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Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern
Sarra Snoussi, Alya El May*, Laurent Coquet, Philippe Chan, Thierry Jouenne, Ahmed Landoulsi and Emmanuelle D?
Corresponding author:
Alya E May
UMR 6270 CNRS, Faculté des Sciences, Université de Rouen, 76821, Mont Saint Aignan Cedex, France
Laboratoire de Biochimie et Biologie Moléculaire, Faculté des Sciences de Bizerte, Université de Carthage, Zarzouna, Bizerte, Tunisie
For all author emails, please .
Proteome Science 2012, 10:6&
doi:10.56-10-6
The electronic version of this article is the complete one and can be found online at:
Received:11 August 2011
Accepted:3 February 2012
Published:3 February 2012
& 2012 S licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Salmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human
gastroenteritis worldwide. Outer membrane proteins whose production is often regulated
by environmental conditions also play important roles in the adaptability of bacterial
pathogens to various environments.
The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h) and
the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE) and LC-MS/MS spectrometry, we compared the
proteome of enriched-outer membrane fraction before and after exposure to a magnetic
field. A total of 11 proteins, displaying more than a two-fold change, were differentially
expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated.
These proteins were involved in the integrity of cell envelope (TolB, Pal), in the
response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF), in the
oxidative stress status (bacterioferritin), in virulence (OmpX, Yfgl) or in motility
(FlgE and UspF). Complementary experiments associated the down-regulation of FlgE
and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore,
the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility
in exposed cells. This decrease could be partly associated with the up-regulation
of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein,
was up-regulated.
Conclusions
SMF (200 mT) seems to maintain the cell envelope integrity and to submit the exposed
cells to an oxidative stress. Some alterations suggest an increase of the ability
of exposed cells to form biofilms.
Keywords: Salmonella; S Outer membrane proteomeBackground
A large number of attempts to explain biological effects of magnetic fields at the
molecular level have been reported for prokaryotes and eukaryotes [-]. Usually, biological materials that are used for such investigations are cells [-], tissues [], and living organisms [,]. Viability and proliferation [,], activity of enzymes [], transport of ions [] and gene transcription [] are the common fields of investigation. All these studies gave contradictory results.
Thus, it has been reported that magnetic field (MF) treatment (10 mT, 50 Hz) on different
strains of Escherichia coli, Leclercia adecarboxylata, and Staphylococcus aureus have induced cell mortality, which was time exposure and/or MF intensity, and strain-dependent
[]. At the opposite, Tsuchiya et al. (1999) [] reported that high MFs (ranging from 5.2 to 6.1 T) were less detrimental. E. coli cells exposed to an extremely low-frequency magnetic field (0.1 T) for 6.5 h exhibited
100 times higher viability as compared to unexposed cells []. Nascimento et al. (2003) [] demonstrated that an increase of glucose transport into E. coli cells was involved in the bacterial growth exacerbation after 8 h of exposure to an
electromagnetic field (0.5 mT, 60 Hz). Consequently, it appears that the MF effects
on bacterial growth and viability are dependent on the applied conditions/parameters
as well as on the strain used.
In other respects, several studies revealed the impact of MF on gene expression. Magnetic
field (1.10 mT, 60 Hz) could stimulate the σ32 expression, a stress promoter transcription factor in E. coli []
, or enhances the transcription of the rpoS gene in E. coli when being inhomogeneous (ranging from 5.2 to 6.1 T) []. It could also stimulate transposition activity mediated by the synthesis and accumulation
of the heat-shock proteins DnaK/J (when it is set at 1.2 mT, 50 Hz) [].
Salmonella spp. is a leading cause of bacterial foodborne disease all over the world, causing
a diversity of illnesses including typhoid fever, gastroenteritis and septicemia.
As a foodborne pathogen, it is a good working prokaryotic model for studying SMF impact.
