Introduction

Globally, most people nowadays consume too much salt in their diets unknowingly from processed foods and even consciously because of its pleasant taste1,2. A WHO report advised reducing salt intake to 5 g/day (2 g/day sodium) as most people consume more salt (9–12 g per day) than the recommended limit and reducing salt intake to this limit could prevent about 2.5 million deaths annually1. Animal studies revealed that an HSD can cause kidney damage via reactive oxygen species-dependent mechanisms. In addition, a high-salt diet also contributes to liver diseases like fibrosis and fatty liver through various mechanisms. Moreover, hypertension caused by HSD can also induce cardiovascular abnormalities by directly affecting endothelial functions and controlling vascular tone3. The gut microbiota drives the metabolism of the host by the interplay of multiple mechanisms like the production of bioactive metabolites, fermentation, and even indirectly by modulating immune responses4. The main bioactive metabolites such as bile acids, amino acids, and peptides are produced by the gut microbiota in response to the circulating signalling molecules in the host5. Trimethylamine-n-oxide (TMAO) is a significant product of the gut microbiota, which induces the expression of scavenger receptors that inhibit reverse cholesterol transport and accumulate cholesterol in macrophages, which accelerates the pathogenesis of atherosclerosis and other coronary artery diseases such as hypertension. Short-chain fatty acids (SCFAs) are a major metabolites of gut microbiota that interact with the olfactory receptor-78 (Olfr78) and the G Protein-Coupled Receptor 4 (GPR4) to regulate the host's blood pressure. Heart failure is usually linked with bacterially derived secondary bile acids and indoxyl sulphate6. Eating high salt over a period leads to a disruption in the microbial ecosystem homeostasis that increases the risk of multiple non-communicable diseases, especially chronic renal disorder, cardiovascular diseases, metabolic disorders, immune-related disorders, and premature death7,8,9,10. Animal studies have displayed that a high-salt diet harms gut health by triggering systemic inflammatory conditions resulting in damage to target organs11. Researchers have highlighted the crucial role of the gut microbiota in contributing to human health and diseases. Now, attention is turning towards intestinal microflora and related metabolites as potential therapeutic targets. Usually, antibiotics are lifesaving therapeutic drugs used to eradicate bacterial pathogens that also induce a change in the intestinal microbiota, disrupting the host immune and nutritional homeostasis and decreasing colonisation resistance12,13. Another study in mice proved that antibiotics cause modulation of gut bacteria and associated circulating LPS levels in obesity by improving insulin tolerance and glucose tolerance in metabolically active tissues14. However, there is growing interest to know as how antibiotics treatment impacts in the backdrop of HSD diet may influence on gut bacteria, haemato-biochemical parameters, and histopathological changes in vital organs. This understanding is highly pragmatic now as the modern diet has higher salt than the recommended level, and antibiotics are commonly given during infections to treat patients. Notwithstanding how antibiotics likely influence the resident gut microbiota by improving or deteriorating the pathophysiology of individuals on an HSD diet, it remains poorly explored. We performed this study to address these gaps by conducting experiments, wherein the mice were fed an HSD diet for 3 weeks and provided Amoxicillin treatment in the 3rd week to follow a typical course of antibiotics. We selected Amoxicillin antibiotic in this study because the primary healthcare settings used it as the most common antibiotic15. In this investigation, we first showed how short-term intake of HSD for 3 weeks caused change in the overall community structure of the gut microbiota, Afterwards, we examined how the short course of Amoxicillin treatment reshaped the gut microbiota by employing 16S rRNA high-throughput sequencing. We also projected the haematological, biochemical profile changes and histopathological effects on vital organs, namely the heart, liver, and kidney in the mouse model.

Methods

Animal model

Twenty-four male C57BL/6 J mice aged 6–8 weeks were kept under standard environmental conditions (22–24 °C, 12 h light/dark cycles) with free access to water and chow at All India Institute of Medical Sciences (AIIMS), New Delhi. All experimental procedures complied with the standards stated in the ARRIVE guidelines and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and were conducted under conditions approved by the Institutional Animal Ethics Committee (IAEC) of AIIMS. All methods were performed in accordance with the relevant guidelines.

Study design

Mice from the same cohort were randomly divided into two groups (N = 12 for each group) and fed CD (0.4% NaCl in chow) and HSD (4% NaCl in chow) for 3 weeks. Half of the animals (N = 6) in each group were given Amoxicillin treatment in the 3rd week at a 50 mg/kg body weight dose, i.e., 0.25 mg/ml in drinking water. The dosage and concentration of Amoxicillin in drinking water was based on published literature16. The treatment/diet group was referred to as CD + Amox as a positive control and HSD + Amox for treated mice. Another half of the animals (N = 6) from each group did not receive Amoxicillin treatment. These diet groups were designated CD for control and HSD for treated mice. The weight of mice was measured weekly and analysed once a week. After 3 weeks, these animals were euthanized to collect samples to see the impact of Amoxicillin on HSD and CD groups.

