Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Bi-directional elucidation of Lactiplantibacillus plantarum (RTA 8) intervention on the pathophysiology of gut-brain axis during Salmonella brain infection

Bi-directional elucidation of Lactiplantibacillus plantarum (RTA 8) intervention on the... Background: There have been reports of patients suffering from typhoid fever, particularly those involving infants and immunocompromised patients, which at times present with Salmonella induced brain infection. Although rare, it has frequently been associated with adverse neurological complications and increased mortality. In this context, the gut-brain axis, involving two-way communication between the gut and the brain, holds immense significance as various gut ailments have been associated with psychiatric complications. In turn, several neurodegenerative diseases have been associated with an altered gut microbiota profile. Given the paucity of effective antimicrobials and increas- ing incidence of multi-drug resistance in pathogens, alternate treatment therapies such as probiotics have gained significant attention in the recent past. Results: In the current study, prophylactic effect of Lactiplantibacillus plantarum (RTA 8) in preventing neurological complications occurring due to Salmonella brain infection was evaluated in a murine model. Along with a significant reduction in bacterial burden and improved histoarchitecture, L. plantarum (RTA 8) administration resulted in amelio- ration in the level of neurotransmitters such as serotonin, norepinephrine and dopamine in the gut as well as in the brain tissue. Simultaneously, increased gene expression of physiologically essential molecules such as mucin (MUC1 and MUC3) and brain-derived neurotrophic factor (BDNF) was also observed in this group. Conclusion: Present study highlights the potential benefits of a probiotic supplemented diet in improving various aspects of host health due to their multi-targeted approach, thereby resulting in multi-faceted gains. Keywords: Salmonella, Neurological complications, Gut-brain axis, Probiotics episodes, neurological abnormalities along with severe Background side effects such as auditory and visual impairments, Neurological complications occurring due to Salmonella mental retardation and poor prognosis leading to high infection of the brain remain a matter of serious concern mortality rates [2]. Previous reports in mouse models [1]. Such infections are associated with frequent relapse have also elaborated on the ineffectiveness of antibiotics in completely curing infections of such kind [3]. Simul- *Correspondence: rishipraveen@yahoo.com; rishiparveen@pu.ac.in 1 taneously, increased incidence and rise in cases of drug Department of Microbiology, Basic Medical Sciences Block I, Panjab University, South Campus, Sector 25, Chandigarh 160014, India resistant Salmonella has further limited the treatment Full list of author information is available at the end of the article arsenal for this dangerous pathogen [4]. Interestingly, © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kaur et al. Gut Pathogens (2022) 14:11 Page 2 of 13 recent evidences have indicated that inflammatory dis - the host pathology and physiology has opened diverse eases/conditions of the gut are linked to psychiatric and areas of targeted therapy which might prove useful in behavioural abnormalities [5]. Additionally, various neu- a variety of related manifestations [14]. In our previous rodegenerative diseases have been associated with an study, administration of Lactiplantibacillus plantarum altered and specific microbiota profile [6]. In the prevail - (RTA 8) was found to be useful in ameliorating Salmo- ing scenario, tackling and treating infections by targeting nella induced brain infection in mouse model [15]. To multi-dimensional aspects of health and disease has led the best of our awareness, this is the first study elaborat - to increasing interest in the field of neurogastroenterol - ing on the prophylactic effect of L. plantarum (RTA 8) in ogy which interlinks diverse aspects of neuroscience, modulating the amount of neurotransmitters and other gastroenterology, immunology, behaviour science, micro- neuroactive molecules such as BDNF along with mucin biology, pharmacology and other related subjects to genes, at the gut-brain axis, thereby preventing Salmo- devise strategies that can improve the host health status nella induced neurological manifestations. at a holistic level [7]. Herein, manipulation of gut-brain axis has proven to be of enhanced significance, given its Results direct implications at the gut as well as the brain [8]. The Efficacy of L. plantarum (RTA 8) at systemic level brain to gut signalling is evidenced by the findings that Significant bacterial burden in vital organs of mice in the different kinds of psychological stressors modulate the infected group indicated systemic spread of the patho- composition as well as the biomass of the gut microbiota. gen. L. plantarum (RTA 8) administration 7 days prior to The CNS has been reported to impact other bodily as well infection significantly reduced Salmonella bio-burden in as gut functions, including the gut microbiota, via ‘the all organs. A 2.9 (p < 0.001) and 2.85 (p < 0.01) fold reduc- emotional motor system (EMS)’ consisting of hypothala- tion was observed in liver and spleen tissues samples of mus–pituitary–adrenal (HPA) axis, parallelly working the treatment group, respectively, in comparison to that branches of the sympathetic as well as parasympathetic of the infected group thereby indicating efficacy of the autonomic nervous system (ANS), and other pathways treatment (Fig. 1). mediating discomfort and pain [9]. The ANS finds direct role in gut functions such as mucus, bicarbonate and acid Efficacy of L. plantarum (RTA 8) at gut‑brain axis secretion along with intestinal motility, immune response Tissue bio‑burden and permeability, coupled with secretion of neuroactive Dissemination of Salmonella from the gut to the brain signalling molecules such as catecholamines, cytokines, was confirmed as significant bacterial burden was serotonin, etc., thereby impacting the microbiota [10]. observed in the brain tissue. Simultaneously, a significant Simultaneously, microbes have been known to influence reduction in bacterial burden in the intestine as well as almost all major pathways associated with the gut-brain the brain tissue samples of mice in L. plantarum (RTA 8) axis. These include the neural pathway by modulating/ treated group was observed. A 2.84 fold (p < 0.01) reduc- producing/regulating the level of neurotransmitters [11]; tion in Salmonella bio-burden was observed in intesti- endocrine pathways and its associated moieties such as nal sections of the treatment group as compared to that neuroendocrine cells, neuroactive substances and neu- in the infected group. Similarly, administration of L. ropeptides; host immune system as well as functioning plantarum (RTA 8) resulted in a significant decrease in and maturation of most components of innate and adap- bacterial counts (6.06 fold reduction, p < 0.001) to 0.77 tive immunity. All these molecules have been reported log CFU/ml in the brain tissue samples relative to the to influence key aspects of brain and behaviour includ - infected group, demonstrating the effectiveness of L. ing neurodegeneration, apoptosis and neurogenesis, plantarum (RTA 8) in preventing Salmonella brain infec- which, coupled with inflammation, further highlight their tion (Fig. 1). importance in maintaining host well-being [12, 13]. Therefore, given the crucial role of gut microbiota in Level of dopamine this bi-directional communication, use of probiotics as a Infection with S. Typhimurium SL1344 significantly bio-compatible treatment strategy has particularly cap- reduced the level of dopamine in murine gut tissue sam- tured the interest of the scientific community. Probiot - ples by 80.9% (p < 0.01) in comparison to the control. L. ics have been reported to influence all the key pathways plantarum (RTA 8) administered group demonstrated that can be influenced by the host microbiota thereby an increase in the level of dopamine, however, the levels presenting as a highly promising candidate for targeting were not found to be significantly different from that of gut-brain axis disorders [13]. Additionally, the capability the infected group. of these bacteria to function as psychobiotics by improv- In comparison to the control group, Salmonella ing the psychological status along with their impact on infected mice demonstrated a significant reduction K aur et al. Gut Pathogens (2022) 14:11 Page 3 of 13 Fig. 1 L. plantarum (RTA 8) administration 7 days prior to infection significantly reduced Salmonella count in all the vital organs. Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing the median, dots representing individual values and ‘x’ representing the mean value of dataset. **p < 0.01 and ***p < 0.001 versus infected by analysis of variance (ANOVA) of 64.1% (p < 0.01) in dopamine levels in the brain tis- plantarum (RTA 8) resulted in a significant increase sues. Treatment with L. plantarum (RTA 8) significantly (p < 0.01) in the hormone levels in comparison to the increased the levels of dopamine in the brain tissues in infected tissue (Fig. 3). comparison to the infected group (p < 0.01). Dopamine levels in the treatment group were found to be restored to the level observed in the control group (Fig. 2). Level of serotonin L. plantarum (RTA 8) administered group demonstrated a significant 34% increase (p < 0.05) in the level of sero- Level of norepinephrine tonin in the gut of mice in comparison to the control Intestinal samples of mice infected with Salmonella dem- group. An 18.4% reduction in the level of serotonin was onstrated a decrease of 46.1% in norepinephrine levels in observed in the intestine tissue samples of mice in the comparison to the control group, however it was found infected group. As compared to the infected group, a to be insignificant. In comparison to the infected group, significant increase was observed in the treatment group mice in the L. plantarum (RTA 8) administered group (p < 0.01). demonstrated a relative increase in norepinephrine levels In the brain tissue sections, mice in the Salmonella but the results were not found to be significant. infected group demonstrated a significant decrease of In contrast to results obtained in the intestinal tis- 21.9% in the level of serotonin as compared to the control sue samples, Salmonella infected group demonstrated group (p < 0.05). Simultaneously, L. plantarum (RTA 8) a very significant reduction of 34.7% in the level of administered group demonstrated a significant increase norepinephrine in brain tissues samples (p < 0.001) in the level of serotonin in comparison to the infected as compared to the control group. Treatment with L. group (p < 0.01) (Fig. 4). Kaur et al. Gut Pathogens (2022) 14:11 Page 4 of 13 Fig. 2 Dopamine levels in the murine brain and intestinal tissue samples. Dopamine levels was observed to be significantly decreased in the infected group and normal levels were found to be restored in murine brain after administration of L. plantarum (RTA 8). Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing the median, dots representing individual values and ‘x’ representing the mean value of dataset. $$ **p < 0.01, *p < 0.05 in comparison to Control. p < 0.01 in comparison to Infected Expression of MUC 1 gene plantarum (RTA 8) administered group in comparison to Expression of MUC 1 gene was found to be significantly the infected group (Fig. 5). upregulated in the group administered with L. plantarum (RTA 8). A 7.6 fold significant increase (p < 0.05) was Expression of BDNF gene observed in this group as compared to the control group. Quantitative RT-PCR studies revealed significant down - Additionally, a 6.7 fold increase (p < 0.01) was observed in regulation of BDNF gene expression in brain tissues of the treatment group as compared to the infected group. Salmonella infected mice. A 6.3 fold reduction (p < 0.001) No significant difference in gene expression was observed in expression was observed in the infected group in between the control and the infected group (Fig. 5). comparison to the control group. L. plantarum (RTA 8) administration resulted in a significant 6.5 fold increase in BDNF expression as compared to the infected group (p < 0.011) (Fig. 5). Expression of MUC 3 gene Expression of MUC 3 gene in the Salmonella infected Histopathological studies group was found to be downregulated significantly (3.1 Histopathological analysis of intestinal tissue sections fold, p < 0.05). Simultaneously, L. plantarum (RTA 8) of the control group depicted normal tissue architec- treated group demonstrated a significant upregulation of ture with normal distribution of surface enterocytes, MUC 3 gene expression (5.5 fold p < 0.05) as compared goblet cells and muscularis mucosa. Inflammatory to the control group. A significant 17.4 fold increase in cells were absent and the crypts and villi demonstrated the expression of MUC 3 gene was observed in the L. K aur et al. Gut Pathogens (2022) 14:11 Page 5 of 13 Fig. 3 Norepinephrine levels in the murine intestinal and brain tissue samples. Decreased level of dopamine was observed in the Salmonella infected group with significantly reduced levels in the brain. L. plantarum (RTA 8) administration was found to increase intestinal norepinephrine levels but significant increase was found in the brain tissue samples. Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing $$ the median, dots representing individual values and ‘x’ representing the mean value of dataset.***p < 0.001 in comparison to Control; p < 0.01 in comparison to Infected normal histoarchitecture (Fig.  6A). However, infected administered with L. plantarum (RTA 8) 7  days prior mice revealed severely damaged villi with focal ulcera- to infection, prevented meningitis as the meninges was tions and inflammation. Crypt hyperplasia was also found to be clear of signs of inflammation. The brain tis - observed. The villi were found to be stunted and the sue in this group showed normal tissue histoarchitecture crypts were found to be enlarged and elongated (Fig. 6B). with only very mild oedema (Fig. 6F). In contrast to this, L. plantarum (RTA 8) administered mice demonstrated normal tissue morphology similar to Discussion that observed in the control tissue samples. The normal In contrast to the previously conducted independent villi to crypt ratio of 5:1 was restored, thereby elaborat- study [15], herein, exclusively the prophylactic poten- ing on the efficacy of L. plantarum (RTA 8) in preventing tial of L. plantarum (RTA 8) in combating Salmonella Salmonella infection (Fig. 6C). induced brain infection was evaluated. It was inferred Brain tissue samples of mice in the control group that decreasing the duration of L. plantarum (RTA 8) revealed normal tissue morphology with the presence administration from 14 to only 7  days prior to infec- of triangular neurons and microglial cells with oligo- tion, did not affect its efficacy in preventing brain infec - dendrocytes; sans inflammation (Fig.  6D). However, oral tion. The same was evidenced by a similar reduction in infection with Salmonella led to brain infection and men- bacterial burden and amelioration in tissue histology, as ingitis which was confirmed histologically by increased observed in the previous study. Other studies employing accumulation of inflammatory cells in the meninges of probiotic strains for similar duration have also reported the brain. Simultaneously, marked oedema was observed similar fold reduction in Salmonella bio-burden [16]. in the cortical tissue (Fig.  6E). On the other hand, mice Infection with S. Typhimurium has been reported to Kaur et al. Gut Pathogens (2022) 14:11 Page 6 of 13 Fig. 4 Serotonin levels in the murine intestinal and brain tissue samples. Significantly decreased levels of serotonin were observed in brain tissues of mice infected with Salmonella. L. plantarum (RTA 8) treated group demonstrated a significant increase in the level of serotonin in the brain as well as the intestinal tissue samples. Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing the median, dots representing $$ individual values and ‘x’ representing the mean value of dataset. *p < 0.05 in comparison to Control; p < 0.01 in comparison to Infected cause destruction of the ileum, similar to that observed the ability of probiotics in reversing pathogen or micro- in our study, thereby resulting in increased translocation bial component such as lipopolysaccharide (LPS) medi- of bacteria to other organs and imbalance in gut micro- ated neurodegeneration by using interventions targeting bial population [17]. Reduction in luminal pH due to pro- the gut microbiota [13, 23]. duction of short chain fatty acids by L. plantarum (RTA Neurotransmitters constitute a vital part of the gut- 8) along with production of IgA and other potent anti- brain communication. As observed in the present study, microbial substances might have resulted in clearance of decreased level of serotonin, more significantly so in the Salmonella [18, 19]. Additionally, the abilities of probiot- brain tissue samples of the Salmonella infected group, ics in occupying receptor binding sites at the epithelial have been associated with increased pathogenesis and surface, strengthening the intestinal microstructure and decreased host survival in case of infections caused by increasing the villus length, might have further blocked pathogens such as Citrobacter rodentium and entero- pathogen adherence [15, 20, 21]. All these factors might haemorrhagic Escherichia coli (EHEC); presence of sero- have conferred colonization resistance to the host against tonin has been shown to decrease the expression of LEE such deadly pathogens, thereby preventing their translo- virulence genes located within the locus of enterocyte cation to other organs such as the brain. Simultaneously, effacement (LEE) pathogenicity island (PI) via the block - direct transmission of information from gut to brain ing of CpxA, a membrane-bound histidine sensor kinases through the vagus nerve might have resulted in activation (HKs) present in bacteria thereby reducing its patho- of Fos immune reactive cells in the hypothalamic region genicity [24]. Significantly increased levels of serotonin of the brain which might have also contributed to effec - in the L. plantarum (RTA 8) group might have resulted tive bacterial clearance from the brain [22]. In conso- in reduced pathogenicity of Salmonella, thereby prevent- nance with our findings, various studies have highlighted ing serious consequences. Other studies have reported K aur et al. Gut Pathogens (2022) 14:11 Page 7 of 13 Fig. 5 Relative fold change in expression of BDNF, MUC1 and MUC3 genes. Fold expression was determined after normalization to GAPDH for each $$ sample. ***p < 0.001, *p < 0.05 in comparison to control; p < 0.01 in comparison to infected the ability of probiotic strains such as E. coli Nissle 1917 of gut microbiota and probiotics in altering the levels in increasing the bioavailability of serotonin in ileal tis- of neurotransmitters in the brain, directly by influenc - sue [25]. The same has been postulated to be a result of ing the ENS and interacting with the glial cells of the SCFA (short chain fatty acids) produced by these gut bac- gut [12, 30]. Administration of cocktail of probiotics or teria acting directly on the enterochromaffin cells which probiotic strain coupled with a prebiotic have been dem- harbour the enzyme tryptophan hydroxylase, resulting onstrated to prevent neurodegeneration while simultane- in serotonin regulation [26]. Similar to our observations, ously increasing the cortical levels of dopamine, as well other studies have also reported increased levels of sero- as preventing degeneration of dopaminergic neurons in tonin in the brain tissues of animals receiving probiotics the substantia nigra pars compacta [31]. Simultaneously, such as Akkermansia muciniphila and Bifidobacterium increased production of butyrate and BDNF/GNDF after breve CCFM1025 [27, 28]. These effects were found to be probiotic administration, as observed in our study, has mediated by the upregulations of TPH1 and TPH2 genes also been implicated in raised dopamine levels [32]. In involved in serotonin biosynthesis in the intestine and another study, administration of Lacticaseibacillus zeae the brain, respectively. LB1 prevented Salmonella infection in Caenorhabditis The significantly reduced levels of dopamine in the elegans via modulation of both serotonin and dopamine, brain and intestinal tissue samples of mice in the Salmo- confirming their role in preventing serious manifesta - nella infected group could be attributed to the ability of tions [33]. Salmonella and its major cell wall component, LPS, in Catecholamines have been reported to induce the activation of microglial cells. This might have resulted in growth of enteric pathogens by acting as quorum sens- a neurotoxic environment thereby increasing the suscep- ing molecules. However, significantly decreased levels tibility of dopaminergic neurons in the substantia nigra of norepinephrine in the brain tissue samples of Sal- and striatum to neuroinflammation induced damage, monella infected mice were observed which could be leading to reduction in its synthesis as well as effective attributed to the role of LPS in reducing the levels of concentration [29]. The normalization of levels of dopa - noradrenaline by affecting the brain physiology [34]. mine in the brain tissue samples of mice in L. plantarum Brain infection with Toxoplasma gondii has also been (RTA 8) treated group could be ascribed to the ability associated with decreased level of norepinephrine Kaur et al. Gut Pathogens (2022) 14:11 Page 8 of 13 Fig. 6 Histological evaluation of brain and intestinal tissue sections. A (X40, scale bar 50 µm) H&E staining of control intestinal tissue depicted normal intensity of goblet cells and lymphocytes in the lamina propria depicting normal tissue morphology. B (X40, scale bar 50 µm) Photomicrographs of Salmonella infected intestinal tissue depicted ileal damage inflicted by Salmonella with heavy influx of inflammatory cells along with crypt elongation (highlighted by arrows). C (X40, scale bar 50 µm) L. plantarum (RTA 8) treated group revealed restoration of normal intestinal tissue morphology as revealed by restoration of villi to crypt ratio and normal density of lymphocytes (highlighted by arrows). D (X40, scale bar 50 µm) Photomicrographs of cortical mice brain region in the control group depicted normal histology revealing presence of triangular neurons and glial cells. E (X40, scale bar 50 µm) Photomicrographs of brain tissue sections in the Salmonella infected group revealed heavy influx of inflammatory cells along the surface and meninges depicting meningitis (highlighted by arrows) along with brain infection characterized by oedema (highlighted by marking a circle). F (X40, scale bar 50 µm) Brain tissue sections of mice administered with L. plantarum (RTA-8) administered group depicted slight oedema with otherwise normal histoarchitecture which might have resulted from changes in the meta- Treatment with L. plantarum (RTA 8) resulted in a bolic enzyme tyrosine hydroxylase, that converts tyros- significant increase in the expression of MUC1 as well ine to catecholamines in noradrenergic neurons [35]. as MUC3 mucin genes in the treatment group. Other Further, administration of L. plantarum (RTA 8) was studies have also reported an increase in mucin produc- observed to normalize the level of norepinephrine and tion after oral administration of probiotic strains such as could be explained by the previously reported ability of VSL#3 or on probiotic exposure to colonic loops and cell probiotics as well as gut microbiota in modulating the lines [40, 41]. The expression of these genes was evaluated level of catecholamines [36]. Interestingly, host nor- given the role of MUC1 in maintaining mucosal barrier epinephrine/epinephrine levels have been implicated during infections such as those caused by Helicobacter in downregulation of two important virulence gene of pylori [42] and of MUC3 in inhibiting the attachment of Salmonella Typhimurium SL1344 i.e., virK and mig14, pathogens such as E. coli in its secreted form [43]. The and simultaneously increasing its sensitivity to host increase in mucin gene expression might be attributed antimicrobial peptide LL-37 (which is a part of the to the ability of probiotic strains in either increasing the innate defence system) suggesting its role in decreasing density of goblet cells or inducing heightened activity virulence of the organism [37]. Probiotic strains such in already differentiated goblet cells [41]. Fermentation as Lacticaseibacillus rhamnosus GG and E. coli CFR 16 products of certain probiotic metabolites have also been have been reported to increase levels of norepinephrine reported to induce mucin expression [44]. Simultane- and restore level of other neurotransmitters via vagus ously, enhanced expression of MUC3 mucin gene might nerve mediated pathways or by directly affecting their have barred the attachment of Salmonella to the intesti- receptors [38, 39]. nal epithelium, thereby preventing brain infection. Other K aur et al. Gut Pathogens (2022) 14:11 Page 9 of 13 co-incubation experiments with L. plantarum strain Methods 299v and Lactobacillus casei GG have been observed to Bacterial strains increase mucin secretion, resulting in reduced adherence Two bacterial strains, namely Salmonella enterica sero- and translocation of gut pathogens such as E. coli [45]. var Typhimurium SL1344 and Lactiplantibacillus plan- A significant decrease in the expression of BDNF gene tarum (RTA 8) were used throughout the study. The was observed in mice suffering from Salmonella infec - former was kindly gifted by Dr. Mrytyunjay Suar, Direc- tion. BDNF has been reported to play an important tor of School of Biotechnology, KIIT University, Odisha role in neurogenesis, neurodegeneration and associated and the latter was kindly provided by Prof. Rupinder behavior [46, 47]. Various studies have reported its role Tewari, Department of Microbial Biotechnology, Pan- in memory and learning owing to its ability in mediating jab University, Chandigarh. Salmonella strain was plastic