Background
Neurological complications occurring due to
Salmonella infection of the brain remain a matter of serious concern [
1]. Such infections are associated with frequent relapse episodes, neurological abnormalities along with severe side effects such as auditory and visual impairments, mental retardation and poor prognosis leading to high mortality rates [
2]. Previous reports in mouse models have also elaborated on the ineffectiveness of antibiotics in completely curing infections of such kind [
3]. Simultaneously, increased incidence and rise in cases of drug resistant
Salmonella has further limited the treatment arsenal for this dangerous pathogen [
4]. Interestingly, recent evidences have indicated that inflammatory diseases/conditions of the gut are linked to psychiatric and behavioural abnormalities [
5]. Additionally, various neurodegenerative diseases have been associated with an altered and specific microbiota profile [
6]. In the prevailing scenario, tackling and treating infections by targeting multi-dimensional aspects of health and disease has led to increasing interest in the field of neurogastroenterology which interlinks diverse aspects of neuroscience, gastroenterology, immunology, behaviour science, microbiology, pharmacology and other related subjects to devise strategies that can improve the host health status at a holistic level [
7]. Herein, manipulation of gut-brain axis has proven to be of enhanced significance, given its direct implications at the gut as well as the brain [
8]. The brain to gut signalling is evidenced by the findings that different kinds of psychological stressors modulate the composition as well as the biomass of the gut microbiota. The CNS has been reported to impact other bodily as well as gut functions, including the gut microbiota, via ‘the emotional motor system (EMS)’ consisting of hypothalamus–pituitary–adrenal (HPA) axis, parallelly working branches of the sympathetic as well as parasympathetic autonomic nervous system (ANS), and other pathways mediating discomfort and pain [
9]. The ANS finds direct role in gut functions such as mucus, bicarbonate and acid secretion along with intestinal motility, immune response and permeability, coupled with secretion of neuroactive signalling molecules such as catecholamines, cytokines, serotonin, etc., thereby impacting the microbiota [
10]. Simultaneously, microbes have been known to influence almost all major pathways associated with the gut-brain axis. These include the neural pathway by modulating/producing/regulating the level of neurotransmitters [
11]; endocrine pathways and its associated moieties such as neuroendocrine cells, neuroactive substances and neuropeptides; host immune system as well as functioning and maturation of most components of innate and adaptive immunity. All these molecules have been reported to influence key aspects of brain and behaviour including neurodegeneration, apoptosis and neurogenesis, which, coupled with inflammation, further highlight their importance in maintaining host well-being [
12,
13].
Therefore, given the crucial role of gut microbiota in this bi-directional communication, use of probiotics as a bio-compatible treatment strategy has particularly captured the interest of the scientific community. Probiotics have been reported to influence all the key pathways that can be influenced by the host microbiota thereby presenting as a highly promising candidate for targeting gut-brain axis disorders [
13]. Additionally, the capability of these bacteria to function as psychobiotics by improving the psychological status along with their impact on the host pathology and physiology has opened diverse areas of targeted therapy which might prove useful in a variety of related manifestations [
14]. In our previous study, administration of
Lactiplantibacillus plantarum (RTA 8) was found to be useful in ameliorating
Salmonella induced brain infection in mouse model [
15]. To the best of our awareness, this is the first study elaborating on the prophylactic effect of
L. plantarum (RTA 8) in modulating the amount of neurotransmitters and other neuroactive molecules such as BDNF along with mucin genes, at the gut-brain axis, thereby preventing
Salmonella induced neurological manifestations.
Discussion
In contrast to the previously conducted independent study [
15], herein, exclusively the prophylactic potential of
L. plantarum (RTA 8) in combating
Salmonella induced brain infection was evaluated. It was inferred that decreasing the duration of
L. plantarum (RTA 8) administration from 14 to only 7 days prior to infection, did not affect its efficacy in preventing brain infection. The same was evidenced by a similar reduction in bacterial burden and amelioration in tissue histology, as observed in the previous study. Other studies employing probiotic strains for similar duration have also reported similar fold reduction in
Salmonella bio-burden [
16]. Infection with
S. Typhimurium has been reported to cause destruction of the ileum, similar to that observed in our study, thereby resulting in increased translocation of bacteria to other organs and imbalance in gut microbial population [
17]. Reduction in luminal pH due to production of short chain fatty acids by
L. plantarum (RTA 8) along with production of IgA and other potent antimicrobial substances might have resulted in clearance of
Salmonella [
18,
19]. Additionally, the abilities of probiotics in occupying receptor binding sites at the epithelial surface, strengthening the intestinal microstructure and increasing the villus length, might have further blocked pathogen adherence [
15,
20,
21]. All these factors might have conferred colonization resistance to the host against such deadly pathogens, thereby preventing their translocation to other organs such as the brain. Simultaneously, direct transmission of information from gut to brain through the vagus nerve might have resulted in activation of Fos immune reactive cells in the hypothalamic region of the brain which might have also contributed to effective bacterial clearance from the brain [
22]. In consonance with our findings, various studies have highlighted the ability of probiotics in reversing pathogen or microbial component such as lipopolysaccharide (LPS) mediated neurodegeneration by using interventions targeting the gut microbiota [
13,
23].