Blood, tissue, and caecal sample collection

After 3 weeks, all animals overnight fasted, and blood samples were taken by cardiac puncture. Then the vital organs, namely the heart, liver, and kidney, were harvested for histopathological examination. Caecal content samples were taken immediately after dissection of mice and frozen in liquid nitrogen at − 80 °C until further 16S rRNA gene sequencing was performed.

Serum biochemistry and haematology

Blood biochemistry was assessed using the serum auto-analyzer Screen Master 3000, Tulip, Alto Santa Cruz, India, with the Coral GPO-PAP kit (CORAL Clinical systems, Goa, India) according to manufacturer's directions. The haematological analysis was conducted using an automated vet haematology counter (Melet Schloesing Laboratories, Guwahati, India) as per the manufacturer’s usage guide.

Histopathology

Vital organ tissues, namely heart, liver, kidney were fixed overnight in 10% neutral buffered formalin. Tissues embedded in paraffin blocks were cut into 4–5 μm thick sections using a microtome. According to standard protocols, tissue sections were subjected to deparaffinization, rehydration, and haematoxylin and eosin (H&E) staining. The digital images were taken by light microscopy (Olympus CX-29: Olympus Optical Co. Ltd, Tokyo, Japan) and camera (Magnus DC 10).

High throughput 16S rRNA gene amplicon sequencing

According to the manufacturer's instructions, the total genomic DNA of gut microbiota was extracted from caecal content samples by using Qiagen DNA Stool Mini Kit. The samples were sent for high throughput 16S rRNA gene amplicon sequencing and genus analysis (DNA Xperts Private Limited, India). After DNA extraction, the samples were expanded by using the universal 16sPCR primer specific for the V3–V4 region included: 341F 5′- CCTAYGGGRBGCASCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′ according to the Illumina Miseq high-throughput sequencer usage guide.

Microbial bioinformatics analysis

Sequence data was analysed using QIIME (v1.9.1) software. FastQC and Trimmomatic were applied to trim and align the paired-end reads with tags with an average read length of 252 bp17. The tags were then clustered as OTUs (Operational Taxonomic Units) with a 97% similarity threshold18. Rarefaction curves were used to ensure the sufficient sequencing depth of samples. The relative abundance of each sample was calculated based on the normalised operational taxonomic units (OTUs). Alpha diversity was estimated by species richness (Chao) and diversity index (Shannon, Simpson). Beta diversity among the group samples was calculated mainly using the unweighted and weighted UniFrac distances, Bray–Curtis, and Jaccard distances. Principal coordinate analysis (PCoA) distance matrices created by QIIME19.

Statistical analysis

GraphPad Prism version 9.2.0 (3.2.0) for Windows, Graph Pad Software, San Diego, California USA, www.graphpad.com" was used for the statistical analysis of experimental data. Values are expressed as the means ± Standard Deviation (SD). Groups were compared by one-way analysis of variance (ANOVA) followed by the Bonferroni test for comparisons between multiple groups with a value of p ≤ 0.05 as the cut-off for statistical significance.

Results

Short term HSD treatment and Amoxicillin treatment to HSD-fed mice did not cause significant difference in weight of mice

In this study, HSD-fed mice did not display a significant difference in weight when compared to CD-fed mice (Fig. 1).. Likewise, we also did not observe a significant weight difference in HSD-fed mice and Amoxicillin treated HSD-fed mice (Fig. 1).. Thus, the present investigation indicates that a high salt diet intake for a short period could not lead to weight gain in mice.

Figure 1
figure 1

Effect of high-salt diet (4% in chow) and Amoxicillin treatment on body weight of mice. As shown by the data, no significant difference in body weight of mice was observed among different groups. Data represent mean ± SD; N = 6 mice/group, One Way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001. CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HSD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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Amoxicillin treatment restores the HSD-promoted thrombocytosis

This study first examined the impact of Amoxicillin treatment in HSD-fed mice on haematological parameters, as shown in Fig. 2 and Table 1. We observed a significant decrease of white blood cells (WBCs) in the HSD-fed mice compared to the CD-fed mice group (p = 0.0016). But the Amoxicillin treatment did not find any significant alteration in WBC values as observed in the HSD + Amox group in comparison to the HSD group. However, we found a significant increase in the thrombocytes in the HSD group in comparison to the CD group (p < 0.0001). Interestingly, in HSD-fed mice treated with Amoxicillin showed a significant decrease in the thrombocytes compared with their HSD-fed counterparts (p < 0.0001). No significant alteration of haematological parameters in the CD + Amox group compared to the CD group was distinguished. Overall, our study also suggests that Amoxicillin treatment could likely restore thrombocyte value to normal range in the pathophysiology associated with HSD. However, Amoxicillin therapy did not impact the value of thrombocytes in the CD fed mice.