changes [48]. Additionally, dysregulation of this routinely cultured in Luria–Bertani (LB) medium and neurotrophic factor has been associated with psychiatric streaked on solid LB agar plates containing antibiotic disorders such as depressive-like behaviour, bipolar dis- streptomycin at a concentration of 50  μg/ml. The Lac - order and schizophrenia [49–51]. Infection studies with tiplantibacillus strain was cultured in de Man, Rogosa T. muris as well as neonatal meningitis caused by Strep- and Sharpe (MRS) medium and maintained on MRS tococcus pneumoniae have also reported similar findings plates. which might be attributed to neurodegeneration caused by neuroinflammation [52]. However, normalization of Animals BDNF expression was observed after administration of Female BALB/c mice, 5–6  weeks old, weighing approx. L. plantarum (RTA 8), thereby highlighting the ability of 22–26  g, were used throughout the study. The animals gut microbes in altering brain behaviour and neurochem- were procured from Central Animal House, Panjab Uni- istry which could be mediated via inflammation depend - versity, Chandigarh and adapted to the conditions of the ent as well as independent pathways. animal room initially for a period of 1  week before ini- tiation of any experiments. Animals were given standard pellet diet along with water ad libitum every day. Conclusions Given the complexity of the interactions and activa- tion of multiple pathways during infection, the role of In vivo study groups direct gut-brain communication holds enhanced signifi - Prior to initiation of experiments, mice were randomly cance. In our previous findings, mice in the Salmonella divided into 3 groups containing 6–8 mice in each group. infected group exhibited depressive-like and anxiety- like behavioural changes [15]. Studies have reported the Control group association of abnormal levels of serotonin, dopamine All mice in this group served as control and each mouse and norepinephrine, as well as the lower levels of BDNF, was administered 0.1 ml of PBS. to various depressive and anxiety disorders [53], which might serve as another plausible reason for the patho- physiology observed therein. Studies using C. jejuni have Infected group also documented that local infection in the gut activates All mice in this group were individually infected with vagal sensory neurons via upregulation of c-Fos thereby 8 10  CFU/ml of S. enterica serovar Typhimurium SL1344 leading to behavioural changes (anxiety and depressive suspended in 0.1 ml of PBS. like behaviour), in the absence of an overt immunologi- cal response [54]. In this context, the importance of these Treatment group molecules in bi-directional communication, and the abil- All mice in this group were administered approx. ity of microbes in influencing the same, have far reach - 9 10 10 –10  CFU/ml of L. plantarum (RTA 8) suspended in ing consequences in the field of gastrointestinal diseases 0.2  ml of PBS daily for a period of 7  days before infect- and psychiatric disorders associated with depression and ing them with 10   CFU/ml of S. enterica serovar Typh- anxiety. It is here that the intervention of probiotics holds imurium SL1344. Thereafter, mice in all the groups were great potential. The crucial role of diet supplemented sacrificed via cervical dislocation on day 7 post infection. with such functional foods including probiotics and syn- Various vital organs of mice i.e. liver, spleen, intestine biotics, have been reported to provide health benefits and brain were rapidly excised, weighted and stored at that go beyond traditional nutrition thereby providing −  60  °C for preparation of homogenates. For qRT-PCR multi-dimensional and holistic health benefits to the studies, the tissue sample was dissected at 0  °C and host. Kaur et al. Gut Pathogens (2022) 14:11 Page 10 of 13 immediately transferred into vials containing RNA later was followed by addition of 0.05 ml water to the test and solution and stored at − 80 °C until use. 0.5  ml alkaline sulphite solution to blank. Thereafter, the fluorescence was measured at 335/410  nm. Values Evaluation of systemic Salmonella dissemination of unknown samples were obtained from the standard Systemic spread of the pathogen from the gut to  other curve of dopamine (0.1–5 μg/ml). organs and efficacy of the treatment with L. plantarum (RTA 8) was evaluated in terms of reduction in Salmo- Estimation of norepinephrine nella bacterial count in vital organs such as the liver and Level of norepinephrine in all the tissue homogenates the spleen as described previously [55]. Briefly, the liver was evaluated using the method as described by Ciar- and spleen tissues samples were rapidly excised and tis- lone [57]. Briefly, 0.1 ml of sodium acetate buffer (pH 6.9) sue homogenates (10%W/V) were prepared in ice cold and 0.05 ml of 0.4 M HCl was added to 0.2 ml of aqueous PBS (0.05 M, pH 7.4). Appropriately diluted samples were extract prepared as described above. This was followed then plated on streptomycin (50  μg/ml) containing LB by addition of 0.1  ml of iodine solution. After 2  min of agar plates with overnight incubation at 37 °C for bacte- incubation, 0.1 ml of alkaline sulphite solution was added rial count assessment. to remove excess iodine. After each addition, all con- tents of the tube were mixed thoroughly. After 1.5  min, Parameters evaluated at the gut‑brain axis 0.1 ml of 10 N acetic acid was also added and mixed well. Intestinal and brain bio‑burden The tubes were then placed in a boiling water bath for Dissemination of Salmonella from gut to brain along a period of 6  min. The reading for NE was recorded at with the efficacy of L. plantarum (RTA 8) treatment was 395/485 nm after cooling the tubes under tap water. Val- evaluated by enumeration of Salmonella bio-burden ues of the unknown samples were extrapolated from the in intestinal and brain tissue homogenates as described standard curve of NE (0.1–5  μg/ml). In case of “blank”, above. the same procedure was followed except that thiosul- phate reagent was added before addition of iodine. Level of neurotransmitters Briefly, a part of the intestinal and brain tissue sample of Estimation of serotonin mice in the above mentioned groups was homogenized Level of serotonin in all the tissue homogenates was eval- in ice cold acidifying butanol solution using glass teflon uated as per the procedure given by Schlumpf et al. [58]. homogenizer (50–75 mg tissue with 5 ml of HCl-butanol) Briefly, 1.2 ml of ortho-phthalaldehyde (OPT) was added for a period of 1  min. Thereafter, the homogenates were as to all the samples as well as blank (0.2  ml of 0.1  N centrifuged at 2000 rpm for 20 min. The supernatant was hydrochloric acid) and mixed thoroughly. Thereafter, all aliquoted and 1  ml of this was then added to tube con- tubes were placed in a boiling water bath for a period of taining a mixture of 0.31  ml of 0.1  M HCl and 2.5  ml of 10  min. After cooling the samples under tap water, the heptane. After shaking vigorously for 10  min the tubes fluorescence was read at 355/470 nm. Values of unknown were then centrifuged again at 2000 rpm for 20 min. The samples were calculated from the standard plot of seroto- aqueous phase (0.2  ml) was then used for assessment of nin (0.1–5 μg/ml). neurotransmitters as mentioned below. All steps were carried out at 0 °C. qRT‑PCR studies Total RNA from intestinal as well as brain tissues sam- Estimation of dopamine ples was extracted using TRIzol reagent (Ambion by Level of dopamine in all the tissue homogenates was Life Technologies). Complementary DNA (cDNA) was evaluated using the method as described by Carlsson and constructed using iScript cDNA Synthesis kit (Bio-Rad). Waldeck [56]. Briefly, 0.5  ml 0.1  M PO buffer (pH 6.5) Real-time PCR was performed using Applied Biosys- was added to 1 ml of above acid extract bringing the total tem StepOne Real-Time PCR system with SYBR Green volume to 3.8 ml by adding water. Thereafter, 0.05 ml of chemistry and SYBR Green Jumpstart Taq Ready Mix 0.02 N of iodine solution (consisting of 0.254 g iodine and (Sigma-Aldrich) to amplify the cDNA template. 5 g KI in 5 ml water diluted to 100 ml) was added to the tubes. Then, after 5 min of incubation, 0.5 ml of alkaline Evaluation of expression of mucin genes sulphite solution was added to test sample but in case Relative expression of two mucin genes, i.e., MUC 1 and of blank, 0.5  ml of 5  N NaOH was added. After another MUC 3, were evaluated in all the groups by performing 5  min, to all the samples, including the blank, 0.6  ml of real time PCR studies. The primer sequences were as fol - 5 N acetic acid was added and all samples were then irra- lows: MUC1 gene (Forward Primer: 5′-CAG TGC CAA diated with UV (240  nm) for a duration of 10  min. This GTC AATAC-3′, Reverse Primer: 5′-TGT TAC TGG AGA K aur et al. Gut Pathogens (2022) 14:11 Page 11 of 13 Statistical analysis AGG TAG -3′) and MUC3 gene (Forward Primer:5′-GTT All the values have been expressed as mean ± SD. Statis- GAT GTC ACC ACT ATG -3′, Reverse Primer: 5′-TGG tical analysis was performed using student’s t test, and TGT TGA GGT TAGAG-3′). They were designed using two-way ANOVA which was followed by pairwise com- NCBI primer designing tool and synthesized by G Bio- parison using Tukey’s test. In all the tests, p ≤ 0.05 was sciences. For RT-PCR analysis, the following programme considered as significant. was employed: initial denaturation was performed at 94 °C for 2 min; which was followed by 40 cycles of dena- turation at 94 °C for 15 s; annealing, extension and fluo - Abbreviations ANS: Autonomic nervous system; BDNF: Brain-derived neurotrophic factor; rescence was read at 55 °C for 1 min with 4 °C of optional cDNA: Complementary DNA; DA: Dopamine; EHEC: Enterohaemorrhagic E. hold. coli; EMS: Emotional motor system; HPA: Hypothalamus–pituitary–adrenal axis; LPS: Lipopolysaccharide; MUC 1: Mucin 1 gene; MUC 3: Mucin 3 gene; OPT: Ortho-phthalaldehyde; SCFA: Short chain fatty acids. Evaluation of expression of BDNF gene Relative expression of BDNF gene was evaluated in all the Acknowledgements groups by performing real time PCR studies. The primer Not applicable. sequences used were: BDNF gene (Forward Primer: Authors’ contributions 5′-CAA GAG TCC CGT CTG TAC TTTAC-3′, Reverse The study was designed and conceived by PR. All experimentations and Primer: 5′-GAC TAG GGA AAT GGG CTT AACA-3′). Prim- investigations were carried out by AK. Formal analysis was done by all the authors. Project administration was done by PR, IPK and KC. PR, IPK and KC ers were synthesized via IDT primer designing tool pro- supervised the study. PR and AK prepared the original draft. Further editing in vided by IDT (Integrated DNA Technologies) and were the manuscript was done by AK, PR, IPK and KC. All authors read and approved synthesized from the same. For RT-PCR analysis, the fol- the final manuscript. lowing programme was employed: Initial denaturation was Funding performed at 94  °C for 2  min; which was followed by 40 Financial assistance provided by Department of Science and Technology cycles of denaturation at 94  °C for 15  s; annealing, exten- (DST ), India under PURSE Grant (P.U.) and University Grants Commission under Basic Scientific Research under Research Fellowship in Sciences for Meritorious sion and fluorescence read at 60  °C for 1  min and 4  °C of Students (RFSMS) scheme Grant No. F.25-1/2014-15(BSR)/7-261/2009 (BSR) for optional hold. carrying out the research work is duly acknowledged. For all the genes: Availability of data and materials Melt curve analysis was performed by heating the All data supporting findings of the study has been included in the manuscript. samples from 55 to 95  °C with an increase of 0.5  °C and Nucleotide sequence of L. planatarum (RTA 8) can be found in GenBank fluorescence was recorded. GAPDH was used as the database under accession number: KJ802485. The NCBI mRNA reference housekeeping gene and the primer sequences were- For sequence number for genes evaluated in the paper are as follows: Mus musculus mucin 1, transmembrane (Muc1): NM_013605.2, https:// www. ncbi. ward Primer: 5′-AAC AGC AAC TCC CAC TCT TC-3′ and nlm. nih. gov/ nucco re/ NM_ 013605.2. Mus musculus mucin 3, intestinal (Muc3): Reverse Primer: 5′-CCT GTT GCT GTA GCC GTA TT-3′. NM_010843.1, https:// www. ncbi. nlm. nih. gov/ nucco re/ NM_ 010843.1. Mus musculus brain derived neurotrophic factor (Bdnf ): NM_007540.4, https:// Three biological replicates as well as three technical rep - www. ncbi. nlm. nih. gov/ nucco re/ NM_ 007540.4. Mus musculus glyceraldehyde- licates were set up for each gene. Relative fold change in 3-phosphate dehydrogenase (Gapdh): NM_001289726.1, https:// www. ncbi. −∆∆ct gene expression was assessed using the 2 method. nlm. nih. gov/ nucco re/ NM_ 00128 9726.1. Histological evaluation of gut and brain tissues Declarations A part of the intestine as well as brain tissues from each Ethics approval and consent to participate group were stored in 10% buffered formalin. Briefly, the The experimental protocols involving animals were approved by the Institu- samples were then dehydrated using different grades tional Animal Ethics Committee of Panjab University, Chandigarh, India under the Approval Number PU/45/99/ CPCSEA/IAEC/2018/225. All the guidelines of alcohol (70%, 80%, 90% and 100% absolute alcohol) laid down by the Committee for the Purpose of Control and Supervision of after which they were washed in xylene for an hour and Experiments on Animals (CPCSEA), Government of India and all applicable dipped in molten paraffin wax for crystallization and fur - international and national institutional guidelines for conducting research on animals were religiously obeyed and strictly adhered to. ther sectioning. The sections were then kept in a water bath at 50 °C to remove wax and for mounting on a glass. Consent for publication Thereafter, the slides were further treated with xylene Not applicable. to remove wax and with ethanol to remove xylene. The Competing interests slides were finally stained with hematoxylin, followed All the authors declare that they have no competing interests. by eosin. Lastly, the slides were mounted in Distyrene Plasticizer Xylene (DPX) for histological investigation Author details Department of Microbiology, Basic Medical Sciences Block I, Panjab Univer- by examining under light microscope. The tissues were sity, South Campus, Sector 25, Chandigarh 160014, India. Universit y I nstitute assessed for signs of inflammation and morphological of Pharmaceutical Sciences, Panjab University, Sector 14, Chandigarh 160014, changes. India. Kaur et al. Gut Pathogens (2022) 14:11 Page 12 of 13 Received: 26 October 2021 Accepted: 17 February 2022 information from gut to brain in STM-infected rats. World J Gastroenterol. 