We have previously described the effect of a static magnetic field (SMF, 200 mT) in
S. Hadar. SMF induced a decrease of the cell viability when applied between 3 and 6
h whereas the growth re-increased after 6 to 9 h of exposure. The analysis of the
differential expression of genes under SMF exposure showed that the expression level
of the 16S-rRNA mRNA remained stable allowing its use as a reference gene. Interestingly,
mRNAs of rpoA, katN, and dnaK genes were over-expressed after 10 h of exposure. This suggests a possible stress
response and adaptation of S. Hadar to SFM []. A more recent study showed that homogenous SMF(159.2 mT), applied for up to 24 h
on different bacteria species spread on agar plates, failed to affect their viability
[]. These contradictory results could be explained by the differences in culture mode
and/or medium composition [], the direction and the homogeneity of the magnetic field reaching the bacteria growing
on an agar plate or in a liquid medium. Indeed, it was shown for E. coli that the adherence to surfaces is dependent on the type of surface and direction of
the magnetic induction towards the surface colonized by the cells (data not published).
Many membrane proteins, especially transporters, play a crucial role in adaptation
of bacteria to environmental stress [] and in their resistance to antibiotics []. However, no study has yet been performed to analyze the changes inflicted on membrane
proteins by SMF and their impact on the bacteria physiology. In this context, the
objectives of this study were to investigate the modifications of S. Hadar outer membrane protein (OMP) patterns induced by a SMF stress (200 mT, 9 h).
Our proteomic investigations showed that SMF exposure may alter some cellular functions,
e.g., the integrity of the cell envelope, the bacterial virulence/motility and modulate
the response to oxidative stress.
Comparison of enriched-outer membrane protein patterns after exposure to SMF
In order to evaluate the impact of SMF on enriched outer membrane (OM) protein fraction
of S. Hadar, we compared the proteomes of enriched-OMP fractions from non-exposed
and 200 mT SMF exposed cells. Typical 2-DE gels obtained from exposed and non-exposed
organisms are shown in Figure . A total of 14 spots exhibited a change in their amount following SMF treatment.
Their location on the 2-DE gel is shown in Figure . These proteins were then identified by LC-MS/MS (Table ) and corresponded to 11 different polypeptides. Thus, in the exposed cells, four
proteins were down-regulated, i.e., the flagellar hook protein (FlgE), the outer membrane
protein assembly complex subunit YfgL (YfgL), the outer membrane protein × (OmpX)
and bacterioferritin (Bfr), whereas 7 proteins were up-regulated, i.e., the dihydrolipoamide
dehydrogenase (Lpd), the outer membrane channel TolC (three TolC isoforms and a TolC
precursor), the translocation protein TolB, the outer membrane protein A (OmpA), the
outer membrane protein W (OmpW), the peptidoglycan-associated outer membrane lipoprotein
(Pal) and the hypothetical protein STY1416.
2-DE gels of enriched-OMP fractions of Salmonella Hadar. A: with no SMF exposure. Arrows and numbers point the proteins differentially expressed
that are mentioned in Table 1, B: after exposure to SMF (200 mT for 9 h). One hundred μg of proteins have been loaded.
Proteins were stained by silver nitrate.
Identification of differentially expressed outer membrane proteins of Salmonella enterica Hadar SH287 exposed to magnetic field (200 mT for 9 h).
Effects of SMF on antibiotic susceptibility
In order to determine the biological effects of SMF on S. Hadar antibiotic susceptibility, a standardized bioassay using the disc diffusion
method was applied. The diameter of inhibition zones was determined for non-exposed
and exposed cells.
The inhibition zone diameters obtained for the different tested antibiotics are given
on Figure . Results pointed out a significant (p & 0.05) decrease of the bacterial susceptibility only for gentamicin. In contrast,
data showed a significant increase (p & 0.05) of the ticarcillin + clavulanic acid mixture efficiency. No significant effect
of SMF exposure was observed for penicillin, cephalotin, tetracycline, erythromycin,
chloramphenicol, nalidixic acid, vancomycin, amoxicillin, nitrofurantoin, norfloxacin,
ceftriaxon, kanamycin, ciprofloxacin and ampicillin sensitivity.
