Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-018-9436-5

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1-

and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced
Diarrhea
Biswaranjan Pradhan 1,2 & Dipanjan Guha 1 & Aman Kumar Naik 1 & Arka Banerjee 1,3 & Subodh Tambat 4 &
Saurabh Chawla 1 & Shantibhusan Senapati 5 & Palok Aich 1

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects
of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their
contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report
how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with
Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the
microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was
able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support
probiotics LA as a treatment option in the case of Salmonella infection.

Keywords Probiotics . Microbiota . Dysbiosis . Inflammation . Lactobacillus . Salmonella

Introduction diversity of microbiota present in a host, gut microbiome is con-

sidered as the second genome, while gut is assumed as the sec-
It has been reported that microbiota and probiotics play important ond brain [2]. Gut microbiota, therefore, has a special place as-
role in improving health and protecting against bacterial over- sociated with the health of the host. Reports are available that has
growth and infection [1]. Because of high abundance and a large aimed at understanding interactions between microorganisms
and host to protect against pathogenic infection by (a) controlling
Electronic supplementary material The online version of this article the overgrowth of the pathogen and pacifying pathogen-
(https://doi.org/10.1007/s12602-018-9436-5) contains supplementary produced toxins [3, 4] and (b) by restoring epithelial integrity
material, which is available to authorized users.
to prevent invasion of the gut mucosa by pathogens [3, 4]. The
mutualistic partnership between microbiota and the host begins
* Palok Aich
palok.aich@niser.ac.in at birth and stabilizes in humans at around the age of 3 [5, 6].
However, change in the composition of gut microbiota
1
School of Biological Sciences, National Institute of Science (dysbiosis) was reported in subjects taking antibiotics [7], infect-
Education and Research (NISER), HBNI, P.O. Bhimpur-Padanpur, ed with a pathogen [8], consuming high calorie diet [9], suffering
Jatni, Khurdha, Odisha 752050, India
from type II diabetes [9], gastrointestinal cancer [10], and inflam-
2
Present address: S. K. Dash Center of Excellence of Biosciences and matory bowel syndrome [11]. We wanted to understand (a) how
Engineering & Technology (SKBET), Indian Institute of Technology
does abundance and diversity of microbiome changes upon
Bhubaneswar, Bhubaneswar, Odisha, India
3
Salmonella challenge and (b) the ability of selected probiotics
Present address: Biozentrum der Universität Basel, 50-70
in clearing pathogen infection and improving health [12, 13].
Klingelbergstrasse, 4056 Basel, Switzerland
4
We used Salmonella enterica serovar typhimurium (ST) for chal-
Bionivid Technology Private Limited, 209, 4th Cross Rd, B
lenge model [14]. ST is the commonly infecting pathogens that
Channasandra, East of NGEF Layout, Kasturi Nagar,
Bengaluru, Karnataka 560043, India can efficiently induce destabilization of gut microbiota popula-
5 tions and cause diarrhea and populate liver. Despite knowing the
Institute of Life Sciences, Nalco Square, Chandrasekharpur,
Bhubaneswar, Odisha 751023, India etiology of Salmonella typhimurium-induced diarrhea, an