2002;8:540–5. 23. Peterson CT. Dysfunction of the microbiota-gut-brain axis in neurode- generative disease: the promise of therapeutic modulation with prebiot- ics, medicinal herbs, probiotics, and synbiotics. J Evid Based Integr Med. 2020;25:1–19. References 24. Kumar A, Russell RM, Pifer R, Menezes-Garcia Z, Cuesta S, Narayanan S, 1. Shaikh AIA, Prabhakar AT. Typhoid fever and its nervous system involve- MacMillan JB, Sperandio V. The serotonin neurotransmitter modulates ment. In: Innate immunity in health and disease. London: Intech Open; virulence of enteric pathogens. Cell Host Microbe. 2020;28:41–53. 2021. p. 1–5. 25. Nzakizwanayo J, Dedi C, Standen G, Macfarlane WM, Patel BA, Jones 2. Hanafi T, Titou H, Kerrouch H, Frikh R, Hjira N. Non-typhoidal Salmonella BV. Escherichia coli Nissle 1917 enhances bioavailability of serotonin in meningitis in an adult patient with HIV Infection: the hostage-taking situ- gut tissues through modulation of synthesis and clearance. Sci Rep. ation. Is there any solution. Clin Med Rev Case Rep. 2020;7:294. 2015;5:1–13. 3. Chaudhuri D, Roy Chowdhury A, Biswas B, Chakravortty D. Salmonella 26. Reigstad CS, Salmonson CE, Rainey JF III, Szurszewski JH, Linden DR, typhimurium infection leads to colonization of the mouse brain and is Sonnenburg JL, Farrugia G, Kashyap PC. Gut microbes promote colonic not completely cured with antibiotics. Front Microbiol. 2018;9:1632. serotonin production through an effect of short-chain fatty acids on 4. Khurshid N, Khan BA, Bukhari SW, Shahid A, Punshi A. Extensively drug- enterochromaffin cells. FASEB J. 2015;29:1395–403. resistant Salmonella Typhi meningitis in a 16-year-old male. Cureus. 27. Yaghoubfar R, Behrouzi A, Ashrafian F, Shahryari A, Moradi HR, Choopani 2019;11:e5961. S, Hadifar S, Vaziri F, Nojoumi SA, Fateh A, Khatami S. Modulation of sero- 5. Serra D, Almeida LM, Dinis TC. The impact of chronic intestinal inflam- tonin signaling/metabolism by Akkermansia muciniphila and its extracel- mation on brain disorders: the microbiota-gut brain axis. Mol Neurobiol. lular vesicles through the gut-brain axis in mice. Sci Rep. 2020;10:1–12. 2019;56:6941–51. 28. Tian P, Wang G, Zhao J, Zhang H, Chen W. Bifidobacterium with the role 6. Gubert C, Kong G, Renoir T, Hannan AJ. Exercise, diet and stress as modu- of 5-hydroxytryptophan synthesis regulation alleviates the symp- lators of gut microbiota: implications for neurodegenerative diseases. tom of depression and related microbiota dysbiosis. J Nutr Biochem. Neurobiol Dis. 2020;134:104621. 2019;66:43–51. 7. Saulnier DM, Ringel Y, Heyman MB, Foster JA, Bercik P, Shulman RJ, Versal- 29. Guzmán DC, Herrera MO, Brizuela NO, Mejía GB, Jiménez FT, García EH, ovic J, Verdu EF, Dinan TG, Hecht G, Guarner F. The intestinal microbiome, Olguín HJ. Assessment of the effects of oseltamivir and indomethacin on probiotics and prebiotics in neurogastroenterology. Gut Microbes. dopamine, 5-HIAA, and some oxidative stress markers in stomach and 2013;4:17–27. brain of Salmonella Typhimurium-infected rats. Neuroendocrinol Lett. 8. Margolis KG, Cryan JF, Mayer EA. The microbiota gut-brain axis: from 2016;37:129–36. motility to mood. Gastroenterology. 2021;160:1486–501. 30. González-Arancibia C, Urrutia-Piñones J, Illanes-González J, Martinez- 9. Holstege G, Bandler R, Saper CB. The emotional motor system. Prog Brain Pinto J, Sotomayor-Zárate R, Julio-Pieper M, Bravo JA. Do your Res. 1996;107:3–6. gut microbes affect your brain dopamine? Psychopharmacology. 10. Mayer EA. The neurobiology of stress and gastrointestinal disease. Gut. 2019;236:1611–22. 2000;47:861–9. 31. Hsieh TH, Kuo CW, Hsieh KH, Shieh MJ, Peng CW, Chen YC, Chang YL, 11. Liu Y, Forsythe P. Vagotomy and insights into the microbiota-gut-brain Huang YZ, Chen CC, Chang PK, Chen KY. Probiotics alleviate the progres- axis. Neurosci Res. 2021;168:20–7. sive deterioration of motor functions in a mouse model of Parkinson’s 12. Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain disease. Brain Sci. 2020;10:206. Res. 2018;1693:128–33. 32. Erickson JT, Brosenitsch TA, Katz DM. Brain-derived neurotrophic factor 13. Westfall S, Lomis N, Kahouli I, Dia SY, Singh SP, Prakash S. Microbiome, and glial cell line-derived neurotrophic factor are required simultane- probiotics and neurodegenerative diseases: deciphering the gut brain ously for survival of dopaminergic primary sensory neurons in vivo. J axis. Cell Mol Life Sci. 2017;74:3769–87. Neurosci. 2001;21:581–9. 14. Liang S, Wu X, Jin F. Gut-brain psychology: rethinking psychology from 33. Liu X, Jiang L, Li L, Yu H, Nie S, Xie M, Gong J. The role of neurotransmitters the microbiota–gut–brain axis. Front Integr Neurosci. 2018;12:33. in the protection of Caenorhabditis elegans for Salmonella infection by 15. Kaur A, Chopra K, Kaur IP, Rishi P. Salmonella strain specificity determines Lactobacillus. Front Cell Infect Microbiol. 2020;10:554052. post-typhoid central nervous system complications: intervention by Lac- 34. Nordgreen J, Munsterhjelm C, Aae F, Popova A, Boysen P, Ranheim B, tiplantibacillus plantarum at gut-brain axis. Front Microbiol. 2020;11:1568. Heinonen M, Raszplewicz J, Piepponen P, Lervik A, Valros A. The effect 16. Asahara T, Shimizu K, Takada T, Kado S, Yuki N, Morotomi M, Tanaka R, of lipopolysaccharide (LPS) on inflammatory markers in blood and Nomoto K. Protective effect of Lactobacillus casei strain Shirota against brain and on behavior in individually-housed pigs. Physiol Behav. lethal infection with multi-drug resistant Salmonella enterica serovar 2018;195:98–111. Typhimurium DT104 in mice. J Appl Microbiol. 2011;110:163–73. 35. Stibbs HH. Changes in brain concentrations of catecholamines and 17. Brenchley JM, Douek DC. Microbial translocation across the GI tract. Annu indoleamines in Toxoplasma gondii infected mice. Ann Trop Med Parasi- Rev Immunol. 2012;30:149–73. tol. 1985;79:153–7. 18. Corr SC, Hill C, Gahan CG. Understanding the mechanisms by which 36. Asano Y, Hiramoto T, Nishino R, Aiba Y, Kimura T, Yoshihara K, Koga Y, Sudo probiotics inhibit gastrointestinal pathogens. Adv Food Nutr Res. N. Critical role of gut microbiota in the production of biologically active, 2009;56:1–15. free catecholamines in the gut lumen of mice. Am J Physiol Gastrointest 19. Ishikawa H, Kutsukake E, Fukui T, Sato I, Shirai T, Kurihara T, Okada N, Liver Physiol. 2012;303:G1288–95. Danbara H, Toba M, Kohda N, Maeda Y. Oral administration of heat- 37. Spencer H, Karavolos MH, Bulmer DM, Aldridge P, Chhabra SR, Winzer K, killed Lactobacillus plantarum strain b240 protected mice against Williams P, Khan CMA. Genome-wide transposon mutagenesis identifies Salmonella enterica serovar Typhimurium. Biosci Biotechnol Biochem. a role for host neuroendocrine stress hormones in regulating the expres- 2010;74:1338–42. sion of virulence genes in Salmonella. J Bacteriol. 2010;192:714–24. 20. Acurcio LB, Wuyts S, de CiccoSandes SH, Santanna FM, Pedroso SHSP, 38. Pandey S, Singh A, Chaudhari N, Nampoothiri LP, Kumar GN. Protection Bastos RW, Dos Reis DC, Vieira AF, Cassali GD, Lebeer S, de Souza MR. against 1,2-di-methylhydrazine-induced systemic oxidative stress and Milk fermented by Lactobacillus paracasei NCC 2461 (ST11) modulates altered brain neurotransmitter status by probiotic Escherichiacoli CFR 16 the immune response and microbiota to exert its protective effects secreting pyrroloquinoline quinone. Curr Microbiol. 2015;70:690–7. against Salmonella Typhimurium infection in mice. Probiotics Antimicrob 39. Kannampalli P, Pochiraju S, Chichlowski M, Berg BM, Rudolph C, Bruckert Proteins. 2020;12:1398–408. M, Miranda A, Sengupta JN. Probiotic Lactobacillus rhamnosus GG (LGG) 21. Mulaw G, Muleta D, Tesfaye A, Sisay T. Protective effect of potential and prebiotic prevent neonatal inflammation-induced visceral hypersen- probiotic strains from fermented Ethiopian food against Salmonella sitivity in adult rats. Neurogastroenterol Motil. 2014;26:1694–704. Typhimurium DT104 in mice. Int J Microbiol. 2020;2020:7523629. 22. Wang X, Wang BR, Zhang XJ, Xu Z, Ding YQ, Ju G. Evidences for vagus nerve in maintenance of immune balance and transmission of immune K aur et al. Gut Pathogens (2022) 14:11 Page 13 of 13 40. Hafez MM. Upregulation of intestinal mucin expression by the pro- biotic bacterium E. coli Nissle 1917. Probiotics Antimicrob Proteins. 2012;4:67–77. 41. Caballero-Franco C, Keller K, De Simone C, Chadee K. The VSL# 3 probiotic formula induces mucin gene expression and secretion in colonic epithe- lial cells. Am J Physiol Gastrointest Liver Physiol. 2007;292:G315–22. 42. Lindén SK, Sheng YH, Every AL, Miles KM, Skoog EC, Florin TH, Sut- ton P, McGuckin MA. MUC1 limits Helicobacter pylori infection both by steric hindrance and by acting as a releasable decoy. PLoS Pathog. 2009;5:e1000617. 43. Liévin-Le Moal V, Servin AL. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clin Microbiol Rev. 2006;19:315–37. 44. Barcelo A, Claustre J, Moro F, Chayvialle JA, Cuber JC, Plaisancié P. Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon. Gut. 2000;46:218–24. 45. Mack DR, Ahrné S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut. 2003;52:827–33. 46. Bath KG, Lee FS. Variant BDNF ( Val66Met) impact on brain structure and function. Cogn Aec ff t Behav Neurosci. 2006;6:79–85. 47. Miranda M, Morici JF, Zanoni MB, Bekinschtein P. Brain-derived neu- rotrophic factor: a key molecule for memory in the healthy and the pathological brain. Front Cell Neurosci. 2019;13:363. 48. Heldt SA, Stanek L, Chhatwal JP, Ressler K. Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories. Mol Psychiatry. 2007;12:656–70. 49. Nieto R, Kukuljan M, Silva H. BDNF and schizophrenia: from neurodevel- opment to neuronal plasticity, learning, and memory. Front Psychiatry. 2013;4:45. 50. Autry AE, Monteggia LM. Brain-derived neurotrophic factor and neu- ropsychiatric disorders. Pharmacol Rev. 2012;64:238–58. 51. Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders. Biol Psychiatry. 2006;59:1116–27. 52. Barichello T, Belarmino E Jr, Comim CM, Cipriano AL, Generoso JS, Savi GD, Stertz L, Kapczinski F, Quevedo J. Correlation between behavioral deficits and decreased brain-derived neurotrofic factor in neonatal men- ingitis. J Neuroimmunol. 2010;223:73–6. 53. Martinowich K, Manji H, Lu B. New insights into BDNF function in depres- sion and anxiety. Nat Neurosci. 2007;10:1089–93. 54. Goehler LE, Gaykema RP, Opitz N, Reddaway R, Badr N, Lyte M. Activation in vagal afferents and central autonomic pathways: early responses to intestinal infection with Campylobacter jejuni. Brain Behav Immun. 2005;19:334–44. 55. Kaur A, Chabba SK, Kaur UJ, Kaur A, Preet S, Rishi P. Management of Staphylococcus mediated systemic infection by enhancing the resurging activity of co-trimoxazole in presence of cryptdin-2. Indian J Microbiol. 2017;57:438–47. 56. Carlsson A, Waldeck B. A fluorimetric method for the determination of dopamine (3-hydroxytyramine). Acta Physiol Scand. 1958;44:293–8. 57. Ciarlone AE. Further modification of a fluorometric method for analyzing brain amines. Microchem J. 1978;23:9–12. 58. Schlumpf M, Lichtensteiger W, Langemann H, Waser PG, Hefti F. A fluo - rometric micromethod for the simultaneous determination of serotonin, noradrenaline and dopamine in milligram amounts of brain tissue. Biochem Pharmacol. 1974;23:2437–46. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : Publisher’s Note fast, convenient online submission Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Gut Pathogens Springer Journals

Bi-directional elucidation of Lactiplantibacillus plantarum (RTA 8) intervention on the pathophysiology of gut-brain axis during Salmonella brain infection

Loading next page...