Effect of SMF (200 mT, 9 h) exposure on susceptibility of Salmonella Hadar to antibiotics. Comparison of the diameter of the inhibition zones of different antibiotics: penicillin
(P), cephalotin (CF), tetracycline (TE), erythromycin (E), chloramphenicol (C), nalidixic
acid (NA), vancomycin (VA), amoxicillin (AMX), nitrofurantoin (F/M), norfloxacin (NOR),
ceftriaxone (CRO), kanamycin (K), ciprofloxacin (CIP), ampicillin (AM), gentamicin
(G) and ticarcillin + clavulanic acid (TIM). The values are the means ± SD (n = 3,
Mann-Whitney U test).
Effects of SMF on bacterial motility
Due to the down-regulation of FlgE by exposed cells, we investigated the effect of
SMF on bacterial motility, i.e., swarming and twitching. The swarming (a flagella-driven
movement) was altered. As shown by the Figure
the zone of motility for the exposed cells is 1 ± 0 cm and 1.5 ± 0.1 cm for the non-exposed
(p & 0.05). SMF had no effect (p & 0.05) on the twitching, a type IV pili-dependent motility (data not shown).
Motility assays of Salmonella enterica Hadar after exposure to SMF (200 mT, 9 h). Swarming test: Cells were inoculated with a needle into the bottom of Nutrient broth
+ agar (0.6%) medium. Plates were incubated at 37°C for 24 h. The values are the means
± SD (n = 3, Mann-Whitney U test). A: E B: Non-exposed.
Discussion
Outer membrane proteins are essential for maintaining the integrity and selective
permeability of bacterial membranes []. They also play an important role in antibiotic resistance []. Due to its cellular location, OM is highly sensitive to environmental conditions
prevailing in the extra-cellular medium. This is important for bacterial pathogenesis
because it enhances the adaptability of pathogens to various environments [,]. We have previously reported a SMF effect on cell viability and gene expression of
S. Hadar []; we now address the question of the SMF impact on the OMPs pattern of this pathogen.
Impact of SMF on cell envelope integrity. The peptidoglycan-associated lipoprotein
(Pal) and the TolB protein were found to be up-regulated after SMF exposure. TolB,
a periplasmic protein, is partially associated with the outer membrane through a specific
interaction with Pal []. The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved
in maintaining the integrity of the outer membrane []. Two possible functions have emerged for the Tol/Pal system: its involvement in cell
envelope biogenesis and/or as a tether in cell division that maintains the appropriate
juxtaposition of the two membranes relative to the peptidoglycan layer []. It could be hypothesized that the SMF increased the expression of the system involving
the TolB and the peptidoglycan-associated lipoprotein Pal in order to maintain the
envelope integrity and thus cell division. This is confirmed by a renewal of the cell
viability observed following 9 h of SMF exposure []. These data suggest a contribution of TolB and Pal proteins in mechanisms of defense
in order to overcome the stress effects of the SMF.
Proteins involved in oxidative stress. Three overexpressed proteins may be related
to the oxidative stress response. The OmpW protein belongs to a family of small OMPs
involved in the transport of small hydrophobic molecules across the outer membrane
[]. It also may be involved in the protection of bacteria against various forms of environmental
stress. Recent studies have indicated that its expression is activated in response
to oxidative stress []. The up-regulated dihydrolipoamide dehydrogenase (Lpd) is a component of the pyruvate
dehydrogenase complex (PDC) that connects glycolysis and tricarboxylic acid cycle
enzymes. PDC carries out the conversion of pyruvate to acetyl-CoA. Haramaki et al.
(1997) [] suggested an antioxidant function for Lpd, related to the role of free lipoic acid
as an antioxidant. The up-regulated STY1416 protein, identified as the universal stress
protein UspF, has been described to confer resistance against oxidative stress. It
has, however, a minor role compared to other members of the Usp family []. Therefore, as shown in eukaryotes [-,], SMF exposure might induce an oxidative stress in Salmonella. The down-regulation of bacterioferritin in cells under magnetic field exposure may
lead to a decrease of iron storage and thus to the impairment of iron detoxification.