effective prevention is yet to be developed. A few probiotic
bacteria were reported to alleviate microbial dysbiosis and main-
tain metabolic and immunological homeostasis in the gut
[15–17]. Current group screened four different bacterial strains
that were either probiotic or potential probiotics and prescribed
widely by clinicians for supplementary therapy [18]. Out of four
strains, we reported that probiotics, Lactobacillus acidophilus
MTCC10307 (LA) and Bacillus clausii MTCC8326 (BC) have
the ability to prime mouse and human macrophages [18]. Out of
four potential probiotic strains LA, BC, Bifidobacterium bifidum
(BF), and Saccharomyces boulardii (SB) that we tried in vitro in
murine macrophages, only LA and BC could alleviate
Salmonella typhimurium MTCC3232 (ST)-induced toxicity effi-
ciently in vitro [18]. In this report, we evaluated (a) effects of ST
challenge in gut microbiome perturbation in C57BL6 and
BALB/c mice, (b) role of LA and BC in (i) restoring gut micro-
biota of Th1-biased C57BL/6, and Th2-biased BALB/c mice in
vivo, (ii) clearing ST infection from the streptomycin pretreated
and untreated mice.
Materials and Methods
Bacterial Dose Titration in Mice
All the animals, used during the present study, were housed in
polysulfone cages with corncob bedding in a controlled envi-
ronment with temperature and humidity ranging between 24
± 3 °C and 40–70% under disease-free conditions. Mice of
two strains C57BL/6 (Th1-biased) and BALB/c (Th2-
biased) were co-housed. Since, mice are coprophagous, co-
housing is needed to ensure that the differences observed, in
gut microbiota between C57BL/6 (Th-1 biased) and BALB/c
(Th-2 biased) animals, was truly intrinsic of the mouse strains
used and not an artifact [19]. All animal experiments were
done as per the guidelines of CPCSEA (Committee for the
Purpose of Control and Supervision of Experiments on
Animals, Govt. of India) and all protocols were approved by
the Institute Animal Ethics Committee constituted by
CPCSEA. Animal House Facility of School of Biological
Sciences, NISER, Bhubaneswar, India is governed and regu-
lated by CPCSEA rules. Animals were exposed to 12-h light
and 12-h dark cycle as a standard practice. Mice of 7 weeks of
age were divided into five groups (n = 3) for each bacterial
dose titration. LA, BC, and ST were collected at log phase
and orally gavaged with 250 μl of saline water at the doses
of 2 × 106, 2 × 107, 2 × 108, and 2 × 109 number of bacteria to
their respective group of mice on day zero. One group of mice
was administered with saline water only as the absolute neg-
ative control. Fresh fecal samples were collected once per day
from each mouse and analyzed for the presence of adminis-
tered bacteria by culturing on nutrient agar plates as well as by
PCR amplification of the genomic DNA isolated from fecal
Probiotics & Antimicro. Prot.
samples using species-specific primers for 3 days. Eating and
drinking pattern, normal cage activities, and body temperature
of mice were recorded every day until any abnormality ob-
served. Based on histopathological analysis bacterial dose was
determined for the further experiments. All experiments were
repeated twice to ensure consistency.
Mice Survivability
Following a dose titration, the doses selected for
probiotics treatment were 2 × 10 9 CFU and 2 × 10 7
CFU for LA and BC, respectively. Similarly, for ST,
the dose selected for infection was 5 × 108 CFU. Mice
were divided into six groups (n = 6 to 9) and orally
gavaged with the bacteria as described in the Table S1
(Supplementary Material). The disease symptoms and
mortality were noted and plotted to get the survivability
plot.
Mice Treatment and Sample Collection
On day 0 (D0), twelve (12) 7-week-old BALB/c or C57BL/6
mice for each treatment condition were treated with either LA
or BC alone or along with ST. A booster dose of LA or BC
was given to respective LA or BC treated mice on day 1 (D1).
A positive control for infection with ST alone (n = 3) and no
treatment mice (n = 3 per time point) for each time point was
also studied. Mortality or morbidity as a symptom of infection
was observed from day 3 (D3) post-ST challenge. Samples for
various studies were collected on D3, D5, and D10 posttreat-
ment or challenge following euthanasia of animals. Animals
were euthanized by cervical dislocation as per standard oper-
ating procedures approved by Institutional Animal Ethics
Committee. Based on the bacterial dose titration data, sample
collection days were planned. Duodenum, jejunum, ileum,
colon, spleen, liver, blood, and fecal samples were collected
from each of the mice after euthanization. Detailed treatment
plans and sample collection are depicted in Fig. S2. Another
batch of mice of 7 weeks of age are divided into different
groups (n = 3) and treated with 7.5 mg/mouse of streptomycin
(Himedia, India) or treated with 20 mg/mouse of streptomy-
cin. A group (n = 6) of untreated mice kept as the time-
matched absolute control. Another group (n = 6) of mice treat-
ed with 20 mg/mouse of streptomycin and not treated/
challenged with probiotics or ST is kept as streptomycin con-
trol. After 24 h, the mice were treated with the probiotics or
challenged with Salmonella. The next day, a booster dose of
probiotics was orally gavaged to the respective group of mice.
The mice were then observed for the disease symptoms and
sacrificed to collect samples for histology and fecal microbial
analysis.
Probiotics & Antimicro. Prot.
RNA Isolation from Gut Wall Tissue (Distal Ileum
and Proximal Colon, n = 3 per Group)
RNA isolation was done using the Rneasy mini kit (Qiagen,
Germany) with minor modifications in the manufacturer’s
protocol. Briefly, 20 mg of tissue was gently homogenized
with 600 μl of buffer RLT in a homogenizer (Himedia,
India). The homogenate was centrifuged for 5 min at
5000 rpm in 4 °C to remove the debris. The supernatant was
passed through the Qiashredder (Qiagen, Germany) at
10000 rpm for 2 min. Six hundred microliters of 70% ethanol
was added to the clear lysate and mixed well by pipetting. The
solution was again centrifuged at 5000 rpm for 5 min to re-
move the pellets formed. The clear lysate-alcohol solution was
passed through the Rneasy mini column (Qiagen, Germany)
for RNA binding to the silica column. The column was then
washed with RW1 and RPE buffer to remove lipids, protein,
and DNA. RNA was then eluted from the matrix with 50 μl
nuclease-free water. RNA quantity and quality was accessed
with Nanodrop 2000 (Thermo Scientific, USA) and
Bioanalyzer2100 (Agilent, USA).
Reverse Transcription and qRT-PCR
cDNA was synthesized from RNA as per the manufacturer’s
instruction using AffinityScript One-Step RT-PCR Kit
(Agilent, USA). qRT-PCR was done using the protocol de-
scribed earlier [20]. The experiment was done with three tech-
nical replicates along with no-template-control and no-primer-
control.
Microarray Experiment and Data Analysis (n = 3,
Technical Replicate 1)
RNA samples for gene expression analysis were labeled using
Agilent Quick-Amp labeling Kit (p/n5190-0444 Agilent,
USA). Time-matched control and treated samples of RNA
(500 ng each) containing RNA spikes were incubated with
reverse transcription mix primed by oligodT with a T7 poly-
merase promoter site at 40 °C for 2 h to synthesize cDNA.
cRNA was generated by in vitro transcription incorporating
Cy3 labeled CTP in time-matched untreated control samples
and Cy5 labeled CTP in test samples. Labeled cRNA samples
were cleaned and aliquots of the samples were assessed for
yield and specific activity. Eight hundred nanograms of each
of Cy3- and Cy5-labeled cRNA sample was mixed,
fragmented, and hybridized onto mouse GE 4x44k microarray
slides. The hybridized slides were washed using Agilent Gene
Expression wash buffers (Part Number 5188–5327, Agilent,
USA). Slides were scanned to obtain images. Scanned images
were uploaded in Feature Extraction software (Version 10.7,
Agilent, USA) to obtain normalized intensity for each spot
corresponding to a gene on the slide. We further obtained
normalized fold change values for all genes present on the
microarray slides using Arraypipe (v2.0) [21]. Differentially
regulated genes under various conditions were functionally
clustered using WEB-based GEne SeT AnaLysis Toolkit [22].
Genomic DNA Isolation from the Gut Content (n = 3,
Technical Replicate 1–2)
Genomic DNA was isolated from the gut contents using the
protocol standardized in our lab. In brief, 200 mg of gut con-
tent from − 80 °C freezer was taken in a 2-ml sterile microfuge
tube and 1 ml of sterile PBS is added to it. The sample was
mixed properly by vortexing and pipetting. It is then centri-
fuged at 4000 rpm for 5 min. To the pellet, 600 μl of lysis
buffer (Tris-HCl 0.1 M, EDTA 20 mM, NaCl 100 mM, 4%
SDS) was added and mixed properly by pipetting. The mix-
ture was kept in 70 °C for 30 min with intermittent mixing by
inverting the microfuge tube several times. The lysate was
centrifuged at 13000 rpm in 4 °C for 15 min. The supernatant
was collected in a new 2-ml microfuge tube. To the clear
lysate, 1 ml of phenol-chloroform-isoamyl alcohol (24:24:1)
was added and mixed properly by inverting several times. The
mixture was centrifuged at 13000 rpm in 4 °C for 15 min. The
colorless upper aqueous layer was collected in a new
microfuge tube. Chilled absolute ethanol was added to the
clear lysate and gently mixed by inverting and rotating simul-
taneously to precipitate gDNA. The precipitate was then
pelleted by centrifugation at 13000 rpm in 4 °C for 15 min.
The pellet was washed twice with 70% ethanol, dried at room
temperature, and solubilized with 100 μl of nuclease-free wa-
ter. RNase treatment was given to the nucleic acid, and the
ethanol precipitation step was repeated to recover the micro-
bial gDNA. The quality and quantity of the gDNA were
accessed with Nanodrop2000 and agarose gel electrophoresis.
Species-Specific Primer Designing
We designed species-specific primers against 16s rRNA gene
to confirm the next generation sequencing data. Sequences of
16s rRNA of the species of interest were collected from NCBI
nucleotide database, aligned with the 16s rRNA database of
bacteria and archaea using BLAST, top 25 hits are taken and
aligned in multiple sequence alignment software MUSCLE.
In the HTML output format of MUSCLE, unique bases in the
sequence of interest were searched. We fixed the unique base
G or C in the sequence of interest as the 3’-OH base and
extended towards the 5′ end till the melting temperature
(Tm) reaches 55 °C (Table S2). We did not fix the length of
the primer constant, as done by many of the online primer
designing software. The primers are then optimized for their
efficiency and specificity through qRT-PCR taking five differ-
ent dilutions of templates.

Histopathological Study
Tissue samples were collected and preserved in 4% parafor-
maldehyde (PFA) buffer solution at room temperature for
minimum 72 h. For histopathological analysis, tissues were
processed for paraffin embedding, and multiple 5-μm sections
were prepared. For hematoxylin and eosin staining, slides
were deparaffinized and hydrated with deionized water
followed by hematoxylin (Sigma) staining for 3 min and eosin
for 2 min. Slides were thoroughly washed in H2O and
dehydrated through sequential alcohol grading, then cleared
in xylene and mounted with permanent mounting media
(Vector Lab). Stained slides were observed under a Leica
DM500 light microscope, and representative images were tak-
en at × 4, × 10, and × 40 magnifications. Images were random-
ly selected from several slides under microscope that represent
the area of interest clearly and accurately without much back-
ground or unwanted surrounding effects. Histological images
were scored as per the method explained in the Table S6.
16S rRNA Gene Profiling Through V3–V4 Sequencing
by NGS
Stringent quality control of the Illumina reads was performed
using NGS QC toolkit to estimate the base call errors. Further,
high-quality reads from all the samples were merged and sub-
jected to de-replication and de-noising. The de-replicated
reads were processed for clustering of OTU’s followed by
chimera filtering. The non-redundant and representative
OTUs were annotated up to species level followed by individ-
ual sample quantitation. All the above analysis was performed
using UPARSE pipeline [23]. Operational taxonomic units
(OTUs) were determined at a threshold of a phylum (80%),
class (90%), order (92%), genus (95%), and species (97%)
level. For our samples, we had performed the analysis with
different tools, such as QIIME [24] and UPARSE [23]. As
UPARSE is giving bacterial taxonomy up to genus level, we
subjected our samples further to get species-level classifica-
tion with MG-RAST [25] tool. OTUs along with their abun-
dance in each sample were parametrically annotated with day
parameters, such as zero, three, five, and ten, and condition
parameters, such as NT, ST, STLA, STBC, LA, and BC with
each of the parameter being representing at least three samples
as criteria for statistical analysis. OTUs abundance along with
parameters was subjected to statistical and multivariate anal-
ysis using METAGENEassist [26]. Unsupervised hierarchical
clustering of OTUs based on parameters was done by apply-
ing Pearson un-centered algorithm with average linkage rule
using a cluster 3.0 software [27] and visualized using a Java
Tree View software [28]. OTUs enrichment analysis was per-
formed independently by plotting rarefaction curves for all the
samples using a MEGAN [29] tool. Parameter specific
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bacterial diversity was identified by calculating alpha diversity
index using a EstimateS [30] tool.
Statistical Analysis
Two-way ANOVA was used to calculate the level of signifi-
cance of different treatment groups with respect to control
groups as well as among various groups. A single asterisk
(*) corresponds to p ≤ 0.05, double aterisks (**) correspond
to p ≤ 0.01, triple asterisks (***) correspond to p ≤ 0.001, and
Bns^ corresponds to not significant. Error bars shown are stan-
dard deviations determined from average values of at least
three replicates. All graphs were prepared and statistical anal-
ysis was done using GraphPad Prism (V5.04, Prism, USA).
Shannon-Weiner Index (H)
Shannon’s index for diversity was calculated based on the
abundance value of gut microbial species in different taxo-
nomic categories [31]. Shannon-Weiner Index (H) is calculat-
ed by using the formula as follows:
n
H ¼ ∑ pi lnpi ;
i¼1
where pi is the proportion of S made up of the ith species. S is
total number of species in the community (aka richness).
H stands for an index of diversity and EH denotes equita-
bility (or evenness) indicating the diversity of the microbial
community. EH is defined as