 
/lp/springer-journals/bi-directional-elucidation-of-lactiplantibacillus-plantarum-rta-8-tUI7C0piz6

References (67)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2022
eISSN
1757-4749
DOI
10.1186/s13099-022-00484-2
Publisher site
See Article on Publisher Site

Abstract

Background: There have been reports of patients suffering from typhoid fever, particularly those involving infants and immunocompromised patients, which at times present with Salmonella induced brain infection. Although rare, it has frequently been associated with adverse neurological complications and increased mortality. In this context, the gut-brain axis, involving two-way communication between the gut and the brain, holds immense significance as various gut ailments have been associated with psychiatric complications. In turn, several neurodegenerative diseases have been associated with an altered gut microbiota profile. Given the paucity of effective antimicrobials and increas- ing incidence of multi-drug resistance in pathogens, alternate treatment therapies such as probiotics have gained significant attention in the recent past. Results: In the current study, prophylactic effect of Lactiplantibacillus plantarum (RTA 8) in preventing neurological complications occurring due to Salmonella brain infection was evaluated in a murine model. Along with a significant reduction in bacterial burden and improved histoarchitecture, L. plantarum (RTA 8) administration resulted in amelio- ration in the level of neurotransmitters such as serotonin, norepinephrine and dopamine in the gut as well as in the brain tissue. Simultaneously, increased gene expression of physiologically essential molecules such as mucin (MUC1 and MUC3) and brain-derived neurotrophic factor (BDNF) was also observed in this group. Conclusion: Present study highlights the potential benefits of a probiotic supplemented diet in improving various aspects of host health due to their multi-targeted approach, thereby resulting in multi-faceted gains. Keywords: Salmonella, Neurological complications, Gut-brain axis, Probiotics episodes, neurological abnormalities along with severe Background side effects such as auditory and visual impairments, Neurological complications occurring due to Salmonella mental retardation and poor prognosis leading to high infection of the brain remain a matter of serious concern mortality rates [2]. Previous reports in mouse models [1]. Such infections are associated with frequent relapse have also elaborated on the ineffectiveness of antibiotics in completely curing infections of such kind [3]. Simul- *Correspondence: rishipraveen@yahoo.com; rishiparveen@pu.ac.in 1 taneously, increased incidence and rise in cases of drug Department of Microbiology, Basic Medical Sciences Block I, Panjab University, South Campus, Sector 25, Chandigarh 160014, India resistant Salmonella has further limited the treatment Full list of author information is available at the end of the article arsenal for this dangerous pathogen [4]. Interestingly, © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kaur et al. Gut Pathogens (2022) 14:11 Page 2 of 13 recent evidences have indicated that inflammatory dis - the host pathology and physiology has opened diverse eases/conditions of the gut are linked to psychiatric and areas of targeted therapy which might prove useful in behavioural abnormalities [5]. Additionally, various neu- a variety of related manifestations [14]. In our previous rodegenerative diseases have been associated with an study, administration of Lactiplantibacillus plantarum altered and specific microbiota profile [6]. In the prevail - (RTA 8) was found to be useful in ameliorating Salmo- ing scenario, tackling and treating infections by targeting nella induced brain infection in mouse model [15]. To multi-dimensional aspects of health and disease has led the best of our awareness, this is the first study elaborat - to increasing interest in the field of neurogastroenterol - ing on the prophylactic effect of L. plantarum (RTA 8) in ogy which interlinks diverse aspects of neuroscience, modulating the amount of neurotransmitters and other gastroenterology, immunology, behaviour science, micro- neuroactive molecules such as BDNF along with mucin biology, pharmacology and other related subjects to genes, at the gut-brain axis, thereby preventing Salmo- devise strategies that can improve the host health status nella induced neurological manifestations. at a holistic level [7]. Herein, manipulation of gut-brain axis has proven to be of enhanced significance, given its Results direct implications at the gut as well as the brain [8]. The Efficacy of L. plantarum (RTA 8) at systemic level brain to gut signalling is evidenced by the findings that Significant bacterial burden in vital organs of mice in the different kinds of psychological stressors modulate the infected group indicated systemic spread of the patho- composition as well as the biomass of the gut microbiota. gen. L. plantarum (RTA 8) administration 7 days prior to The CNS has been reported to impact other bodily as well infection significantly reduced Salmonella bio-burden in as gut functions, including the gut microbiota, via ‘the all organs. A 2.9 (p < 0.001) and 2.85 (p < 0.01) fold reduc- emotional motor system (EMS)’ consisting of hypothala- tion was observed in liver and spleen tissues samples of mus–pituitary–adrenal (HPA) axis, parallelly working the treatment group, respectively, in comparison to that branches of the sympathetic as well as parasympathetic of the infected group thereby indicating efficacy of the autonomic nervous system (ANS), and other pathways treatment (Fig. 1). mediating discomfort and pain [9]. The ANS finds direct role in gut functions such as mucus, bicarbonate and acid Efficacy of L. plantarum (RTA 8) at gut‑brain axis secretion along with intestinal motility, immune response Tissue bio‑burden and permeability, coupled with secretion of neuroactive Dissemination of Salmonella from the gut to the brain signalling molecules such as catecholamines, cytokines, was confirmed as significant bacterial burden was serotonin, etc., thereby impacting the microbiota [10]. observed in the brain tissue. Simultaneously, a significant Simultaneously, microbes have been known to influence reduction in bacterial burden in the intestine as well as almost all major pathways associated with the gut-brain the brain tissue samples of mice in L. plantarum (RTA 8) axis. These include the neural pathway by modulating/ treated group was observed. A 2.84 fold (p < 0.01) reduc- producing/regulating the level of neurotransmitters [11]; tion in Salmonella bio-burden was observed in intesti- endocrine pathways and its associated moieties such as nal sections of the treatment group as compared to that neuroendocrine cells, neuroactive substances and neu- in the infected group. Similarly, administration of L. ropeptides; host immune system as well as functioning plantarum (RTA 8) resulted in a significant decrease in and maturation of most components of innate and adap- bacterial counts (6.06 fold reduction, p < 0.001) to 0.77 tive immunity. All these molecules have been reported log CFU/ml in the brain tissue samples relative to the to influence key aspects of brain and behaviour includ - infected group, demonstrating the effectiveness of L. ing neurodegeneration, apoptosis and neurogenesis, plantarum (RTA 8) in preventing Salmonella brain infec- which, coupled with inflammation, further highlight their tion (Fig. 1). importance in maintaining host well-being [12, 13]. Therefore, given the crucial role of gut microbiota in Level of dopamine this bi-directional communication, use of probiotics as a Infection with S. Typhimurium SL1344 significantly bio-compatible treatment strategy has particularly cap- reduced the level of dopamine in murine gut tissue sam- tured the interest of the scientific community. Probiot - ples by 80.9% (p < 0.01) in comparison to the control. L. ics have been reported to influence all the key pathways plantarum (RTA 8) administered group demonstrated that can be influenced by the host microbiota thereby an increase in the level of dopamine, however, the levels presenting as a highly promising candidate for targeting were not found to be significantly different from that of gut-brain axis disorders [13]. Additionally, the capability the infected group. of these bacteria to function as psychobiotics by improv- In comparison to the control group, Salmonella ing the psychological status along with their impact on infected mice demonstrated a significant reduction K aur et al. Gut Pathogens (2022) 14:11 Page 3 of 13 Fig. 1 L. plantarum (RTA 8) administration 7 days prior to infection significantly reduced Salmonella count in all the vital organs. Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing the median, dots representing individual values and ‘x’ representing the mean value of dataset. **p < 0.01 and ***p < 0.001 versus infected by analysis of variance (ANOVA) of 64.1% (p < 0.01) in dopamine levels in the brain tis- plantarum (RTA 8) resulted in a significant increase sues. Treatment with L. plantarum (RTA 8) significantly (p < 0.01) in the hormone levels in comparison to the increased the levels of dopamine in the brain tissues in infected tissue (Fig. 3). comparison to the infected group (p < 0.01). Dopamine levels in the treatment group were found to be restored to the level observed in the control group (Fig. 2). Level of serotonin L. plantarum (RTA 8) administered group demonstrated a significant 34% increase (p < 0.05) in the level of sero- Level of norepinephrine tonin in the gut of mice in comparison to the control Intestinal samples of mice infected with Salmonella dem- group. An 18.4% reduction in the level of serotonin was onstrated a decrease of 46.1% in norepinephrine levels in observed in the intestine tissue samples of mice in the comparison to the control group, however it was found infected group. As compared to the infected group, a to be insignificant. In comparison to the infected group, significant increase was observed in the treatment group mice in the L. plantarum (RTA 8) administered group (p < 0.01). demonstrated a relative increase in norepinephrine levels In the brain tissue sections, mice in the Salmonella but the results were not found to be significant. infected group demonstrated a significant decrease of In contrast to results obtained in the intestinal tis- 21.9% in the level of serotonin as compared to the control sue samples, Salmonella infected group demonstrated group (p < 0.05). Simultaneously, L. plantarum (RTA 8) a very significant reduction of 34.7% in the level of administered group demonstrated a significant increase norepinephrine in brain tissues samples (p < 0.001) in the level of serotonin in comparison to the infected as compared to the control group. Treatment with L. group (p < 0.01) (Fig. 4). Kaur et al. Gut Pathogens (2022) 14:11 Page 4 of 13 Fig. 2 Dopamine levels in the murine brain and intestinal tissue samples. Dopamine levels was observed to be significantly decreased in the infected group and normal levels were found to be restored in murine brain after administration of L. plantarum (RTA 8). Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing the median, dots representing individual values and ‘x’ representing the mean value of dataset. $$ **p < 0.01, *p < 0.05 in comparison to Control. p < 0.01 in comparison to Infected Expression of MUC 1 gene plantarum (RTA 8) administered group in comparison to Expression of MUC 1 gene was found to be significantly the infected group (Fig. 5). upregulated in the group administered with L. plantarum (RTA 8). A 7.6 fold significant increase (p < 0.05) was Expression of BDNF gene observed in this group as compared to the control group. Quantitative RT-PCR studies revealed significant down - Additionally, a 6.7 fold increase (p < 0.01) was observed in regulation of BDNF gene expression in brain tissues of the treatment group as compared to the infected group. Salmonella infected mice. A 6.3 fold reduction (p < 0.001) No significant difference in gene expression was observed in expression was observed in the infected group in between the control and the infected group (Fig. 5). comparison to the control group. L. plantarum (RTA 8) administration resulted in a significant 6.5 fold increase in BDNF expression as compared to the infected group (p < 0.011) (Fig. 5). Expression of MUC 3 gene Expression of MUC 3 gene in the Salmonella infected Histopathological studies group was found to be downregulated significantly (3.1 Histopathological analysis of intestinal tissue sections fold, p < 0.05). Simultaneously, L. plantarum (RTA 8) of the control group depicted normal tissue architec- treated group demonstrated a significant upregulation of ture with normal distribution of surface enterocytes, MUC 3 gene expression (5.5 fold p < 0.05) as compared goblet cells and muscularis mucosa. Inflammatory to the control group. A significant 17.4 fold increase in cells were absent and the crypts and villi demonstrated the expression of MUC 3 gene was observed in the L. K aur et al. Gut Pathogens (2022) 14:11 Page 5 of 13 Fig. 3 Norepinephrine levels in the murine intestinal and brain tissue samples. Decreased level of dopamine was observed in the Salmonella infected group with significantly reduced levels in the brain. L. plantarum (RTA 8) administration was found to increase intestinal norepinephrine levels but significant increase was found in the brain tissue samples. Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing $$ the median, dots representing individual values and ‘x’ representing the mean value of dataset.***p < 0.001 in comparison to Control; p < 0.01 in comparison to Infected normal histoarchitecture (Fig.  6A). However, infected administered with L. plantarum (RTA 8) 7  days prior mice revealed severely damaged villi with focal ulcera- to infection, prevented meningitis as the meninges was tions and inflammation. Crypt hyperplasia was also found to be clear of signs of inflammation. The brain tis - observed. The villi were found to be stunted and the sue in this group showed normal tissue histoarchitecture crypts were found to be enlarged and elongated (Fig. 6B). with only very mild oedema (Fig. 6F). In contrast to this, L. plantarum (RTA 8) administered mice demonstrated normal tissue morphology similar to Discussion that observed in the control tissue samples. The normal In contrast to the previously conducted independent villi to crypt ratio of 5:1 was restored, thereby elaborat- study [15], herein, exclusively the prophylactic poten- ing on the efficacy of L. plantarum (RTA 8) in preventing tial of L. plantarum (RTA 8) in combating Salmonella Salmonella infection (Fig. 6C). induced brain infection was evaluated. It was inferred Brain tissue samples of mice in the control group that decreasing the duration of L. plantarum (RTA 8) revealed normal tissue morphology with the presence administration from 14 to only 7  days prior to infec- of triangular neurons and microglial cells with oligo- tion, did not affect its efficacy in preventing brain infec - dendrocytes; sans inflammation (Fig.  6D). However, oral tion. The same was evidenced by a similar reduction in infection with Salmonella led to brain infection and men- bacterial burden and amelioration in tissue histology, as ingitis which was confirmed histologically by increased observed in the previous study. Other studies employing accumulation of inflammatory cells in the meninges of probiotic strains for similar duration have also reported the brain. Simultaneously, marked oedema was observed similar fold reduction in Salmonella bio-burden [16]. in the cortical tissue (Fig.  6E). On the other hand, mice Infection with S. Typhimurium has been reported to Kaur et al. Gut Pathogens (2022) 14:11 Page 6 of 13 Fig. 4 Serotonin levels in the murine intestinal and brain tissue samples. Significantly decreased levels of serotonin were observed in brain tissues of mice infected with Salmonella. L. plantarum (RTA 8) treated group demonstrated a significant increase in the level of serotonin in the brain as well as the intestinal tissue samples. Data has been represented in the form of box and whiskers plot with whiskers representing the maximum and minimum values, boxes representing the 25th (1st quartile) and 75th percentile (3rd quartile), line representing the median, dots representing $$ individual values and ‘x’ representing the mean value of dataset. *p < 0.05 in comparison to Control; p < 0.01 in comparison to Infected cause destruction of the ileum, similar to that observed the ability of probiotics in reversing pathogen or micro- in our study, thereby resulting in increased translocation bial component such as lipopolysaccharide (LPS) medi- of bacteria to other organs and imbalance in gut micro- ated neurodegeneration by using interventions targeting bial population [17]. Reduction in luminal pH due to pro- the gut microbiota [13, 23]. duction of short chain fatty acids by L. plantarum (RTA Neurotransmitters constitute a vital part of the gut- 8) along with production of IgA and other potent anti- brain communication. As observed in the present study, microbial substances might have resulted in clearance of decreased level of serotonin, more significantly so in the Salmonella [18, 19]. Additionally, the abilities of probiot- brain tissue samples of the Salmonella infected group, ics in occupying receptor binding sites at the epithelial have been associated with increased pathogenesis and surface, strengthening the intestinal microstructure and decreased host survival in case of infections caused by increasing the villus length, might have further blocked pathogens such as Citrobacter rodentium and entero- pathogen adherence [15, 20, 21]. All these factors might haemorrhagic Escherichia coli (EHEC); presence of sero- have conferred colonization resistance to the host against tonin has been shown to decrease the expression of LEE such deadly pathogens, thereby preventing their translo- virulence genes located within the locus of enterocyte cation to other organs such as the brain. Simultaneously, effacement (LEE) pathogenicity island (PI) via the block - direct transmission of information from gut to brain ing of CpxA, a membrane-bound histidine sensor kinases through the vagus nerve might have resulted in activation (HKs) present in bacteria thereby reducing its patho- of Fos immune reactive cells in the hypothalamic region genicity [24]. Significantly increased levels of serotonin of the brain which might have also contributed to effec - in the L. plantarum (RTA 8) group might have resulted tive bacterial clearance from the brain [22]. In conso- in reduced pathogenicity of Salmonella, thereby prevent- nance with our findings, various studies have highlighted ing serious consequences. Other studies have reported K aur et al. Gut Pathogens (2022) 14:11 Page 7 of 13 Fig. 5 Relative fold change in expression of BDNF, MUC1 and MUC3 genes. Fold expression was determined after normalization to GAPDH for each $$ sample. ***p < 0.001, *p < 0.05 in comparison to control; p < 0.01 in comparison to infected the ability of probiotic strains such as E. coli Nissle 1917 of gut microbiota and probiotics in altering the levels in increasing the bioavailability of serotonin in ileal tis- of neurotransmitters in the brain, directly by influenc - sue [25]. The same has been postulated to be a result of ing the ENS and interacting with the glial cells of the SCFA (short chain fatty acids) produced by these gut bac- gut [12, 30]. Administration of cocktail of probiotics or teria acting directly on the enterochromaffin cells which probiotic strain coupled with a prebiotic have been dem- harbour the enzyme tryptophan hydroxylase, resulting onstrated to prevent neurodegeneration while simultane- in serotonin regulation [26]. Similar to our observations, ously increasing the cortical levels of dopamine, as well other studies have also reported increased levels of sero- as preventing degeneration of dopaminergic neurons in tonin in the brain tissues of animals receiving probiotics the substantia nigra pars compacta [31]. Simultaneously, such as Akkermansia muciniphila and Bifidobacterium increased production of butyrate and BDNF/GNDF after breve CCFM1025 [27, 28]. These effects were found to be probiotic administration, as observed in our study, has mediated by the upregulations of TPH1 and TPH2 genes also been implicated in raised dopamine levels [32]. In involved in serotonin biosynthesis in the intestine and another study, administration of Lacticaseibacillus zeae the brain, respectively. LB1 prevented Salmonella infection in Caenorhabditis The significantly reduced levels of dopamine in the elegans via modulation of both serotonin and dopamine, brain and intestinal tissue samples of mice in the Salmo- confirming their role in preventing serious manifesta - nella infected group could be attributed to the ability of tions [33]. Salmonella and its major cell wall component, LPS, in Catecholamines have been reported to induce the activation of microglial cells. This might have resulted in growth of enteric pathogens by acting as quorum sens- a neurotoxic environment thereby increasing the suscep- ing molecules. However, significantly decreased levels tibility of dopaminergic neurons in the substantia nigra of norepinephrine in the brain tissue samples of Sal- and striatum to neuroinflammation induced damage, monella infected mice were observed which could be leading to reduction in its synthesis as well as effective attributed to the role of LPS in reducing the levels of concentration [29]. The normalization of levels of dopa - noradrenaline by affecting the brain physiology [34]. mine in the brain tissue samples of mice in L. plantarum Brain infection with Toxoplasma gondii has also been (RTA 8) treated group could be ascribed to the ability associated with decreased level of norepinephrine Kaur et al. Gut Pathogens (2022) 14:11 Page 8 of 13 Fig. 6 Histological evaluation of brain and intestinal tissue sections. A (X40, scale bar 50 µm) H&E staining of control intestinal tissue depicted normal intensity of goblet cells and lymphocytes in the lamina propria depicting normal tissue morphology. B (X40, scale bar 50 µm) Photomicrographs of Salmonella infected intestinal tissue depicted ileal damage inflicted by Salmonella with heavy influx of inflammatory cells along with crypt elongation (highlighted by arrows). C (X40, scale bar 50 µm) L. plantarum (RTA 8) treated group revealed restoration of normal intestinal tissue morphology as revealed by restoration of villi to crypt ratio and normal density of lymphocytes (highlighted by arrows). D (X40, scale bar 50 µm) Photomicrographs of cortical mice brain region in the control group depicted normal histology revealing presence of triangular neurons and glial cells. E (X40, scale bar 50 µm) Photomicrographs of brain tissue sections in the Salmonella infected group revealed heavy influx of inflammatory cells along the surface and meninges depicting meningitis (highlighted by arrows) along with brain infection characterized by oedema (highlighted by marking a circle). F (X40, scale bar 50 µm) Brain tissue sections of mice administered with L. plantarum (RTA-8) administered group depicted slight oedema with otherwise normal histoarchitecture which might have resulted from changes in the meta- Treatment with L. plantarum (RTA 8) resulted in a bolic enzyme tyrosine hydroxylase, that converts tyros- significant increase in the expression of MUC1 as well ine to catecholamines in noradrenergic neurons [35]. as MUC3 mucin genes in the treatment group. Other Further, administration of L. plantarum (RTA 8) was studies have also reported an increase in mucin produc- observed to normalize the level of norepinephrine and tion after oral administration of probiotic strains such as could be explained by the previously reported ability of VSL#3 or on probiotic exposure to colonic loops and cell probiotics as well as gut microbiota in modulating the lines [40, 41]. The expression of these genes was evaluated level of catecholamines [36]. Interestingly, host nor- given the role of MUC1 in maintaining mucosal barrier epinephrine/epinephrine levels have been implicated during infections such as those caused by Helicobacter in downregulation of two important virulence gene of pylori [42] and of MUC3 in inhibiting the attachment of Salmonella Typhimurium SL1344 i.e., virK and mig14, pathogens such as E. coli in its secreted form [43]. The and simultaneously increasing its sensitivity to host increase in mucin gene expression might be attributed antimicrobial peptide LL-37 (which is a part of the to the ability of probiotic strains in either increasing the innate defence system) suggesting its role in decreasing density of goblet cells or inducing heightened activity virulence of the organism [37]. Probiotic strains such in already differentiated goblet cells [41]. Fermentation as Lacticaseibacillus rhamnosus GG and E. coli CFR 16 products of certain probiotic metabolites have also been have been reported to increase levels of norepinephrine reported to induce mucin expression [44]. Simultane- and restore level of other neurotransmitters via vagus ously, enhanced expression of MUC3 mucin gene might nerve mediated pathways or by directly affecting their have barred the attachment of Salmonella to the intesti- receptors [38, 39]. nal epithelium, thereby preventing brain infection. Other K aur et al. Gut Pathogens (2022) 14:11 Page 9 of 13 co-incubation experiments with L. plantarum strain Methods 299v and Lactobacillus casei GG have been observed to Bacterial strains increase mucin secretion, resulting in reduced adherence Two bacterial strains, namely Salmonella enterica sero- and translocation of gut pathogens such as E. coli [45]. var Typhimurium SL1344 and Lactiplantibacillus plan- A significant decrease in the expression of BDNF gene tarum (RTA 8) were used throughout the study. The was observed in mice suffering from Salmonella infec - former was kindly gifted by Dr. Mrytyunjay Suar, Direc- tion. BDNF has been reported to play an important tor of School of Biotechnology, KIIT University, Odisha role in neurogenesis, neurodegeneration and associated and the latter was kindly provided by Prof. Rupinder behavior [46, 47]. Various studies have reported its role Tewari, Department of Microbial Biotechnology, Pan- in memory and learning owing to