This observation suggests a contribution of the bacterioferritin down-regulation in
the oxidative stress status, a correlation being established between iron homeostasis
involving bacterioferritin and the oxidative stress response [-].
Impact of SMF on the Salmonella virulence. Two proteins involved in virulence, OmpX and YfgL, were down-regulated
under SMF. The integral OmpX belongs to a family of virulence-related membrane proteins
which promotes mammalian cell adhesion and invasion and helps to defend against the
human complement system []. This small channel could also play a role in the antibiotic resistance as it is
up-regulated in conditions where the Omp35 and Omp36 major porins in Enterobacter aerogenes have a decreased expression []. However, we did not detect an increasing expression of the Salmonella major porins, as we could expect concomitantly to the under-expression of OmpX. In
other respects, the outer membrane lipoprotein YfgL is involved in virulence of Salmonella as it plays a crucial role in the regulation of the expression of all Salmonella TTSS systems (Type Three Secretion Systems) that occurs during host infection. Indeed,
three TTSS (TTSS-1, TTSS-2, and flagella) allow the bacterium to cross the intestinal
barrier and to disseminate systemically []. The deletion of yfgL gene in S. enterica serovar Enteritidis led to the transcriptional down-regulation of the flagellar genes
encoding the TTSS structural proteins and of the effector proteins secreted by these
TTSS []. This down-expression of both ompX and YfgL suggests a possible impairment of the
virulence under a SMF exposure (200 mT, 9 h).
Impact of SMF on the Salmonella motility. For a lot of pathogens, virulence and motility are often intimately linked
by complex regulatory networks []. Thus, we observed a down-regulation of the hook flagellar protein FlgE by exposed
cells. In accordance with this result, SMF-exposed cells exhibited a decrease in the
swarming motility, a flagella-driven movement. The flagellar hook, a constituent of
the bacterial motile flagellum, is a short connection between the flagellar motor
and the long filament acting as a helical propeller. It is made of about 120 copies
of a single protein, FlgE, and its function is essential for dynamic and efficient
bacterial motility and taxis []. Confirming this bacterial motility decrease was the up-regulation of the STY1416
protein, which was shown to exhibit a negative effect on bacterial motility [].
Effects of SMF on antibiotic susceptibility. As pointed out by proteomic data, SMF
exposure induced an overexpression of the TolC protein. This protein is the outer
membrane component of the efflux pump AcrAB-TolC, an efflux system known to play an
important role in the multidrug resistance in Salmonella []. TolC is required for the functioning of seven drug efflux systems in S. enterica serovar Typhimurium []. In this context, we compared the susceptibility of S. Hadar to different antibiotics before and after SMF exposure. Our data showed no
influence of SMF on antibiotic susceptibility, except for gentamicin that underwent
a significant decrease of its effectiveness and for ticarcillin + clavulanic acid
mixture that acts in an unexpected increasing manner (Figure ). Efflux pumps have already been described as involved in the resistance to aminoglycoside
in Pseudomonas aeruginosa []. These pumps usually play an essential role in export of different structurally unrelated
substrates ranging from antimicrobials, heavy metals, and detergents to large toxins
[]. If we cannot exclude a role of TolC accumulation in the decrease of susceptibility
of exposed cells to gentamicin, it was actually not sufficient to promote an effect
on other antibiotic susceptibility. We can consequently imagine that the decrease
of susceptibility to gentamicin by SMF exposure might also result from other mechanisms.
Indeed, gentamicin is a cationic antibiotic which binds reversibly to anionic sites
of the bacterial cell membrane dependent on concentration []. Its binding to anionic phospholipids (such as cardiolipids) of the OM facilitates
the rupture of this membrane []. The membrane lipid composition and modification, especially cardiolipid's proportion,
should therefore be examined after SMF exposure.