EH ¼ H H max
;
where Hmax = lnS.
Results
Selection of Probiotics
Previously, our group screened four potential probiotic strains
(that are most commonly prescribed) LA, BC, BF, and SB in
vitro with respect to their cytotoxicity to the murine and hu-
man macrophage/monocyte cells and their potential to induce
innate immune gene expression [18]. BF was found to be
cytotoxic to the RAW 264.7 cells above the multiplicity of
infection (MOI) of 10 and SB was unable to induce expression
of select innate immune genes even at an MOI of 100 [18]. LA
and BC imparted insignificant cytotoxicity to the macrophage
cells and were able to induce the expression of select innate
immune genes that helped to clear Salmonella infection in
vitro [18]. LA and BC primed macrophages were significantly
Probiotics & Antimicro. Prot.
(p < 0.001) protected against the ST-induced cytotoxicity up
to 12-h post-challenge. Therefore, based on previous reports
by our group, LA and BC were selected for the present study
[18]. In this report, we studied the efficacy of LA and BC
against Salmonella challenge in vivo in mouse model. The
differential immune biasness of BALB/c and C57BL/6 mouse
may influence the population structure of the gut microbiota.
Therefore, we checked the cecal microbial composition of
both types of mouse through 16S rRNA sequencing.
Gut Microbial Composition of BALB/C and C57BL/6
Mice Are Different
Genetic makeup and the immunological bias of C57BL/6 and
BALB/c mice are different. We also observed significant dif-
ferences in gut microbial abundance even after co-housing the
Th1- and Th2-biased mice. It is expected that differential mi-
crobiota of these two mice strains may lead to different re-
sponses following treatment with LA or BC as well as for
challenge with ST. Results from 16S rRNA sequencing for
both types of mice of 7 weeks of age are shown in Fig. 1.
Our results show, at the genus level, the BALB/c microbiota is
more diverse, consisting of 39.7% moryella, 14.4%
Fig. 1 Gut microbial compositions at genus and phylum levels (inset) for
BALB/c (a) and C57BL/6 (b) mouse are shown. The gut microbial di-
versity of BALB/c mouse is higher (Shannon index = 6.75) compared to
C57BL/6 mouse (Shannon index = 5.82). Standard deviations of the data
lachnospiracea, 8.7% flavonifractor, 6.3% anaerostipes,
3.2% alistipes, 2.8% desulfovibrio, and several other low
abundant genera (Fig. 1a). C57BL/6 microbiota is less di-
verse, consisting of 54.9% prevotella, 16.7% oscillospira,
12.2% bacteroides, 3.9% ruminococcus, 3.4% odoribacter,
and several other genera in small percentages (Fig. 1b). The
major phyla that constitute BALB/c gut microbiota are 83.7%
Firmicutes, 11% Bacteroides, and 3.3% Proteobacteria (inset
to Fig. 1a). Similarly, C57BL/6 microbiota consists of 49.5%
Firmicutes, 46.2% Bacteroides, and 3.4% Spirochaetes (inset
to Fig. 1b). Detailed information on the abundance of gut
microbiota in BALB/c and C57BL/6 at the level of class,
family, and order is shown in Fig. 1c–e. Clostridia is the major
class in the gut microbiota of both the mice models.
Deferribacteres is the second major class of bacteria present
in both mice models. However, its abundance is more in the
C57BL/6 mice compared to BALB/c mice. Clostridiales is the
major order present in the gut microbiota of BALB/c. In case
of C57BL/6 mice, Clostridiales and Deferribacterales consti-
tute the majority of the order present in the gut microbiota.
The major family constituting the gut microbiota of BALB/c
mice is Lachnospiraceae, whereas in the case of C57BL/6
mice the major family is Prevotellaceae. The differential gut
are shown as error bars. Gut microbial compositions of BALB/c and
C57BL/6 mouse are also represented at the level of class (c), order (d),
and family (e)

microbiota in Th1- and Th2-biased co-housed mouse may be
attributed to their immune biasness. The differential gut mi-
crobiota may utilize the abundant food sources differently,
imparting differential colonization of the introduced
probiotics and pathogen. Our next aim is to see the site and
duration of colonization of the treated probiotic strains as well
as the challenged pathogenic bacteria.
ST, LA, and BC Were Able to Colonize in Distal Ileum
and Proximal Colon of Mice Gut
The strains of probiotics (LA and BC), as well as pathogen ST,
were tested in vitro using murine macrophage RAW 264.7 for
their immune-modulatory and cytotoxic ability. It was report-
ed that the two strains of LA and BC, that were used, could
clear ST infection in vitro [18]. Results from the current in
vivo ST challenge studies with streptomycin treated and un-
treated BALB/c and C57BL/6 mice following treatment with
or without these strains of LA and BC are reported and
discussed. Figure 2a–d showed colonization ability of LA
and BC in the gut of BALB/c and C57BL/6 mouse. In both
mice strains, colonization of LA and BC is significantly higher
in colon and ileum compared to colonization in duodenum
and jejunum. BALB/c and C57BL/6 mice were treated with
LA and BC through oral gavage with doses ranging from
Fig. 2 Colonization of LA (a, c) and BC (b, d) in mouse gut is shown.
LA is able to colonize in the BALB/c (a) and C57BL/6 (c) mouse gut
when orally gavaged at a dose of 2 × 109 CFU/mouse. BC is also able to
colonize in the BALB/c (b) and C57BL/6 (d) mouse gut when orally
gavaged at a dose of 2 × 107 CFU of BC. There was no adverse effect
Probiotics & Antimicro. Prot.
0.02–2 × 109 CFU of bacteria per mouse. LA is able to colo-
nize in the BALB/c (Fig. 2a) and C57BL/6 (Fig. 2c) mouse
gut, orally gavaged up to a dose of 2 × 109 CFU/mouse with-
out any significant adverse effects in the animal. Bacterial
presence in the fecal samples of mice as well as health and
behavior of the mice was tracked for several days to find the
optimal dose of LA and BC. Both LA and BC were able to
colonize in the distal ileum and proximal colon (Fig. 2a–d).
However, when LA was being tolerated by the mice up to the
dose of 2 × 109 CFU/mouse, BC-induced bloating (Fig. 2g),
and villi atrophy (Fig. 2h) in mice when administered beyond
the dose of 2 × 107 CFU/mouse. BC could also colonize in the
BALB/c (Fig. 2b) and C57BL/6 (Fig. 2d) mouse gut when
orally gavaged up to a dose of 2 × 107 CFU of BC. However,
LA-treated mouse was found to have normal gut morphology
(Fig. 2e) and histological structure (Fig. 2f), even at a dose of
2 × 109 CFU/mouse. We have chosen lower dosage (2 × 107
CFU/mouse) of BC to work with for further studies, while
kept the higher dosage (2 × 109 CFU/mouse) for LA, since
equivalent dose (2 × 107 CFU/mouse) of LA did not have
any significant protection against salmonella challenge as de-
scribed in the next section. We tracked the health of mice with
respect to their feeding, drinking, grooming, and playful ac-
tivities. Histology of the gut, spleen, and liver of the mice
about to die was performed to quantify the level of
like bloating rather intestine look normal (e) and no inflammation in the
gut as shown by histopathology (f) when treated with 2 × 109 CFU of LA
(e, f). The higher dose of BC (2 × 108) is detrimental to the mouse and
induced bloating (g) and inflammation (h) in the mouse gut
Probiotics & Antimicro. Prot.