Figure 2
figure 2

Short-term effect of high-salt diet (4% in chow) followed by Amoxicillin treatment on haematological parameters in mice. HSD treatment for 3 weeks in mice increases the thrombocytes concentration. Amoxicillin treatment in the 3rd week in HSD fed mice brought the elevated level of the thrombocytes near to control value. Values are means ± SD for 6 samples in each group. Statistically significance of differences was evaluated by one way ANOVA followed by Bonferroni test (*p < 0.05, **p < 0.01, ****p < 0.0001). CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HSD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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Table 1 Short term effect high salt (4% in chow) diet followed by Amoxicillin treatment on hepatological parameters in mice.
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Amoxicillin treatment declines the HSD-promoted blood glucose level

Next, we demonstrate the effect of Amoxicillin in HSD-fed mice on biochemical parameters involved in metabolic syndrome (Fig. 3 and Table 2). Our data showed a significant increase in blood glucose and total cholesterol (p < 0.0001) in HSD-fed mice compared to the CD-fed mice. Amoxicillin treatment in HSD-fed mice significantly reduced glucose levels (p < 0.0001) but found no significant change in cholesterol levels. Amoxicillin treatment in the standard chow diet group showed no alteration in cholesterol and glucose level. The results of the study suggest that Amoxicillin treatment could likely restore blood glucose levels towards normal range in the HSD induced pathophysiology.

Figure 3
figure 3

Short-term effect of high-salt diet (4% in chow) followed by Amoxicillin treatment on biochemical parameters in mice. HSD treatment for 3 weeks in mice showed a significant increase in total cholesterol, glucose, creatinine and a significant decrease in blood urea level. Amoxicillin treatment in the last one week in HSD fed mice caused restoration of the glucose, triglycerides, creatinine and blood urea levels towards the control value. Values are means ± SD for 6 samples in each group. Statistical significance of differences was evaluated by one way ANOVA followed by Bonferroni test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HSD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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Table 2 Short term effect high salt (4% in chow) diet followed by Amoxicillin treatment on biochemical parameters in mice.
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Amoxicillin treatment decreases the HSD-promoted blood creatinine level

We then examined the effect of Amoxicillin treatment on blood creatinine level in HSD-fed mice (Fig. 3 and Table 2). Our data observed a significant increase in creatinine in HSD-fed mice compared to CD-fed mice (p < 0.0001). However, Amoxicillin-treated HSD-fed mice showed a significant reduction in creatinine (p < 0.0001) level compared to HSD-fed mice. There was no significant difference in creatinine level between the Amoxicillin-treated CD-fed mice and the CD-fed mice. Our study suggests that Amoxicillin treatment in HSD-fed mice could likely decrease the creatinine towards the normal range.

Amoxicillin treatment restores the HSD-decreased blood urea level

In addition, we investigated the effect of Amoxicillin on blood urea level in HSD-fed mice (Fig. 3 and Table 2). Our data showed a significantly reduced urea level in HSD-fed mice compared to CD-fed mice (p < 0.0001). Amoxicillin treatment in HSD-fed mice substantially increases the urea concentration compared to HSD-fed mice (p < 0.0001). However, Amoxicillin-treated CD-fed mice also showed a significant decrease in urea concentration (p = 0.0029). Our data suggest that Amoxicillin treatment in HSD-fed could restore urea level to a normal range.