Electromagnetic fields have been hypothesized to affect the membrane permeability
through modifications on channel-forming proteins []. Since we did not detect any differential expression of the OmpC and OmpF porins,
we could hypothesize that the SMF may alter kinetics of beta-lactamase inhibition
by clavulanic acid, thus reducing beta-lactamase activity.
Accumulation of major outer membrane protein OmpA. We observed the up-regulation of
OmpA by SMF exposed cells. This OMP is a multifaceted protein which can function as
an adhesin and invasin, participate in biofilm formation, act as both an immune target
and evasin, and serve as a receptor for several bacteriophages []. The structure of OmpA has given many insights into the possible porous nature of
this protein. However, the definition of molecules that utilize this channel into
the cell has not been resolved []. OmpA is the predominant cell surface antigen in Enterobacteria, found in about 100,000 copies per cell []. Furthermore, OmpA-sal modulates the adaptive immune responses to S. enterica serovar Typhimurium by activating dendritic cells and initiates an adaptive immune
response, two important properties to be considered in the development of effective
S. enterica serovar Typhimurium vaccines []. OmpA expression is controlled by two stress-responsive ribonucleolytic mechanisms,
and the environmental stimuli regulating OmpA expression could be transduced through
these pathways in response to membrane stress []. Concomitantly to the disappearance of the flagellum, the overexpression of this
adhesion involved in the first steps of biofilm formation raises questions about a
possible enhancement of the adhesion capacities of the bacterium.
Conclusions
Using a proteomic approach we obtained an overview of S. Hadar OMPs, which were differentially expressed under SMF stress. These proteins
were involved in the integrity of cell envelope (TolB, Pal), in the response to oxidative
stress (OmpW, dihydrolipoamide dehydrogenase, UspF), in the oxidative stress status
(bacterioferritin), in virulence (OmpX, Yfgl) or in motility (FlgE and UspF). Complementary
experiments associated the down-regulation of FlgE and UspF with an alteration of
swarming motility under SMF. Furthermore, we showed a decrease of gentamicin susceptibility,
associated with the up-regulation of TolC. This study enhances knowledge of the biological
effects of the SMF. Whereas the cell envelope integrity seems to be maintained, exposed
cells are submitted to an oxidative stress. Some alterations suggest an increase of
the ability of exposed cells to form biofilms. Experiments aiming to confirm this
hypothesis are currently in progress.
Bacterial growth and magnetic field exposure
The bacterium Salmonella enterica subsp. enterica serovar Hadar (isolate 287) (6, 8: Z10: e, n, X) was provided by the
Institut Pasteur de Tunis (Tunisia) and stored at -80°C. Nutrient broth (Pronadisa,
Hispanlab, Madrid, Spain) and nutrient agar (15 g/L) were used for cultivation of
Precultures were performed overnight at 37°C in 10 mL of culture medium in 18-mm diameter
tubes and then diluted to the same initial concentrations corresponding to 0.1 OD600
in 70 ml in an Erlenmeyer-shaped glass double phial (external diameter, 6 external
height, 8 internal diameter, 4 internal height, 7 cm). The SMF was generated
by a Helmholtz coil, i.e., two bobbins (diameter 20 cm, length 13 cm, each), powered
by a transformer. The two bobbins were separated by 6 cm. Magnetic induction was perpendicular
to the double glass phial and thus to the bacterial suspension. The induction of SMF
was measured and standardized using a Teslameter (CA 42, Chauvin Arnoux). The bobbins
were water-cooled, and the temperature inside was regulated at the value of the laboratory
temperature (≈ 25°C). The temperature was maintained at 37°C inside the glass double
phial by water circulation, using an incubator system composed of pump and resistance.
The samples were positioned in the center between the two bobbins. The SMF induction
was 200 ± 8 mT. The background SMF value was 60 ± 5 μT.
Bacterial cultures were exposed to magnetic field during 9 subsequently, the culture
was centrifuged at 2000 g for 10 min. For the control experiments, the bacterial cultures
were similarly positioned, except that the magnetic field was turned off.