infection-mediated inflammation and tissue damage. Results
also revealed that ST was able to colonize the mice gut for at
least 3-day post-challenge (Fig. S1). ST infection load was
found to be higher in the C57BL/6 mice compared to the
BALB/c mice. The optimal ST infective dose per mouse was
determined to be 5 × 108 CFU of bacteria based on mice sur-
vivability (Fig. 3) and histopathology. In addition, we evalu-
ated the efficiency of LA at a low (2 × 107 CFU per mouse)
and a high dose (2 × 109 CFU per mouse) in ameliorating the
effects of ST infection in mouse. The data revealed that LA at
the dose of 2 × 107 CFU per mouse is ineffective in protecting
the ST infected mouse. But LA at a dose of 2 × 109 CFU per
mouse is effective in protecting the mouse against ST infec-
tion (Fig. 3b). Therefore, the optimized doses selected for the
further studies were 2 × 109 CFU/mouse for LA and 2 × 107
CFU/mouse for BC. The symptoms of diarrhea and mortality
in the mice have been observed post 4 days of ST infection.
Therefore, the study is designed to sacrifice a group of mice on
day 3 and on day 5, posttreatment to find out the kinetics of the
infection and curing processes. We also followed the STLA-
treated group of mice until day 10 to find out the degree of
clearance of ST from the mouse by the probiotic LA. The
detailed study plan is depicted graphically in Fig. S2 and the
doses of different bacteria per mice as well as sample collec-
tion plan are listed in Table S1. The survivability of the mouse
was determined following ST challenge and probiotic
treatment.
LA and BC Protects BALB/c Mice from ST Infection,
but Only LA (Not BC) Was Able to Protect C57BL/6
Mice from ST Infection
The rate of mortality of the mouse increased with increasing
CFU of ST infection (Fig. 3a). BALB/c mice infected with ST
are significantly (p < 0.001) protected following treatment
with LA at CFU of 2 × 10 9, but not at CFU of 2 × 107

Fig. 3 The rate of mortality of the

mouse increased with increasing
CFU of ST infection (a). BALB/c
mice infected with ST is signifi-
cantly (p < 0.001, n = 6) protected
following treatment with LA at a
CFU of 2 × 109, but not at a CFU
of 2 × 107 (b). Similarly, C57BL/
6 mice infected with ST is signif-
icantly (p < 0.001, n = 6)
protected after treatment with LA
at a CFU of 2 × 109, but not at a
CFU of 2 × 107(c). BC could
protect BALB/c mice (b) but not
C57BL/6 mice (c) significantly
(p < 0.001, n = 6) from ST infec-
tion. BALB/c-mice pretreated
with streptomycin at a dose of
7.5 mg/mouse (d) and 20 mg/
mouse (e) is also significantly (p
< 0.001, n = 3) protected from ST
infection after treatment with LA
but not with BC. Similarly,
C57BL/6-mice pretreated with
streptomycin at a dose of 7.5 mg/
mouse (f) and 20 mg/mouse (g) is
also significantly (p < 0.01, n = 3)
protected from ST infection after
treatment with LA but not with
BC
 Probiotics & Antimicro. Prot.

(Fig. 3b). Similarly, C57BL/6 mice infected with ST is signif-
icantly (p < 0.001) protected against Salmonella challenge fol-
lowing treatment with LA at a CFU of 2 × 109, but not at a
CFU of 2 × 107 (Fig. 3c). BC can protect BALB/c mice
(Fig. 3b) but not C57BL/6 mice (Fig. 3c) significantly (p <
0.001) against ST challenge. BALB/c mice pretreated with
streptomycin at a dose of 7.5 mg/mouse (Fig. 3d) and
20 mg/mouse (Fig. 3e) was also significantly (p < 0.001)
protected against ST challenge following treatment with LA
but not with BC. Similarly, C57BL/6 mice pretreated with
streptomycin at a dose of 7.5 mg/mouse (Fig. 3f) and
20 mg/mouse (Fig. 3g) was also significantly (p < 0.01)
protected against ST challenge following treatment with LA
but not with BC. In addition, we compared the pathological
scores of the group of ST infected mice with or without pre-
treatment with streptomycin. The rate of mortality of the ST-
infected mice pretreated with streptomycin is higher compared
to the non-streptomycin model of ST-infected mice (Fig. 3).
The pathology score of the ST-infected mouse model with
streptomycin pretreatment is significantly (p ≤ 0.05) higher
compared to non-streptomycin model of mouse (Fig. S3).
This phenomenon is similar in both the Th1 and Th2
immune-biased mice strains (Fig. S3).
Microbial Dysbiosis in Mice Following Treatment
with ST and STBC but Not with STLA
Changes in abundance (%) of gut microbiota and diversity on
day 3 and day 5 following various treatment conditions in
BALB/c and C57BL/6 mice are shown in Fig. 4. Figure 4a,
b shows changes in BALB/c and changes for C57BL/6 are
shown in Fig. 4c, d. Figure 4e shows kinetics of changes in the
diversity of gut microbiome. It is observed that abundance of
bacterial genus Moryella decreased and abundance of
Lachnospirace increased in BALB/c on day 3 and day 5 fol-
lowing ST challenge (Fig. 4a–b). The abundance of bile me-
tabolizing Flavonifractor was increased significantly (p ≤
0.01) on day 5 in the BALB/c mice treated with ST and
STBC (Fig. 4b). Reduced abundance of Oscillospira and
Prevotella was observed in the microbiota of C57BL/6 mice