Amoxicillin treatment depletes HSD induced richness and diversity of especially of different gut bacteria

After initial quality control filtering, we generated a dataset of 1,169,822 high-quality sequences with an average of 292,455 reads per group sample. The high-quality sequences were clustered with a consistency of 97% similarity level and obtained a total number of 22,980 OTUs, as shown in Table 3. The Shannon–Wiener curve of all four samples already reached a plateau (Fig. 4), suggesting that the sequencing was deep enough to capture the entire spectrum of bacterial diversity and the majority of information pertaining to dominant phylotypes in each group. Estimators of the alpha and beta diversity are summarised in Tables 3 and 4 and shown in Fig. 4. Next, we calculated and compared the Chao1, Shannon, and Simpson indices of alpha diversity and observed species as outlined in Table 4 for each group. We found significantly higher alpha diversity estimators like Chao1 (richness) and Shannon (diversity of rare species) in the HSD group than in the Amoxicillin-treated HSD and CD-fed mice. The alpha diversity indices illustrated that HSD increased the richness and diversity of organisms, especially different gut bacteria. Amoxicillin treatment in HSD-fed mice showed a reduction in richness and diversity, especially of specific gut bacteria. There was no statistically significant difference for the Simpson index, indicating a remarkable similarity in general microbial diversity in all four group samples. Beta diversity measures of microbiota (shown in Table 4 and Fig. 4) based on principal coordinates analysis (PCoA) of weighted Unifrac distance and Bray–Curtis dissimilarity showed a distinguished separation among four groups, suggesting that the composition of microbiota was significantly distinct from each other.

Table 3 Short term effect of high-salt (4% in chow) diet followed by Amoxicillin treatment on alpha diversity.
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Figure 4
figure 4

Short-term effect of high-salt diet (4% in chow) followed by Amoxicillin treatment on gut bacteria diversity in mice. Rarefaction curves (a) and Shannon–Wiener curves (b) achieved a plateau, suggesting that the number of OTUs was sufficient to capture the authentic bacterial communities in each sample. Comparisons for alpha-diversity such as observed species index (a), Shannon (b) and Simpson (c) index and beta-diversity (with unweighted (d), weighted (e) Unifrac distance matrix and Bray–Curtis dissimilarity matrix (e) showed marked differences in the bacterial communities among the groups. The percent variation explained by each group is indicated on the axis. CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HSD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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Table 4 Short term effect of high-salt diet (4% in chow) followed by Amoxicillin treatment on beta diversity.
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Amoxicillin treatment reshapes the specific gut microbial community associated with the improvement in the pathophysiology of metabolic syndrome and increases the risk of some opportunistic gut pathogens

Next, we performed pairwise differential alteration analysis on gut microbiota to identify specific gut bacteria with potential clinical importance in the pathophysiology of metabolic syndrome and the subsequent impact of Amoxicillin on the modulation of these gut microbiota. We conducted core bacteria analysis at the phylum level (Table 5, Fig. 5) that demonstrates a significant decrease in Firmicutes (p = 0.0028), Bacteroidetes (p < 0.0001) and an increase of Proteobactera (p < 0.0001) in HSD-fed mice compared to standard CD-fed mice. However, Amoxicillin treatment for one week in HSD-fed mice significantly further decreased Firmicutes (p = 0.0009) and increased Bacteroidetes (p < 0.0001). Similarly, Amoxicillin treatment in CD-fed mice also showed a decrease in Firmicutes (p < 0.0001) considerably and increase in Bacteroidetes (p < 0.0001). The relative abundance of Proteobacteria increased dramatically in HSD-fed mice compared to CD-fed mice (p < 0.0001). Amoxicillin-treated HSD-fed mice showed a significant increase in the abundance of Proteobacteria (p < 0.0001). Similarly, Amoxicillin treatment in CD-fed mice caused a significant increase in the abundance of Proteobacteria (p < 0.0001). The Firmicutes/ Bacteroidetes (F/B) ratio was significantly higher in HSD-fed as compared to mice CD-fed mice Amoxicillin treatment in HSD-fed mice substantially reduces the F/B ratio compared to HSD (p < 0.0001). Similarly, Amoxicillin-treated CD mice showed a significant decrease in the F/B ratio (p < 0.0001).

Table 5 Short term effect of high salt (4% in chow) diet followed by Amoxicillin treatment on the mean relative abundance of major bacterial groups at phylum level in mice.
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Figure 5
figure 5