Outer membrane protein extract preparation
Enriched-outer-membrane extracts were prepared from bacterial pellets using ultracentrifugation
and a sarkosyl extraction method [,]. Briefly, free cells were harvested for 15 min at 2000 g, the pellet was then resuspended
in 20 mM Tris-HCl and 0,7% lysozyme. The suspension was sonicated (pulses 1 s on/1
s off, time 1 min, amplification 25%, Vibra Cell 75115, Bioblock Scientific, Illkirch,
France), incubated for 30 min at 37°C and then centrifuged 10 min at 10,000 g to separate
the supernatant (S) from the cellular debris (C).
Membrane proteins (C0) were pelleted at 60,000 g for 45 min at 20°C (Beckman Coulter TL100 ultracentrifuge)
and the supernatant (S0) was considered as soluble proteins. The pellet C0 was suspended in 4 ml (for 1 L of culture) of Tris-HCl 10 mM and 0.3% sarkosyl for
30 min at room temperature. An ultracentrifugation (60,000 g for 45 min at 4°C) allows
the separation of an enriched-inner membrane protein fraction in the supernatant (S1) and enriched-outer membrane protein fraction in the pellet (C1). Pellet was re-suspended in 2 ml of Tris- HCl 10 mM, assayed (Bio-Rad protein assay,
Biorad, France) and stored at -20°C.
Two-dimensional gel electrophoresis
For the first dimension (IEF), 100 μg of proteins were solubilized in 400 μL IEF buffer
composed of 7 M urea, 2 M thiourea, 0.1% (w/v) ASB, 14, 2 mM tributyl phosphine and
0.4% (w/v) Coomassie blue. The first-dimension separation was carried out with immobilized
pH gradients (Immobiline DryStrip pH 4-7 non-linear, 18 Amersham Pharmacia Biotech).
IEF was performed within the IEF cell (Bio-Rad) as follows: active rehydratation for
12 h at 50 V, 250 V for 15 min, gradient from 250 V to 10,000 V for 3 h and final
focusing for 12 h at 10,000 V. Strips containing focused proteins were then stored
at -20°C. After the IEF step, the strip was equilibrated in a buffer containing 1%
dithiothreitol for 10 min. A second equilibration step was performed for 10 min in
equilibration buffer containing 4% iodoacetamide. The second dimension of separation
was ensured by sodium dodecyl sulfate polyacrylamide gel electrophoresis using 12.5%
polyacrylamide resolving gel (width 16 cm, length 20 cm, thickness 0.75 cm). Experiments
were carried out using the protean II Xi vertical systems (Biorad). Proteins were
visualized after silver staining. Images of gels were acquired using the ProXPRESS
Proteomic Imaging System (Perkin Elmer) with a resolution of 100 μm.
Protein identification
Protein spots were manually excised from 2-D gels. Excised spots were washed three
times with water, once with acetonitrile (CH3CN) and dried for 2 h. Trypsin digestion was performed overnight with a dedicated
automated system (MultiPROBE II, PerkinElmer). The gel fragments were subsequently
incubated twice for 15 min in a H2O/CH3CN solution, once for 15 min in 1% (v/v) Formic Acid and once with 100% ACN to allow
extraction of peptides from the gel pieces. Peptide extracts were then dried and dissolved
in starting buffer for chromatographic elution, consisting of 3% CH3CN and 0.1% HCOOH in water. Peptides were enriched and separated using a nano-LC1200
system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube
interface (Agilent Technologies, Massy, France). The fragmentation data were interpreted
using the Data Analysis program (version 3.4, Bruker Daltonic, Billerica, MA, USA).
For protein identification, MS/MS peak lists were extracted, converted into mgf-format
files and compared with the Salmonella protein database using the MASCOT Daemon (version 2.1.3; Matrix Science, London, UK)
search engine. The searches were performed with variable modifications for oxidation
of methionines, carbamidomethylation and carboxymethylation and with a maximum of
one missed cleavage. MS/MS spectra were searched with a mass tolerance of 1.6 Da for
precursor ions and 0.8 for MS/MS fragments. Only peptides matching an individual ion
score & 51 were considered. Proteins with two or more unique peptides matching the
protein sequence were automatically considered as a positive identification.