Fig. 4 The gut microbial

composition at genus level of
BALB/c mice treated with either
LA or BC at day 3 (a) and at day 5
(b) following challenge with ST.
The gut microbial composition in
genus level of C57BL/6 mice
treated with either LA or BC at
day 3 (c) and at day 5 (d) follow-
ing challenge with ST. Heatmap
for BALB/c and C57BL/6 (e) be-
tween treatment groups is being
made based on weighted unifrac
approach. STLA is grouped along
with untreated (NT) for both mice
models. This implies that LA is
aiding in normalizing the gut
microbiome of the mice disrupted
with ST infection
Probiotics & Antimicro. Prot.
on day 3 posttreatment with ST and STBC in comparison to
time-matched untreated mice (Fig. 4c d). From the species-
level analysis of the C57BL/6 gut microbiota, we found that
the short-chain-fatty-acids-producing bacterial species in-
creased in the STLA-treated mice group. The bacteria from
the genus Moryella and Lachnospirace metabolize complex
carbohydrates, and produce short chain fatty acids (SCFAs)
that maintains gut homeostasis via signaling through anti-
inflammatory pathways. Reduced SCFA producers in ST
and STBC-treated mice indicated the inflammatory condition
in their gut. This was further validated by gut microbial dis-
tribution calculated by Shannon diversity index (Fig. 4e). For
both C57BL/6 and BALB/c, STLA treatment increased the
gut microbial diversity. This implies that LA is aiding in nor-
malizing the gut microbiome of the mice disrupted with ST
infection but BC failed to do that. Species-level information
on day 5 for BALB/c following treatment with STLA revealed
significant fold increases (values shown in bracket by the bac-
teria) with respect to (WRT) untreated time-matched (day 5)
mice in abundance of Butyricicoccus pullicaecorum (6.3-
fold), Candidatus arthromitus (41.6-fold), Clostridium
scindens (41.6-fold), and Faecalibacterium prausnitzii
(143.2-fold) (Table 1). These significantly increased bacteria
are known to ferment complex carbohydrates, helps in an
expansion of T-reg cells and produce anti-inflammatory
SCFAs, such as butyrate, propionate, acetate, isobutyrate,
isovalerate, and valerate. The presence of all these species in
the fecal sample was confirmed through qRT-PCR using
species-specific primers designed against 16 s rRNA. The
bacteria that became highly abundant (numbers increased)
on day 5 in C57BL/6 mice following STLA treatment are
Clostridium lepttum (13.7-fold), Ruminococcus gnavus (8.8-
fold), and Acetatifactor muris (6846 folds), and similarly on
day 3, posttreatment Herbinix hemicellulosilytica (17.8) and
Helicobacter typhlonius (6.3) (Table 2). C57BL/6 mice treat-
ed with STBC have increased numbers of inflammatory-
inducing sulfate-reducing bacteria, such as Desulfovibrio
intestinalis (2.6-fold), and Helicobacter typhlonius (17.4-fold)
(Table 2). The species-specific primers used to validate the
Table 1 BALB/c microbiota changes in species level
OTU_ID Species name Fold changes wrt time-matched NT mice
D3STLA
gi_ Butyricicoccus pullicaecorum 2.6
343205971
gi_ Candidatus arthromitus − 2.0
444304121
gi_ Clostridium scindens 1.7
265678482
gi_ Faecalibacterium prausnitzii − 4.6
265678656
presence of abovementioned bacteria in the mice gut is listed
in Table S2. Together, these data implicated that LA treatment
of the ST-infected mice relieved inflammation by stimulating
SCFA producers in gut microbiota, whereas BC treatment to
the ST-infected mice have no such effect. In addition, BC
treatment to ST-infected C57BL/6 mice aids in destabilizing
the microbiota by stimulating the growth of a few
inflammation-inducing sulfate reducers. The effect of LA or
BC administration on infected mice is well defined, to study
the effect of LA or BC on host response; we studied genome-
wide gene expression in the gut wall tissue taken from the
distal ileum and proximal colon of the mice to access response
to the changed microbiota in the mouse gut. It is important to
note that BALB/c and C57BL/6 mice behaved differently in
terms of microbiota rearrangement following treatment with
LA and BC. For BALB/c, on D3 post LA treatment gut mi-
crobial composition was similar to that of untreated mice,
whereas on D5 following LA treatment Lachnospiracea,
Moryella, and Flavonifractor group of bacteria changed sig-
nificantly. BC treatment on the contrary significantly affected
abundance of Lachnospiracea and Moryella on both days 3
and 5 posttreatment compared to untreated mice. In C57BL/6
on the other genus that were significantly altered compared to
untreated mice were Oscillospira and Prevotella. Interestingly,
on D3, following treatment with BC composition of microbi-
ota was more similar to D3 following challenge with ST. Our
results laid the foundation for BC as less effective in treating
dysbiosis following challenge with ST.
Gene Expression in the Gut Wall Tissue in the Distal
Ileum and Proximal Colon of Treated Mice
Gut wall tissue comprised of a mixed population of cells,
including intestinal epithelial cells, goblet cells, M cells, en-
terochromaffin cells, and immune cells, such as macrophages,
dendritic cells, B cells, T cells, and NKT cells. Gut microbiota
and the gut tissues are in continuous crosstalk through the
interaction of various secretory molecules with gut tissue re-
ceptors, and the respective activation of various metabolic and
D3STBC D5STLA D5STBC Impt function
− 3.0 6.3 3.4 Anti-inflammatory
− 1.2 41.6 1.2 Innate immune response
− 1.4 16.7 − 1.8 Expansion of T-reg cells
− 3.4 143.2 5.8 SCFA, butyrate
 Probiotics & Antimicro. Prot.

Table 2 C57BL/6 microbiota changes in species level

OTU_ID Species name Fold changes wrt time-matched NT mice

D3STLA D3STBC D5STLA D5STBC

denovo9209 Acetatifactor muris 1 1 6846 6 Bioremediation

denovo1769 Ruminococcus gnavus − 2.8 − 1.6 8.8 36.6 SCFA, acetate, butyrate
denovo8429 Herbinix hemicellulosilytica 17.8 1.5 1 2.2 Cellolose metabolism
denovo3701 Mucispirillum schaedleri 1 6 − 2.1 18 Maintain ENS and anti-diabetic
denovo2541 Helicobacter typhlonius 6.3 17.4 −3 3.3 Antiinflammatory
denovo465 Clostridium leptum − 1.6 30.3 13.7 − 44.5 Expansion of T-reg cells

immunological signaling pathways required for gut homeosta- enhanced renal glucose reabsorption or synthesis of complex
sis. Therefore, it is necessary to monitor the gene expression in lipids or long chain fatty acids that might have been compro-
gut wall tissue following its response to microbial perturba- mised as a result of ST. STLA-treated mice also expressed the
tion, rather than analyzing intestinal epithelial cells alone. We toxin-clearing cytochrome p450 gene Cyp26b1 to clear the
extracted total RNA from mice gut wall tissue from the distal toxins that might have been produced as a result of ST chal-
ileum and proximal colon to analyze host gene expression. lenge. Overexpression of Tnf and Il1b in STBC treated mice,
The genes were considered to be differentially expressed if
their fold changes are more than 1.5 with respect to time-
matched untreated controls and if the p value is less than
0.05. A detailed number of differentially expressed genes that
are either conserved or unique following various treatment
and challenge conditions are shown in Venn diagram (Fig.
S4). Following analysis of the microarray data, we have found
that lipopolysaccharide mediated TLR pathway is enriched in
BALB/c mice after 3 days of ST challenge and for C57BL/6
mice after 5 days of ST challenge, which is also validated
through qRT-PCR (Fig. 5). We have observed several proin-
flammatory cytokines like Il1b, Il6, Nfkb1, Tnf gets upregu-
lated in ST challenged mice and downregulated in STLA-
treated mice (Fig. 5a, b). STLA treatment can reduce proin-
flammatory response in C57BL/6 mice compared to ST-
treated mice group. Primer sequences of the genes used for
validation in qRT-PCR are listed in Table S3.
Genes were clustered together based on their function and
the pathways they represent. Several pathways came up fol-
lowing our analysis, and we focused mainly on pathways
associated with immunological, metabolic, and gut barrier
functions. Some of the functionally important genes and their
expression values in terms of fold changes on day 3, treated
groups with respect to time-matched untreated controls of
BALB/c mice are listed in Table 3. Results, from day 3 sug-
gested higher expression of mucin genes (Muc13, Muc16, and
Muc20) and glucose transporters (Slc2a2 and Slc2a7). Results
also revealed a higher expression of Slc27a5 gene that sug-
gested significantly more synthesis of long chain fatty acid Fig. 5 Genes of TLR pathway differentially expressed in mice after
following LA-treated ST-challenged mice compared to LA- 3 days of treatment with ST, STBC, and STLA in BALB/c and C57BL/
untreated ST-challenged mice (Table 3). Upregulation of these 6 mice (c) and after 5 days of treatment with ST, STBC, and STLA in
BALB/c and C57BL/6 mice (b). Note that the higher expression of in-
genes in STLA-treated mice but downregulation in ST chal- flammatory cytokines il1b and tnf in ST infected C57BL/6 mice, which is
lenged mice suggest repair or maintenance of the (a) gut mu- decreased significantly (p < 0.01, n = 3) following treatment with LA
cosa and (b) epithelial tissue barrier function as well as
Probiotics & Antimicro. Prot.

