiF) and the FEI (Forschungskreis
der Ernhrungsindustrie e.V., Bonn), Project AiF 15835N, and by the
Frderverein fr Fleischforschung e.V, Kulmbach. Dr. Claudia Held and
Dr. Armin Ehrenreich from the Institute for Microbiology at the Techni-
cal University of Munich are thanked for help with the microarray
analyses.
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