Gel analysis
Scanned gel images (ProX PerkinElmer) were imported into the image analysis
software Progenesis SameSpots v3.0 (Nonlinear Dynamics). Bacterial culture and protein
extraction experiments were performed 3 times per condition. For each experimental
condition, two 2-D gels were matched together to form a reference image. The protein
spot volumes were automatically normalized in the software. A list of spots which
changed in abundance on the different gels was generated. In our study, only normalized
spots exhibiting variations with a fold of at least 2 and with p-value (Anova) & 0.05, q-value (exclusion of false positive) & 0.05 and P (Power Analysis) & 0.8 were selected as
being differentially expressed. Principal component analysis (PCA) was applied to
analyze the similarity of protein patterns among gels and the expression profiles
of protein spots fulfilling the above criteria.
Bioinformatic tools
A prediction of unknown protein cellular location was obtained from the genome annotation
of Salmonella (accessible at
Antibiotic susceptibility
Antimicrobial susceptibility tests were performed using the agar diffusion method
on nutrient agar. A volume of 300 μl of each bacterial suspension (3-3.6 × 108 CFU/ml) was spread on agar plates. Antibiotic disks were disposed manually. Antibiograms
were performed using the following antibiotics (BioMérieux, France): penicillin 10
U (P), cephalotin 30 μg (CF), tetracycline 30 μg (TE), erythromycin 15 μg (E), chloramphenicol
30 μg (C30), nalidixic acid 30 μg (NA), vancomycin 30 μg (VA), (ticarcillin + clavulanic
acid) 75 + 10 μg (TIM), amoxicillin 25 μg (AMX), nitrofurantoin 300 μg (F/M), norfloxacin
10 μg (NOR), ceftriaxone 30 μg (CRO), kanamycin 30 μg (K), ciprofloxacin 5 μg (CIP),
ampicillin 10 μg (AM) and gentamicin 10 μg (GM). After incubation for 18 h at 37°C,
inhibition zone diameters were measured by the standard methods.
Antibiograms were performed for exposed and non-exposed cells. For control experiments
(i.e., non-exposed organisms), the bacterial cultures were similarly positioned except
that MF was turned off. Experiments were performed in triplicate.
Bacterial motility
Twitching (type IV pili) assays were performed with Nutrient broth (Pronadisa, Hispanlab, Madrid, Spain:
5 g polypepton and 3 g meat extract per liter of distilled water, pH 7) solidified
with 1.5% agar. Twitch plates were briefly dried and strains were stab-inoculated
with a sharp toothpick to the bottom of the Petri dish from an overnight-growth. After
incubation at 37°C for 24 h, the zone of motility at the agar/Petri dish interface
was determined [,].
Swarming (flagella and type IV pili). Medium used for assay is a 0.6% agar Nutrient broth. Swarm plates were briefly dried
and strains were stab inoculated with a sharp toothpick to the bottom of the Petri
dish from an overnight-grown then. After incubation at 37°C for 24 h, the zone of
motility at the agar/Petri dish interface was measured [,].
Statistical analysis
The differences between control and exposed cells were determined using the nonparametric
Mann-Whitney U test. Means were given with ± SD and the level of significance was set at p & 0.05.
Abbreviations
LC-MS/MS: liquid chromatography-tand MF: SMF:
s OM: OMPs: out IEF: iso-electro-focalisation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS carried out proteomic studies, analyzed data, and dr AEM designed
SMF studies and dr LQ performed trypsin digestion and identification
of the proteins, PC performed mass spectrometry experiments, TJ, AL supervised this
study and ED designed OM extraction method, proteomic studies and drafted the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
This work was supported by the Ministry of Higher Education, Scientific Research and
Technology (Tunisia).
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