Table 3 Important genes expressed in BALB/c after 3 days of treatment

GeneSymbol Gene Name D3BC D3LA D3ST D3STBC D3STLA

Adh4 Alcohol Dehydrogenase 4 11.6 1.5 4.2 4.7 7.0
Maob Monoamine Oxidase B 2.0 -1.1 1.9 3.1 2.9
UDP Glucuronosyltransferase Family 2
Ugt2a3 Member A3 9.6 1.2 5.4 7.9 6.3
UDP Glucuronosyltransferase Family 1
Ugt1a6a Member A6 -2.8 -1.2 -1.2 -3.0 3.4
Gzmg Granzyme G 1.3 1.7 1.9 1.1 140.8
Slc27a5 Solute Carrier Family 27 Member 5 12.5 4.1 1.8 5.6 5.6
Nme6 NDP Kinase 6 1.2 1.2 1.3 -1.0 124.7
Oas1h 2'-5'-Oligoadenylate Synthetase 1 -1.1 1.0 1.2 1.0 31.0
Protein Phosphatase 1 Regulatory
Ppp1r12b Subunit 12B -5.4 -1.3 -1.3 1.0 29.1
Ptger4 Prostaglandin E Receptor 4 -2.6 1.4 1.4 -1.5 22.6
Slc2a2 Solute Carrier Family 2 Member 2 202.2 -1.2 1.8 40.2 22.1
Slc2a7 Solute Carrier Family 2 Member 7 7.0 6.4 2.4 3.3 15.9
Mpo Myeloperoxidase 1.2 1.2 9.7 -1.1 1.3
Lect2 Leukocyte Cell Derived Chemotaxin 2 5.0 -1.5 7.4 25.6 5.6
C-Type Lectin Domain Family 4
Cd209c Member M -1.0 -1.0 1.4 -1.3 1.2
Hdc Histidine Decarboxylase 2.6 1.9 1.1 7.4 3.1
Trim6 Tripartite Motif Containing 6 5.7 7.6 4.3 3.2 5.6
Cytochrome P450 Family 26 Subfamily
Cyp26b1 B Member 1 4.3 1.2 7.0 8.4 1.8
Muc 5ac Mucin 5Ac -1.4 3.4 -1.1 1.6 2.4
Muc 5B Mucin 5B -1.4 1 -1.3 -1.7 2.7
Muc 13 Mucin 13 -1.8 1.3 -3.4 -1.1 1.8
Muc16 Mucin 16 3.3 1.5 -1.2 3.0 1.8
Muc20 Mucin 20 1.3 2.3 -2.0 1.4 1.9
Tjp1 Tight Junction Protein 1 2.6 2.2 -1.7 -3.0 2.4
Hdac1 Histone deacetylase 1 1.3 1.1 1.5 -1.1 -1.2
Hdac3 Histone deacetylase 3 1.5 -1.0 1.2 1.4 1.5
Hdac6 Histone deacetylase 6 1.9 1.3 1.4 1.5 1.2
Hdac7 Histone deacetylase 7 1.3 -1.3 -1.0 1.2 1.1
Hdac8 Histone deacetylase 8 7.7 1.2 1.2 3.3 -1.0
Hdac9 Histone deacetylase 9 2.6 -2.6 -3.4 1.4 -2.5

but in STLA-treated mice, suggesting induction of a proin-
flammatory condition in STBC-treated mice but not in
STLA (Fig. 5a and Table S4).
Similarly, expression of a few more relevant important
genes and their expression values in terms of fold changes
on day 3 in mice with various treatment conditions were
compared with respect to time-matched untreated control
group of C57BL/6 mice is shown in Table 4. The high
expression level of the defensins, DEFA4 (42.4 folds) and
DEFA1 (25.1 folds), in STBC, treated mice suggests that
tissue damage occurred in the gut [32]. Il12b and Il17a
expression in STLA-treated mice suggest the upregulation
 Probiotics & Antimicro. Prot.

Table 4 Important genes expressed in C57BL/6 after 3 days of treatment

GeneSymbol Gene Name D3BC D3LA D3ST D3STBC D3STLA

Defa4 Defensin Alpha 4 -2.1 -1.9 -1.1 42.4 7.7
Defa1 Defensin Alpha 1 2.3 1.0 1.3 25.1 7.6
Tumor Necrosis Factor
Tnfsf4 Superfamily Member 4 -1.8 4.5 7.7 3.1 7.2
Itga5 Integrin Subunit Alpha 5 -5.5 -1.3 3.7 7.0 6.4
Il17f Interleukin 17F 2.4 4.0 1.2 3.1 4.5
Il19 Interleukin 19 2.2 -1.3 1.2 -1.6 4.1
Tlr6 Toll Like Receptor 6 13.2 5.1 2.5 8.9 3.6
Il17a Interleukin 17A 2.3 1.5 -1.3 1.9 3.5
Transforming Growth Factor
Tgfb3 Beta 3 -1.0 1.3 1.3 1.4 2.4
C-C Motif Chemokine Receptor
Ccr4 4 3.3 4.1 4.5 2.0 2.1
C-X-C Motif Chemokine Ligand
Cxcl1 1 1.2 2.2 1.8 -1.8 2.0
Il12b Interleukin 12B -1.1 1.2 -1.1 1.4 1.9
Ctse Cathepsin E 1.7 10.8 5.3 11.6 1.9
Cytotoxic T-Lymphocyte
Ctla4 Associated Protein 4 2.3 1.9 -1.1 2.5 1.7
Csf3 Colony Stimulating Factor 3 3.9 2.5 3.5 1.7 1.7
C3 Complement Component 3 1.3 1.4 1.0 1.7 1.4
Il6 Interleukin 6 1.7 1.6 9.2 1.8 -1.3
Ccl2 C-C Motif Chemokine Ligand 2 -1.1 1.9 -1.1 1.1 1.1
Fcer1g Fc Fragment Of IgE Receptor Ig -1.3 -1.4 -1.2 1.5 -1.1
Cathelicidin Antimicrobial
Camp Peptide 1.4 2.2 -1.1 1.8 -1.1
Il1b Interleukin 1 Beta 1.9 2.0 14.8 2 1.9
Tnf Tumor Necrosis Factor 1.4 1.4 2.2 1.1 1.5
Fpr2 Formyl Peptide Receptor 2 1.0 -2.1 1.2 2.1 -1.2
Ccl7 C-C Motif Chemokine Ligand 7 1.4 2.6 1.4 4.2 -1.3
Muc13 Mucin 13 4.0 5.9 1.2 1.8 2.5
Muc16 Mucin 16 8.0 3.8 -1.9 4.7 6.9
Tjp1 Tight junction protein 1 1.9 2.4 1.2 1.5 3.2
Hdac6 Histone deacetylase 6 1.6 1.6 1.3 1.5 1.0

of proinflammatory signals for pathogen clearance. The
requirement of Il17a was reported before to suppress
Salmonella invasion [33, 34]. Downregulation of Il1b
and Tnf in STLA-treated mice, but upregulation in
STBC-treated mice suggested the presence of severe in-
flammation in the STBC group, whereas inflammation
observed in the STLA group of mice (Fig. 5b and Table
S5) was minimal. Together, these data suggested that LA-
induced anti-inflammation and BC-induced inflammation
in ST infected mice, findings which are in accordance
with our data on the microbiota. However, the inflamma-
tory damage to the gut by ST and STBC needs to be
further explored for confirmation and establishing the
mechanism.
Probiotics & Antimicro. Prot.
BALB/C and C57BL/6 Mice Treated with ST and STBC
Had Intensive Damage to Ileum, Colon, Spleen,
and Liver
Histopathological scoring of the eosin and hematoxylin
(H&E) stained transverse slices of BALB/c and C57BL/6
mice ileum, colon, spleen, and liver were performed using
the method explained in the materials and methods section.
The histological images were scored for the level of damage
based on the scoring method listed in Table S6. Extensive
Fig. 6 Representative images of the histology of colon from BALB/c and
C57BL/6 mice treated with ST, LA, BC, and their combinations (a). The
histological images (n = 3) were scored for the pathological assessment
based on the criteria explained in Table S6 and normalized to the scale
ranging from zero to four. The histology scores of various treatment
groups of mice are represented graphically (b). The treatment plan is
damage, immune infiltration into the tissue, tissue abscises,
and immune infiltrations into the red pulp area of the spleen
are observed in the BALB/c mice treated with ST and STBC
(Fig. S5 and S6). Few of the representative colon images of
the mouse across treatment conditions are presented in Fig. 6a.
This was further supported by the higher pathological score of
3.4 ± 0.2, 1.9 ± 0.2, 2.6 ± 0.2, 1.9 ± 0.3 for the ileum, colon,
spleen, and liver respectively in BALB/c mice treated with ST
compared to the NT (Fig. 6b). Similarly, a pathological score
of 3.6 ± 0.1, 2.6 ± 0.2, 2.4 ± 0.4, and 2.1 ± 0.5 for the ileum,
mentioned in Fig. S2. The pathological scores of the histology images
of different treatment groups were plotted and tested for statistical signif-
icance (two-way ANOVA) among the treatment groups. Note that ST and
STBC treated groups of mice have significantly (p < 0.001) higher pa-
thology score compared to the NT- and STLA-treated groups of mice