Neurotransmitters constitute a vital part of the gut-brain communication. As observed in the present study, decreased level of serotonin, more significantly so in the brain tissue samples of the
Salmonella infected group, have been associated with increased pathogenesis and decreased host survival in case of infections caused by pathogens such as
Citrobacter rodentium and enterohaemorrhagic
Escherichia coli (EHEC); presence of serotonin has been shown to decrease the expression of LEE virulence genes located within the locus of enterocyte effacement (LEE) pathogenicity island (PI) via the blocking of CpxA, a membrane-bound histidine sensor kinases (HKs) present in bacteria thereby reducing its pathogenicity [
24]. Significantly increased levels of serotonin in the
L. plantarum (RTA 8) group might have resulted in reduced pathogenicity of
Salmonella, thereby preventing serious consequences. Other studies have reported the ability of probiotic strains such as
E. coli Nissle 1917 in increasing the bioavailability of serotonin in ileal tissue [
25]. The same has been postulated to be a result of SCFA (short chain fatty acids) produced by these gut bacteria acting directly on the enterochromaffin cells which harbour the enzyme tryptophan hydroxylase, resulting in serotonin regulation [
26]. Similar to our observations, other studies have also reported increased levels of serotonin in the brain tissues of animals receiving probiotics such as
Akkermansia muciniphila and
Bifidobacterium breve CCFM1025 [
27,
28]. These effects were found to be mediated by the upregulations of TPH1 and TPH2 genes involved in serotonin biosynthesis in the intestine and the brain, respectively.
The significantly reduced levels of dopamine in the brain and intestinal tissue samples of mice in the
Salmonella infected group could be attributed to the ability of
Salmonella and its major cell wall component, LPS, in activation of microglial cells. This might have resulted in a neurotoxic environment thereby increasing the susceptibility of dopaminergic neurons in the substantia nigra and striatum to neuroinflammation induced damage, leading to reduction in its synthesis as well as effective concentration [
29]. The normalization of levels of dopamine in the brain tissue samples of mice in
L. plantarum (RTA 8) treated group could be ascribed to the ability of gut microbiota and probiotics in altering the levels of neurotransmitters in the brain, directly by influencing the ENS and interacting with the glial cells of the gut [
12,
30]. Administration of cocktail of probiotics or probiotic strain coupled with a prebiotic have been demonstrated to prevent neurodegeneration while simultaneously increasing the cortical levels of dopamine, as well as preventing degeneration of dopaminergic neurons in the substantia nigra pars compacta [
31]. Simultaneously, increased production of butyrate and BDNF/GNDF after probiotic administration, as observed in our study, has also been implicated in raised dopamine levels [
32]. In another study, administration of
Lacticaseibacillus zeae LB1 prevented
Salmonella infection in
Caenorhabditis elegans via modulation of both serotonin and dopamine, confirming their role in preventing serious manifestations [
33].
Catecholamines have been reported to induce the growth of enteric pathogens by acting as quorum sensing molecules. However, significantly decreased levels of norepinephrine in the brain tissue samples of
Salmonella infected mice were observed which could be attributed to the role of LPS in reducing the levels of noradrenaline by affecting the brain physiology [
34]. Brain infection with
Toxoplasma gondii has also been associated with decreased level of norepinephrine which might have resulted from changes in the metabolic enzyme tyrosine hydroxylase, that converts tyrosine to catecholamines in noradrenergic neurons [
35]. Further, administration of
L. plantarum (RTA 8) was observed to normalize the level of norepinephrine and could be explained by the previously reported ability of probiotics as well as gut microbiota in modulating the level of catecholamines [
36]. Interestingly, host norepinephrine/epinephrine levels have been implicated in downregulation of two important virulence gene of
Salmonella Typhimurium SL1344 i.e.,
virK and
mig14, and simultaneously increasing its sensitivity to host antimicrobial peptide LL-37 (which is a part of the innate defence system) suggesting its role in decreasing virulence of the organism [
37]. Probiotic strains such as
Lacticaseibacillus rhamnosus GG and
E. coli CFR 16 have been reported to increase levels of norepinephrine and restore level of other neurotransmitters via vagus nerve mediated pathways or by directly affecting their receptors [
38,
39].
Treatment with
L. plantarum (RTA 8) resulted in a significant increase in the expression of MUC1 as well as MUC3 mucin genes in the treatment group. Other studies have also reported an increase in mucin production after oral administration of probiotic strains such as VSL#3 or on probiotic exposure to colonic loops and cell lines [
40,
41]. The expression of these genes was evaluated given the role of MUC1 in maintaining mucosal barrier during infections such as those caused by
Helicobacter pylori [
42] and of MUC3 in inhibiting the attachment of pathogens such as
E. coli in its secreted form [
43]. The increase in mucin gene expression might be attributed to the ability of probiotic strains in either increasing the density of goblet cells or inducing heightened activity in already differentiated goblet cells [
41]. Fermentation products of certain probiotic metabolites have also been reported to induce mucin expression [
44]. Simultaneously, enhanced expression of MUC3 mucin gene might have barred the attachment of
Salmonella to the intestinal epithelium, thereby preventing brain infection. Other co-incubation experiments with
L. plantarum strain 299v and
Lactobacillus casei GG have been observed to increase mucin secretion, resulting in reduced adherence and translocation of gut pathogens such as
E. coli [
45].
A significant decrease in the expression of BDNF gene was observed in mice suffering from
Salmonella infection. BDNF has been reported to play an important role in neurogenesis, neurodegeneration and associated behavior [
46,
47]. Various studies have reported its role in memory and learning owing to its ability in mediating plastic changes [
48]. Additionally, dysregulation of this neurotrophic factor has been associated with psychiatric disorders such as depressive-like behaviour, bipolar disorder and schizophrenia [
49‐
51]. Infection studies with
T. muris as well as neonatal meningitis caused by
Streptococcus pneumoniae have also reported similar findings which might be attributed to neurodegeneration caused by neuroinflammation [
52]. However, normalization of BDNF expression was observed after administration of
L. plantarum (RTA 8), thereby highlighting the ability of gut microbes in altering brain behaviour and neurochemistry which could be mediated via inflammation dependent as well as independent pathways.
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