its ability in mediating jab University, Chandigarh. Salmonella strain was plastic changes [48]. Additionally, dysregulation of this routinely cultured in Luria–Bertani (LB) medium and neurotrophic factor has been associated with psychiatric streaked on solid LB agar plates containing antibiotic disorders such as depressive-like behaviour, bipolar dis- streptomycin at a concentration of 50  μg/ml. The Lac - order and schizophrenia [49–51]. Infection studies with tiplantibacillus strain was cultured in de Man, Rogosa T. muris as well as neonatal meningitis caused by Strep- and Sharpe (MRS) medium and maintained on MRS tococcus pneumoniae have also reported similar findings plates. which might be attributed to neurodegeneration caused by neuroinflammation [52]. However, normalization of Animals BDNF expression was observed after administration of Female BALB/c mice, 5–6  weeks old, weighing approx. L. plantarum (RTA 8), thereby highlighting the ability of 22–26  g, were used throughout the study. The animals gut microbes in altering brain behaviour and neurochem- were procured from Central Animal House, Panjab Uni- istry which could be mediated via inflammation depend - versity, Chandigarh and adapted to the conditions of the ent as well as independent pathways. animal room initially for a period of 1  week before ini- tiation of any experiments. Animals were given standard pellet diet along with water ad libitum every day. Conclusions Given the complexity of the interactions and activa- tion of multiple pathways during infection, the role of In vivo study groups direct gut-brain communication holds enhanced signifi - Prior to initiation of experiments, mice were randomly cance. In our previous findings, mice in the Salmonella divided into 3 groups containing 6–8 mice in each group. infected group exhibited depressive-like and anxiety- like behavioural changes [15]. Studies have reported the Control group association of abnormal levels of serotonin, dopamine All mice in this group served as control and each mouse and norepinephrine, as well as the lower levels of BDNF, was administered 0.1 ml of PBS. to various depressive and anxiety disorders [53], which might serve as another plausible reason for the patho- physiology observed therein. Studies using C. jejuni have Infected group also documented that local infection in the gut activates All mice in this group were individually infected with vagal sensory neurons via upregulation of c-Fos thereby 8 10  CFU/ml of S. enterica serovar Typhimurium SL1344 leading to behavioural changes (anxiety and depressive suspended in 0.1 ml of PBS. like behaviour), in the absence of an overt immunologi- cal response [54]. In this context, the importance of these Treatment group molecules in bi-directional communication, and the abil- All mice in this group were administered approx. ity of microbes in influencing the same, have far reach - 9 10 10 –10  CFU/ml of L. plantarum (RTA 8) suspended in ing consequences in the field of gastrointestinal diseases 0.2  ml of PBS daily for a period of 7  days before infect- and psychiatric disorders associated with depression and ing them with 10   CFU/ml of S. enterica serovar Typh- anxiety. It is here that the intervention of probiotics holds imurium SL1344. Thereafter, mice in all the groups were great potential. The crucial role of diet supplemented sacrificed via cervical dislocation on day 7 post infection. with such functional foods including probiotics and syn- Various vital organs of mice i.e. liver, spleen, intestine biotics, have been reported to provide health benefits and brain were rapidly excised, weighted and stored at that go beyond traditional nutrition thereby providing −  60  °C for preparation of homogenates. For qRT-PCR multi-dimensional and holistic health benefits to the studies, the tissue sample was dissected at 0  °C and host. Kaur et al. Gut Pathogens (2022) 14:11 Page 10 of 13 immediately transferred into vials containing RNA later was followed by addition of 0.05 ml water to the test and solution and stored at − 80 °C until use. 0.5  ml alkaline sulphite solution to blank. Thereafter, the fluorescence was measured at 335/410  nm. Values Evaluation of systemic Salmonella dissemination of unknown samples were obtained from the standard Systemic spread of the pathogen from the gut to  other curve of dopamine (0.1–5 μg/ml). organs and efficacy of the treatment with L. plantarum (RTA 8) was evaluated in terms of reduction in Salmo- Estimation of norepinephrine nella bacterial count in vital organs such as the liver and Level of norepinephrine in all the tissue homogenates the spleen as described previously [55]. Briefly, the liver was evaluated using the method as described by Ciar- and spleen tissues samples were rapidly excised and tis- lone [57]. Briefly, 0.1 ml of sodium acetate buffer (pH 6.9) sue homogenates (10%W/V) were prepared in ice cold and 0.05 ml of 0.4 M HCl was added to 0.2 ml of aqueous PBS (0.05 M, pH 7.4). Appropriately diluted samples were extract prepared as described above. This was followed then plated on streptomycin (50  μg/ml) containing LB by addition of 0.1  ml of iodine solution. After 2  min of agar plates with overnight incubation at 37 °C for bacte- incubation, 0.1 ml of alkaline sulphite solution was added rial count assessment. to remove excess iodine. After each addition, all con- tents of the tube were mixed thoroughly. After 1.5  min, Parameters evaluated at the gut‑brain axis 0.1 ml of 10 N acetic acid was also added and mixed well. Intestinal and brain bio‑burden The tubes were then placed in a boiling water bath for Dissemination of Salmonella from gut to brain along a period of 6  min. The reading for NE was recorded at with the efficacy of L. plantarum (RTA 8) treatment was 395/485 nm after cooling the tubes under tap water. Val- evaluated by enumeration of Salmonella bio-burden ues of the unknown samples were extrapolated from the in intestinal and brain tissue homogenates as described standard curve of NE (0.1–5  μg/ml). In case of “blank”, above. the same procedure was followed except that thiosul- phate reagent was added before addition of iodine. Level of neurotransmitters Briefly, a part of the intestinal and brain tissue sample of Estimation of serotonin mice in the above mentioned groups was homogenized Level of serotonin in all the tissue homogenates was eval- in ice cold acidifying butanol solution using glass teflon uated as per the procedure given by Schlumpf et al. [58]. homogenizer (50–75 mg tissue with 5 ml of HCl-butanol) Briefly, 1.2 ml of ortho-phthalaldehyde (OPT) was added for a period of 1  min. Thereafter, the homogenates were as to all the samples as well as blank (0.2  ml of 0.1  N centrifuged at 2000 rpm for 20 min. The supernatant was hydrochloric acid) and mixed thoroughly. Thereafter, all aliquoted and 1  ml of this was then added to tube con- tubes were placed in a boiling water bath for a period of taining a mixture of 0.31  ml of 0.1  M HCl and 2.5  ml of 10  min. After cooling the samples under tap water, the heptane. After shaking vigorously for 10  min the tubes fluorescence was read at 355/470 nm. Values of unknown were then centrifuged again at 2000 rpm for 20 min. The samples were calculated from the standard plot of seroto- aqueous phase (0.2  ml) was then used for assessment of nin (0.1–5 μg/ml). neurotransmitters as mentioned below. All steps were carried out at 0 °C. qRT‑PCR studies Total RNA from intestinal as well as brain tissues sam- Estimation of dopamine ples was extracted using TRIzol reagent (Ambion by Level of dopamine in all the tissue homogenates was Life Technologies). Complementary DNA (cDNA) was evaluated using the method as described by Carlsson and constructed using iScript cDNA Synthesis kit (Bio-Rad). Waldeck [56]. Briefly, 0.5  ml 0.1  M PO buffer (pH 6.5) Real-time PCR was performed using Applied Biosys- was added to 1 ml of above acid extract bringing the total tem StepOne Real-Time PCR system with SYBR Green volume to 3.8 ml by adding water. Thereafter, 0.05 ml of chemistry and SYBR Green Jumpstart Taq Ready Mix 0.02 N of iodine solution (consisting of 0.254 g iodine and (Sigma-Aldrich) to amplify the cDNA template. 5 g KI in 5 ml water diluted to 100 ml) was added to the tubes. Then, after 5 min of incubation, 0.5 ml of alkaline Evaluation of expression of mucin genes sulphite solution was added to test sample but in case Relative expression of two mucin genes, i.e., MUC 1 and of blank, 0.5  ml of 5  N NaOH was added. After another MUC 3, were evaluated in all the groups by performing 5  min, to all the samples, including the blank, 0.6  ml of real time PCR studies. The primer sequences were as fol - 5 N acetic acid was added and all samples were then irra- lows: MUC1 gene (Forward Primer: 5′-CAG TGC CAA diated with UV (240  nm) for a duration of 10  min. This GTC AATAC-3′, Reverse Primer: 5′-TGT TAC TGG AGA K aur et al. Gut Pathogens (2022) 14:11 Page 11 of 13 Statistical analysis AGG TAG -3′) and MUC3 gene (Forward Primer:5′-GTT All the values have been expressed as mean ± SD. Statis- GAT GTC ACC ACT ATG -3′, Reverse Primer: 5′-TGG tical analysis was performed using student’s t test, and TGT TGA GGT TAGAG-3′). They were designed using two-way ANOVA which was followed by pairwise com- NCBI primer designing tool and synthesized by G Bio- parison using Tukey’s test. In all the tests, p ≤ 0.05 was sciences. For RT-PCR analysis, the following programme considered as significant. was employed: initial denaturation was performed at 94 °C for 2 min; which was followed by 40 cycles of dena- turation at 94 °C for 15 s; annealing, extension and fluo - Abbreviations ANS: Autonomic nervous system; BDNF: Brain-derived neurotrophic factor; rescence was read at 55 °C for 1 min with 4 °C of optional cDNA: Complementary DNA; DA: Dopamine; EHEC: Enterohaemorrhagic E. hold. coli; EMS: Emotional motor system; HPA: Hypothalamus–pituitary–adrenal axis; LPS: Lipopolysaccharide; MUC 1: Mucin 1 gene; MUC 3: Mucin 3 gene; OPT: Ortho-phthalaldehyde; SCFA: Short chain fatty acids. Evaluation of expression of BDNF gene Relative expression of BDNF gene was evaluated in all the Acknowledgements groups by performing real time PCR studies. The primer Not applicable. sequences used were: BDNF gene (Forward Primer: Authors’ contributions 5′-CAA GAG TCC CGT CTG TAC TTTAC-3′, Reverse The study was designed and conceived by PR. All experimentations and Primer: 5′-GAC TAG GGA AAT GGG CTT AACA-3′). Prim- investigations were carried out by AK. Formal analysis was done by all the authors. Project administration was done by PR, IPK and KC. PR, IPK and KC ers were synthesized via IDT primer designing tool pro- supervised the study. PR and AK prepared the original draft. Further editing in vided by IDT (Integrated DNA Technologies) and were the manuscript was done by AK, PR, IPK and KC. All authors read and approved synthesized from the same. For RT-PCR analysis, the fol- the final manuscript. lowing programme was employed: Initial denaturation was Funding performed at 94  °C for 2  min; which was followed by 40 Financial assistance provided by Department of Science and Technology cycles of denaturation at 94  °C for 15  s; annealing, exten- (DST ), India under PURSE Grant (P.U.) and University Grants Commission under Basic Scientific Research under Research Fellowship in Sciences for Meritorious sion and fluorescence read at 60  °C for 1  min and 4  °C of Students (RFSMS) scheme Grant No. F.25-1/2014-15(BSR)/7-261/2009 (BSR) for optional hold. carrying out the research work is duly acknowledged. For all the genes: Availability of data and materials Melt curve analysis was performed by heating the All data supporting findings of the study has been included in the manuscript. samples from 55 to 95  °C with an increase of 0.5  °C and Nucleotide sequence of L. planatarum (RTA 8) can be found in GenBank fluorescence was recorded. GAPDH was used as the database under accession number: KJ802485. The NCBI mRNA reference housekeeping gene and the primer sequences were- For sequence number for genes evaluated in the paper are as follows: Mus musculus mucin 1, transmembrane (Muc1): NM_013605.2, https:// www. ncbi. ward Primer: 5′-AAC AGC AAC TCC CAC TCT TC-3′ and nlm. nih. gov/ nucco re/ NM_ 013605.2. Mus musculus mucin 3, intestinal (Muc3): Reverse Primer: 5′-CCT GTT GCT GTA GCC GTA TT-3′. NM_010843.1, https:// www. ncbi. nlm. nih. gov/ nucco re/ NM_ 010843.1. Mus musculus brain derived neurotrophic factor (Bdnf ): NM_007540.4, https:// Three biological replicates as well as three technical rep - www. ncbi. nlm. nih. gov/ nucco re/ NM_ 007540.4. Mus musculus glyceraldehyde- licates were set up for each gene. Relative fold change in 3-phosphate dehydrogenase (Gapdh): NM_001289726.1, https:// www. ncbi. −∆∆ct gene expression was assessed using the 2 method. nlm. nih. gov/ nucco re/ NM_ 00128 9726.1. Histological evaluation of gut and brain tissues Declarations A part of the intestine as well as brain tissues from each Ethics approval and consent to participate group were stored in 10% buffered formalin. Briefly, the The experimental protocols involving animals were approved by the Institu- samples were then dehydrated using different grades tional Animal Ethics Committee of Panjab University, Chandigarh, India under the Approval Number PU/45/99/ CPCSEA/IAEC/2018/225. All the guidelines of alcohol (70%, 80%, 90% and 100% absolute alcohol) laid down by the Committee for the Purpose of Control and Supervision of after which they were washed in xylene for an hour and Experiments on Animals (CPCSEA), Government of India and all applicable dipped in molten paraffin wax for crystallization and fur - international and national institutional guidelines for conducting research on animals were religiously obeyed and strictly adhered to. ther sectioning. The sections were then kept in a water bath at 50 °C to remove wax and for mounting on a glass. Consent for publication Thereafter, the slides were further treated with xylene Not applicable. to remove wax and with ethanol to remove xylene. The Competing interests slides were finally stained with hematoxylin, followed All the authors declare that they have no competing interests. by eosin. Lastly, the slides were mounted in Distyrene Plasticizer Xylene (DPX) for histological investigation Author details Department of Microbiology, Basic Medical Sciences Block I, Panjab Univer- by examining under light microscope. The tissues were sity, South Campus, Sector 25, Chandigarh 160014, India. Universit y I nstitute assessed for signs of inflammation and morphological of Pharmaceutical Sciences, Panjab University, Sector 14, Chandigarh 160014, changes. India. Kaur et al. Gut Pathogens (2022) 14:11 Page 12 of 13 Received: 26 October 2021 Accepted: 17 February 2022 information from gut to brain in STM-infected rats. World J Gastroenterol. 