colon, spleen, and liver respectively in BALB/c mice treated
with STBC suggested an inflammatory state in this group of
mice (Fig. 6b). Tissues of the abovementioned organs collect-
ed from mice treated with LA, BC, and STLA have lower
pathological scores compared to the ST-treated mice in the
groups (Fig. 6b).
Extensive damage, immune infiltration into the tissue, tis-
sue abscises, and immune infiltrations into the red pulp area
are also observed in the C57BL/6 mice treated with ST and
STBC. This was further supported by a higher pathological
score of 3.2 ± 0.2, 2.3 ± 0.3, 2.7 ± 0.2, and 3.2 ± 0.3 for the
ileum, colon, spleen, and liver respectively in C57BL/6 mice
treated with ST compared to the NT (Fig. 6b). Similarly, a
pathological score of 3.6 ± 0.2, 3.6 ± 0.1, 2.7 ± 0.4, and 2.6
± 0.6 for the ileum, colon, spleen, and liver respectively in
C57BL/6 mice treated with STBC suggests an inflammatory
state in this group of mice (Fig. 6b). Tissues of the
abovementioned organs collected from mice treated with
LA, BC, and STLA have lower pathological scores compared
to the ST treated mice in the groups (Fig. 6). Microbial
dysbiosis and inflammatory gene expression in ST- and
STBC-treated BALB/c and C57BL/6 mice were further sup-
ported with significantly higher pathological scores in these
groups of mice with respect to time-matched untreated control
groups of mice.
Discussion
Microbial Diversity and its Resilience to Perturbation
It has been reported that microbial diversity is reduced in the
gut from the patients suffering from Crohn’s disease [35],
ulcerative colitis [36], irritable bowel syndrome [37],
Clostridium difficile associated diarrhea [38], and antibiotic-
associated diarrhea [39]. Diversity in the microbial composi-
tion is, therefore, a major factor in maintaining health. Fransen
et al. reported that BALB/c gut microbiota was more diverse
than that of C57BL/6 mice [40], which corroborated with the
current report. We, therefore, propose that because of greater
microbial diversity, BALB/c mice might be less vulnerable to
perturbation by ST than C57BL/6 mice. Our 16s rRNA pro-
filing data from the mice gut microbiota suggested that ST
perturbed the microbiota of C57BL/6 mice more than that of
the BALB/c mice. Surprisingly, the microbial perturbation
was more pronounced in STBC-treated mice than in ST-
treated mice. There was little perturbation of microbiota in
STLA-, LA-, and BC-treated mice implying that LA and BC
by itself did not perturb or alter the microbiota composition
significantly and LA was restoring ST-perturbed microbiota to
a normal level (Fig. 4). Our species-level information revealed
an increase in the number of bacteria that enhances anti-
inflammatory responses in BALB/c and C57BL/6 mice either
Probiotics & Antimicro. Prot.
via production of SCFA or other routes. Bacterial species,
include Butyricoccus pullicaecorum, Clostridium scindens,
Candidatus arthromitus, and Faecalibacterium prausnitzii,
in STLA-treated BALB/c mice and Clostridium leptum,
Herbinix hemicellulosilytica, Ruminococcus gnavus,
Acetatifactor muris, Mucisspirillum schaedleri, and
Helicobactor typholnis. SCFAs usually induce anti-
inflammatory responses in the intestinal epithelial cells of
mice by inhibiting HDACs, followed by NF-kB suppression
and downregulation of TNF and IL1B [41]. The rise in SCFA
producing bacteria in both mice species following LA treat-
ment might be suggestive of a plausible mechanism by which
treatment of mice with LA could alleviate ST-induced inflam-
mation in the gut. Such pronounced increase following BC
treatment was not evident from the current results. Dysbiosis
was evident in ST and STBC-treated mice with reduced mi-
crobial diversity compared to untreated mice; however, little
perturbation in STLA-, LA-, and BC-treated mice was ob-
served. To validate, we evaluated inflammatory and physio-
logical status in the gut by gene expression analysis using
expression microarray. The inflammatory status of the gut
was also visualized through H & E stained transverse sections
of the gut, as well as spleen and liver.
ST and STBC Treated Mice Exhibit Severe
Inflammation in Gut Compared to STLA-Treated Mice
SCFAs are inhibitors of histone deacetylases (HDACs) and
ligands for G-protein coupled receptors (GPCRs), and thereby
act as signaling molecules that influence the expansion and
function of hematopoietic and non-hematopoietic cell line-
ages. SCFA-driven inhibition of − 1 s tends to promote a
tolerogenic, antiinflammatory cell phenotype that is crucial
for maintaining immune homeostasis [42]. Exposure of pe-
ripheral blood mononuclear cells and neutrophils to SCFAs
inhibits HDAC and inactivated nuclear factor–kB (NF-kB),
and downregulated production of the proinflammatory cyto-
kine, tumor necrosis factor (TNF) [42, 43]. We observed acti-
vation of Hdac1, Hdac3, Hdac6, Hdac7, Hdac8, and Hdac9
expression by BC and STBC in BALB/c mice followed by
activation of Tnfsf10, Tnfsf13b, and Tnfsf14 through NF-kB
pathway leading to inflammation and immune cell infiltration
in STBC-treated mice. On the contrary, LA-treated BALB/c
mice downregulated HDACs and TNFs to induce low inflam-
mation and immune cell infiltration in STLA-treated mice.
Upregulated HDACs and TNFs by BC and ST might explain
the synergistic activity of ST and BC with respect to inflam-
mation in BALB/c mice, whereas HDAC downregulation by
LA antagonized the ST-induced inflammation [44]. However,
moderate activation (low-fold changes) of chemokines like
Ccl17, Ccl24, Ccrl2, CXCL12, and interleukins, such as Il2,
Il6, Il22, Il22ra1, and Il24 in STLA-treated BALB/c mice
indicated the immune activation for ST clearance without
Probiotics & Antimicro. Prot.
inducing major inflammation. Higher activation (fold changes
greater than five) of the aforementioned chemokines and cy-
tokines, along with Tnf and Il1b in STBC-treated C57BL/6
mice, indicated immune activation for ST clearance, high in-
flammation might have caused the severe immune infiltration
and damage to the gut mucosa. Overexpression of formyl
peptide receptor 1 (Fpr1) in STBC-treated BALB/c mice,
which are known to be aberrantly expressed during inflamma-
tion [45], corroborated with the inflammatory condition ob-
served in these groups of mice. SCFAs are also essential for
the maintenance of mucosal immunity by fortifying the intes-
tinal epithelial cell barrier function. It was reported that in
response to SCFAs, intestinal epithelial goblet cells increased
transcription of mucin genes [45, 46]. Results from the current
transcriptome study suggested that, while ST downregulates
mucins, including Muc5Ac, Muc5B, Muc13, Muc16, and
Muc20, LA restored expression of the Mucin genes in
STLA-treated BALB/c mice. Apart from mucins other pro-
teins contributing to IEC barrier function, are intercellular
junction proteins, such as occludins, zonula occludens 1
(known as Tjp1), and E-cadherins. Downregulation of tight
junction protein 2 (Tjp2) in STBC and ST-treated mice im-
plied compromised intestinal barrier in these groups of mice.
However, Tjp2 is overexpressed in STLA and LA-treated
mice, indicating the high integrity of the intestinal barrier in
these groups of mice. Downregulation of E-cadherins, such as
Cdh10, Cdh17, Cdh22, Cdhr1, Cdhr2, and Cdhr5 in ST- and
STBC-treated mice, indicates a compromised gut barrier in
these groups of mice.
Activation of Hdac6 by BC and STBC in C57BL/6 mice
followed by activation of Tnf, Tnfsf4, Il1b, and Tnfrsf4
through NF-kB pathway corroborated with high inflammation
and immune cell infiltration observed in STBC-treated groups
of mice. However, LA-treated C57BL/6 mice had downregu-
lated HDACs and TNFs, which was corroborated by low in-
flammation and low immune cell infiltration in STLA-treated
mice. Upregulated HDACs and TNFs by treatment with BC
and ST might explain the synergistic activity of ST and BC
with respect to inflammation in C57BL/6 mice, whereas
HDAC downregulation by LA could alleviate the ST-
induced inflammation. Moderate activation (low-fold
changes) of chemokines, such as Ccl2, Ccl7, Ccr4, Cxcl1,
Cxcl13, and interleukins, like Il12b, Il6, Il17a, Il17f, Il19,
and Il22 in STLA-treated C57BL/6 mice, indicated the im-
mune activation for ST clearance without inducing major in-
flammation [47, 48]. Higher activation of the aforementioned
chemokines and cytokines along with Tnf and Il1b in STBC-
treated C57BL/6 mice indicated immune activation for ST
clearance, but over inflammation might have compromised
survival. Aberrant expression of formyl peptide receptors
(FPRs) has been well established in states of inflammation,
autoimmune diseases, neurodegenerative disorders, and can-
cer [44]. Overexpression of Fpr1 and Fpr2 in ST-treated mice
corroborated the inflammatory status of ST-treated mice after
5 days. Expression mucin genes (Muc13, Muc16, and Muc20)
in LA and STLA-treated mice indicated an intact IEC barrier,
while downregulation of those same genes in ST- and STBC-
treated mice indicated compromised IEC barrier. Tjp1 was
moderately expressed in LA- and STLA-treated mice group
but was downregulated in ST- and STBC-treated mice to com-
promise of IEC barrier integrity in ST- and STBC-treated
mice. E-cadherin (Cdh2, Cdh7, Cdh12, Cdh20, Cdhr2, and
Cdhr4) were downregulated in ST- and STBC-treated mice,
indicating compromised gut barrier integrity in these groups
of mice. It is also important to note that there are reports that
suggested efficacy of treatment of probiotics, in general and
LA and BC, in particular, against pathogen challenge or infec-
tion [49–56]. It was reported that secreted compounds of BC
could alleviate infection because of C. difficile and BC could
have human use as well as act against viral infection by acti-
vating TLR-2 dependent mechanism [49, 52, 54, 57].
Similarly, LA could be effective against breast cancer, allevi-
ate inflammation following Salmonella infection by Tgf-β
signaling pathway and promote gut immunity as well can treat
respiratory infections [50, 51, 53, 55, 56]. The current report
revealed differential efficacy of LA and BC in treating
Salmonella infection in BALB/c and C57BL/6 mice.
Together, the microbiota, host transcriptome, and host
histopathological data suggested that BALB/c mice mi-
crobiota were less perturbed than that of C57BL/6 mice
with ST infection. LA antagonizes the ST infection
completely by inducing SCFA producing microbial spe-
cies like B. pullicaecorum, C. scindens, C. arthromitus, C.
tertium, C. xylanolyticum, and F. prausnitzii in BALB/c
mice. However, BC could not induce these SCFA produc-
ing bacteria significantly; therefore, STBC-treated BALB/
c mice survived, but with severe inflammation in the gut.
Downregulated Tnf and Il1b in STLA-treated mice but
upregulated Tnf and Il1b in STBC-treated mice, in addi-
tion to evidence from histological data, was in accordance
with the previously mentioned observations. Further ex-
pression of several mucins, zonula occludens 1, and sev-
eral E-cadherins in STLA-treated mice indicated the intact
IEC barrier function, which was supported by gut histo-
logical data (Fig. S7, the graphical summary).
The microbiota of C57BL/6 mice was readily perturbed by
the ST-induced growth of sulfate-reducing proteobacteria D.
intestinalis and inflammation-inducing proteobacteria H.
typhlonius. However, LA-treated mice have increased
antiinflammatory SCFA producing bacteria, such as C. lepum,
C . p o l y s a c c h a r o l y t i c u m , P. d e n t a l i s , a n d H .
hemicellulosilytica through which consequently neutralized
the ST-induced inflammation in mice gut. The overactivation
of HDACs by BC and ST in C57BL/6 mice induces NF-kB
mediated production of the inflammation-inducing Tnf and
Il1b. The inflammation is again accompanied by

compromised IEC barrier function through downregulation of
mucins, Tjp1, and several E-cadherins in STBC-treated
C57BL/6 mice, which is evident from the histology of the
mice gut (Fig. S8, the graphical summary).
Microbial diversity has been reported to be depleted in
various inflammatory intestinal diseases [40, 58, 59]. It has
been observed that strain difference, strain dependent differ-
ence in secretoryIgA (sIgA) production as well as conserved
signatures of salmonellosis in mouse and human hinted at
important strategies of Salmonella infection [40, 58, 59].
However, a common treatment or prevention was yet to be
established. The present study reported that LA could be a
strain independent solution for Salmonella infection. Here,
also we reported that gut microbiota is depleted by ST-
induced diarrhea accompanied by severe gut inflammation.
Therefore, depletion of microbial diversity can be regarded
as a marker for several gut-related diseases. While probiotics
were supplemented successfully offset the dysbiosis in gut
microbiota caused by antibiotics, it should be kept in mind
that a few probiotics species exist, such as BC, which may
exacerbate inflammation during ST infection. We conclude
that each species and each strain of probiotics should be pre-
screened for their compatibility with a particular type of infec-
tion or disease; further study in this area is highly needed and
future insights may serve as promising prospects for probiotic
use in clinical settings. Since current study is done in mice, we
cannot directly corroborate this with humans as well as the
SCFA associated microbiome identified in Th1 and Th2 mice
need to be validated in humans under appropriate immune
bias.
Acknowledgments We acknowledge and we are thankful to Mr. Madan
Mohan Mallick and Mr. Susanta Kumar Swain, the technicians of Dr.
Shantibhusan Senapati’s laboratory for making the histological slides.
We deeply appreciate Bionivid, Xcelris Lab and SciGenome for timely
execution of 16S rRNA sequencing. We acknowledge Mr. Sibabrata
Sarangi, attendant, NISER animal house for taking care of the animals
during the experiments. We heartily appreciate Mr. David Alexander
Datzkiw for proofreading this manuscript.
Data Submission The transcriptomics data discussed in this publication
have been deposited in NCBI’s Gene Expression Omnibus and are acces-
sible through GEO series accession number GSE98353 (https://www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?token=excdimugvlubnkj&acc=
GSE98353).
The 16S rDNA sequencing data discussed in this publication have
been deposited in NCBI’s Sequence Read Archive and are accessible
through Bio project number PRJNA388784 (http://www.ncbi.nlm.nih.
gov/bioproject/388784) and PRJNA392028 (https://www.ncbi.nlm.nih.
gov/bioproject/PRJNA392028).
Author Contributions P.A. and B.P. planned the study and designed the
experiments. B.P. executed most of the experiments and D.G., A.K.N,
A.B. assisted in the experiments with supervision from P.A., S.S., S.C.,
B.P., and D.G. analyzed the histopathological data. P.A., B.P., and D.G.
analyzed the microarray data. S.T., B.P., P.A., and A.B. analyzed the
microbiome data. BP prepared the first draft of the manuscript. P.A.
overall supervised the work and finalized the manuscript.
Probiotics & Antimicro. Prot.
Funding Information This study is provided by the Department of
Biotechnology (DBT), Government of India for partially funding this
project through extramural support. The funders had no role in study
design, data collection, and interpretation, or the decision to submit the
work for publication
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflicts of
interest with the contents of this article.
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