2002;8:540–5. 23. Peterson CT. Dysfunction of the microbiota-gut-brain axis in neurode- generative disease: the promise of therapeutic modulation with prebiot- ics, medicinal herbs, probiotics, and synbiotics. J Evid Based Integr Med. 2020;25:1–19. References 24. Kumar A, Russell RM, Pifer R, Menezes-Garcia Z, Cuesta S, Narayanan S, 1. Shaikh AIA, Prabhakar AT. Typhoid fever and its nervous system involve- MacMillan JB, Sperandio V. The serotonin neurotransmitter modulates ment. In: Innate immunity in health and disease. London: Intech Open; virulence of enteric pathogens. Cell Host Microbe. 2020;28:41–53. 2021. p. 1–5. 25. Nzakizwanayo J, Dedi C, Standen G, Macfarlane WM, Patel BA, Jones 2. Hanafi T, Titou H, Kerrouch H, Frikh R, Hjira N. Non-typhoidal Salmonella BV. Escherichia coli Nissle 1917 enhances bioavailability of serotonin in meningitis in an adult patient with HIV Infection: the hostage-taking situ- gut tissues through modulation of synthesis and clearance. Sci Rep. ation. Is there any solution. Clin Med Rev Case Rep. 2020;7:294. 2015;5:1–13. 3. Chaudhuri D, Roy Chowdhury A, Biswas B, Chakravortty D. Salmonella 26. Reigstad CS, Salmonson CE, Rainey JF III, Szurszewski JH, Linden DR, typhimurium infection leads to colonization of the mouse brain and is Sonnenburg JL, Farrugia G, Kashyap PC. Gut microbes promote colonic not completely cured with antibiotics. Front Microbiol. 2018;9:1632. serotonin production through an effect of short-chain fatty acids on 4. Khurshid N, Khan BA, Bukhari SW, Shahid A, Punshi A. Extensively drug- enterochromaffin cells. FASEB J. 2015;29:1395–403. resistant Salmonella Typhi meningitis in a 16-year-old male. Cureus. 27. Yaghoubfar R, Behrouzi A, Ashrafian F, Shahryari A, Moradi HR, Choopani 2019;11:e5961. S, Hadifar S, Vaziri F, Nojoumi SA, Fateh A, Khatami S. Modulation of sero- 5. Serra D, Almeida LM, Dinis TC. The impact of chronic intestinal inflam- tonin signaling/metabolism by Akkermansia muciniphila and its extracel- mation on brain disorders: the microbiota-gut brain axis. Mol Neurobiol. lular vesicles through the gut-brain axis in mice. Sci Rep. 2020;10:1–12. 2019;56:6941–51. 28. Tian P, Wang G, Zhao J, Zhang H, Chen W. Bifidobacterium with the role 6. Gubert C, Kong G, Renoir T, Hannan AJ. Exercise, diet and stress as modu- of 5-hydroxytryptophan synthesis regulation alleviates the symp- lators of gut microbiota: implications for neurodegenerative diseases. tom of depression and related microbiota dysbiosis. J Nutr Biochem. Neurobiol Dis. 2020;134:104621. 2019;66:43–51. 7. Saulnier DM, Ringel Y, Heyman MB, Foster JA, Bercik P, Shulman RJ, Versal- 29. Guzmán DC, Herrera MO, Brizuela NO, Mejía GB, Jiménez FT, García EH, ovic J, Verdu EF, Dinan TG, Hecht G, Guarner F. The intestinal microbiome, Olguín HJ. Assessment of the effects of oseltamivir and indomethacin on probiotics and prebiotics in neurogastroenterology. Gut Microbes. dopamine, 5-HIAA, and some oxidative stress markers in stomach and 2013;4:17–27. brain of Salmonella Typhimurium-infected rats. Neuroendocrinol Lett. 8. Margolis KG, Cryan JF, Mayer EA. The microbiota gut-brain axis: from 2016;37:129–36. motility to mood. Gastroenterology. 2021;160:1486–501. 30. González-Arancibia C, Urrutia-Piñones J, Illanes-González J, Martinez- 9. Holstege G, Bandler R, Saper CB. The emotional motor system. Prog Brain Pinto J, Sotomayor-Zárate R, Julio-Pieper M, Bravo JA. Do your Res. 1996;107:3–6. gut microbes affect your brain dopamine? Psychopharmacology. 10. Mayer EA. The neurobiology of stress and gastrointestinal disease. Gut. 2019;236:1611–22. 2000;47:861–9. 31. Hsieh TH, Kuo CW, Hsieh KH, Shieh MJ, Peng CW, Chen YC, Chang YL, 11. Liu Y, Forsythe P. Vagotomy and insights into the microbiota-gut-brain Huang YZ, Chen CC, Chang PK, Chen KY. Probiotics alleviate the progres- axis. Neurosci Res. 2021;168:20–7. sive deterioration of motor functions in a mouse model of Parkinson’s 12. Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain disease. Brain Sci. 2020;10:206. Res. 2018;1693:128–33. 32. Erickson JT, Brosenitsch TA, Katz DM. Brain-derived neurotrophic factor 13. Westfall S, Lomis N, Kahouli I, Dia SY, Singh SP, Prakash S. Microbiome, and glial cell line-derived neurotrophic factor are required simultane- probiotics and neurodegenerative diseases: deciphering the gut brain ously for survival of dopaminergic primary sensory neurons in vivo. J axis. Cell Mol Life Sci. 2017;74:3769–87. Neurosci. 2001;21:581–9. 14. Liang S, Wu X, Jin F. Gut-brain psychology: rethinking psychology from 33. Liu X, Jiang L, Li L, Yu H, Nie S, Xie M, Gong J. The role of neurotransmitters the microbiota–gut–brain axis. Front Integr Neurosci. 2018;12:33. in the protection of Caenorhabditis elegans for Salmonella infection by 15. Kaur A, Chopra K, Kaur IP, Rishi P. Salmonella strain specificity determines Lactobacillus. Front Cell Infect Microbiol. 2020;10:554052. post-typhoid central nervous system complications: intervention by Lac- 34. Nordgreen J, Munsterhjelm C, Aae F, Popova A, Boysen P, Ranheim B, tiplantibacillus plantarum at gut-brain axis. Front Microbiol. 2020;11:1568. Heinonen M, Raszplewicz J, Piepponen P, Lervik A, Valros A. The effect 16. Asahara T, Shimizu K, Takada T, Kado S, Yuki N, Morotomi M, Tanaka R, of lipopolysaccharide (LPS) on inflammatory markers in blood and Nomoto K. Protective effect of Lactobacillus casei strain Shirota against brain and on behavior in individually-housed pigs. Physiol Behav. lethal infection with multi-drug resistant Salmonella enterica serovar 2018;195:98–111. Typhimurium DT104 in mice. J Appl Microbiol. 2011;110:163–73. 35. Stibbs HH. Changes in brain concentrations of catecholamines and 17. Brenchley JM, Douek DC. Microbial translocation across the GI tract. Annu indoleamines in Toxoplasma gondii infected mice. Ann Trop Med Parasi- Rev Immunol. 2012;30:149–73. tol. 1985;79:153–7. 18. Corr SC, Hill C, Gahan CG. Understanding the mechanisms by which 36. Asano Y, Hiramoto T, Nishino R, Aiba Y, Kimura T, Yoshihara K, Koga Y, Sudo probiotics inhibit gastrointestinal pathogens. Adv Food Nutr Res. N. Critical role of gut microbiota in the production of biologically active, 2009;56:1–15. free catecholamines in the gut lumen of mice. Am J Physiol Gastrointest 19. Ishikawa H, Kutsukake E, Fukui T, Sato I, Shirai T, Kurihara T, Okada N, Liver Physiol. 2012;303:G1288–95. Danbara H, Toba M, Kohda N, Maeda Y. Oral administration of heat- 37. Spencer H, Karavolos MH, Bulmer DM, Aldridge P, Chhabra SR, Winzer K, killed Lactobacillus plantarum strain b240 protected mice against Williams P, Khan CMA. Genome-wide transposon mutagenesis identifies Salmonella enterica serovar Typhimurium. Biosci Biotechnol Biochem. a role for host neuroendocrine stress hormones in regulating the expres- 2010;74:1338–42. sion of virulence genes in Salmonella. J Bacteriol. 2010;192:714–24. 20. Acurcio LB, Wuyts S, de CiccoSandes SH, Santanna FM, Pedroso SHSP, 38. Pandey S, Singh A, Chaudhari N, Nampoothiri LP, Kumar GN. Protection Bastos RW, Dos Reis DC, Vieira AF, Cassali GD, Lebeer S, de Souza MR. against 1,2-di-methylhydrazine-induced systemic oxidative stress and Milk fermented by Lactobacillus paracasei NCC 2461 (ST11) modulates altered brain neurotransmitter status by probiotic Escherichiacoli CFR 16 the immune response and microbiota to exert its protective effects secreting pyrroloquinoline quinone. Curr Microbiol. 2015;70:690–7. against Salmonella Typhimurium infection in mice. Probiotics Antimicrob 39. Kannampalli P, Pochiraju S, Chichlowski M, Berg BM, Rudolph C, Bruckert Proteins. 2020;12:1398–408. M, Miranda A, Sengupta JN. Probiotic Lactobacillus rhamnosus GG (LGG) 21. Mulaw G, Muleta D, Tesfaye A, Sisay T. Protective effect of potential and prebiotic prevent neonatal inflammation-induced visceral hypersen- probiotic strains from fermented Ethiopian food against Salmonella sitivity in adult rats. Neurogastroenterol Motil. 2014;26:1694–704. Typhimurium DT104 in mice. Int J Microbiol. 2020;2020:7523629. 22. Wang X, Wang BR, Zhang XJ, Xu Z, Ding YQ, Ju G. Evidences for vagus nerve in maintenance of immune balance and transmission of immune K aur et al. Gut Pathogens (2022) 14:11 Page 13 of 13 40. Hafez MM. Upregulation of intestinal mucin expression by the pro- biotic bacterium E. coli Nissle 1917. Probiotics Antimicrob Proteins. 2012;4:67–77. 41. Caballero-Franco C, Keller K, De Simone C, Chadee K. The VSL# 3 probiotic formula induces mucin gene expression and secretion in colonic epithe- lial cells. Am J Physiol Gastrointest Liver Physiol. 2007;292:G315–22. 42. Lindén SK, Sheng YH, Every AL, Miles KM, Skoog EC, Florin TH, Sut- ton P, McGuckin MA. MUC1 limits Helicobacter pylori infection both by steric hindrance and by acting as a releasable decoy. PLoS Pathog. 2009;5:e1000617. 43. Liévin-Le Moal V, Servin AL. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clin Microbiol Rev. 2006;19:315–37. 44. Barcelo A, Claustre J, Moro F, Chayvialle JA, Cuber JC, Plaisancié P. Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon. Gut. 2000;46:218–24. 45. Mack DR, Ahrné S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut. 2003;52:827–33. 46. Bath KG, Lee FS. Variant BDNF ( Val66Met) impact on brain structure and function. Cogn Aec ff t Behav Neurosci. 2006;6:79–85. 47. Miranda M, Morici JF, Zanoni MB, Bekinschtein P. Brain-derived neu- rotrophic factor: a key molecule for memory in the healthy and the pathological brain. Front Cell Neurosci. 2019;13:363. 48. Heldt SA, Stanek L, Chhatwal JP, Ressler K. Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories. Mol Psychiatry. 2007;12:656–70. 49. Nieto R, Kukuljan M, Silva H. BDNF and schizophrenia: from neurodevel- opment to neuronal plasticity, learning, and memory. Front Psychiatry. 2013;4:45. 50. Autry AE, Monteggia LM. Brain-derived neurotrophic factor and neu- ropsychiatric disorders. Pharmacol Rev. 2012;64:238–58. 51. Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders. Biol Psychiatry. 2006;59:1116–27. 52. Barichello T, Belarmino E Jr, Comim CM, Cipriano AL, Generoso JS, Savi GD, Stertz L, Kapczinski F, Quevedo J. Correlation between behavioral deficits and decreased brain-derived neurotrofic factor in neonatal men- ingitis. J Neuroimmunol. 2010;223:73–6. 53. Martinowich K, Manji H, Lu B. New insights into BDNF function in depres- sion and anxiety. Nat Neurosci. 2007;10:1089–93. 54. Goehler LE, Gaykema RP, Opitz N, Reddaway R, Badr N, Lyte M. Activation in vagal afferents and central autonomic pathways: early responses to intestinal infection with Campylobacter jejuni. Brain Behav Immun. 2005;19:334–44. 55. Kaur A, Chabba SK, Kaur UJ, Kaur A, Preet S, Rishi P. Management of Staphylococcus mediated systemic infection by enhancing the resurging activity of co-trimoxazole in presence of cryptdin-2. Indian J Microbiol. 2017;57:438–47. 56. Carlsson A, Waldeck B. A fluorimetric method for the determination of dopamine (3-hydroxytyramine). Acta Physiol Scand. 1958;44:293–8. 57. Ciarlone AE. Further modification of a fluorometric method for analyzing brain amines. Microchem J. 1978;23:9–12. 58. Schlumpf M, Lichtensteiger W, Langemann H, Waser PG, Hefti F. A fluo - rometric micromethod for the simultaneous determination of serotonin, noradrenaline and dopamine in milligram amounts of brain tissue. Biochem Pharmacol. 1974;23:2437–46. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : Publisher’s Note fast, convenient online submission Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

Journal

Gut PathogensSpringer Journals

Published: Mar 2, 2022

Keywords: Salmonella; Neurological complications; Gut-brain axis; Probiotics

There are no references for this article.