Bar chart (a) and heat map (b) showed the short-term effect of a high-salt diet (4% in chow) followed by Amoxicillin treatment on the relative abundance of the most represented phylum for each group. HSD treatment for 3 weeks in mice caused a significant increase of Firmicutes to Bacteroidetes (F/B) ratio and Proteobacteria. During the last week, Amoxicillin treatment in HSD fed mice mice caused a significant decrease in the Firmicutes to Bacteroidetes (F/B) ratio and as well as increased of Proteobacteria. Color heat map (4b) analysis illustrating the top 15 mean abundances of the bacterial community taxa assigned to phyla. The color intensity in each sample is normalized to represent its relative ratio in the four groups. The color scale of higher (dark red) and lower (dark blue) shows the relative abundances of bacterial communities. CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HSD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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We observed a total of 45, 53, 71, and 69 families (Table 6 and Fig. 6) in the CD, CD + Amox, HSD, and HSD + Amox group samples, respectively. There was a significant increase in the abundance of Erysipelotrichiciae, Desulfovibrionaceae, Coriobacteriaceae and F16 (p < 0.0001) in HSD-fed mice. Amoxicillin-treated HSD-fed mice showed a significant decrease in Erysipelotrichaceae, Desulfovibrionaceae, Coriobacteriaceae, and F16 in comparison to HSD-fed mice (p < 0.0001). Amoxicillin treatment in CD-fed mice also caused a significant decrease in the Erysipelotrichaceae, Desulfovibrionaceae, and Coriobacteriaceae compared to CD-fed mice (p < 0.0001). HSD-fed mice also showed significant increases in Verrucomicrobiaceae and Enterobacteriaceae (p < 0.0001) compared to CD-fed mice. Amoxicillin treatment in HSD-fed mice further significantly increased Verrucomicrobiaceae and Enterobacteriaceae (p < 0.0001). HSD-fed mice showed a significant increase in Enterobacteriaceae (p < 0.0001) but did not alter Bacteroidaceae in comparison to the CD-fed mice. Amoxicillin resentment caused a significant increase in Enterobacteriaceae and Bacteroidaceae (p < 0.0001). Similarly, Amoxicillin-treated CD-fed mice also showed a significant increase in the abundance of Enterobacteriaceae and Bacteroidaceae (p < 0.0001).

Table 6 Short term effect of high-salt (4% in chow) diet followed by amoxicillin treatment on the mean relative abundance of major bacterial groups at family level in mice.
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Figure 6
figure 6

Bar chart (a) and heat map (b) showed the short-term effect of a high-salt diet (4% in chow) followed by Amoxicillin treatment on the relative abundance of the most represented family for each group. Amoxicillin-treatment in HSD-fed mice found a significant decrease in Erysipelotrichaceae, Desulfovibrionaceae, Coriobacteriaceae, F16 and a significant increase in Bacteroidaceae, Enterobacteriaceae and Verrucomicrobiaceae. Color heat map (b) analysis illustrating the top 12 mean abundances of the bacterial community taxa assigned to family. The color intensity in each sample is normalized to represent its relative ratio in the four groups. The color scale of higher (dark red) and lower (dark blue) shows the relative abundances of bacterial communities. CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HFD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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We observed, at the genus level, a total of 21, 26, 44, and 47 genera (Table 7 and Fig. 7) in CD, CD + Amox, HSD, and HSD + Amox group samples, respectively. The species detected in these groups are presented in Table 8. There was a significant increase in the relative abundance of gut bacteria in HSD-fed mice such as Lactobacillus, Streptococcus, Unc Clostridiaceae, Clostridium, [Ruminococcus], Unc Erysipelotrichaceae Allobaculum, Neisseria, Desulfovibrio, Haemophilus, Unc Coriobacteriaceae, Unc F16 and Unc RF 39 in HSD-fed mice (p < 0.0001) compared to CD-fed mice. HSD-fed mice also showed an increase in the abundance of Ruminococcus gnavus, Neisseria subflava, and a significant decrease of Lactobacillus reuteri (p < 0.0001). However, Amoxicillin treatment in HSD-fed mice caused the significant decrease in Lactobacillus, Streptococcus, Unc Clostridiaceae, Clostridium, [Ruminococcus], Unc Erysipelotrichaceae, Allobaculum, Desulfovibrio, Unc Coriobacteriaceae, and Unc F16 (p < 0.0001). Amoxicillin treatment in HSD-fed mice did not cause any changes in the abundance of Neisseria, and Haemophilus. Amoxicillin-treated HSD-fed mice also showed a significant decrease in Ruminococcus gnavus compared to the HSD-fed mice at the species level. Even Amoxicillin-treatment to CD-fed mice also showed a significant decrease in Lactobacillus, [Ruminococcus] (p < 0.0001), Unc Erysipelotrichaceae, Desulfovibrio and Unc Coriobacteriaceae (p < 0.0001), (p < 0.0001). Amoxicillin treatment in CD-fed mice also caused a significant decrease in Ruminococcus gnavus (p < 0.0001) compared to CD-fed mice.

Table 7 Short term effect of high-salt (4% in chow) diet followed by Amoxicillin treatment on the mean relative abundance of major bacterial groups at genus level in mice.
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Figure 7
figure 7

Bar chart (a) and heat map (b) showed the short-term effect of a high-salt diet (4% in chow) on the relative abundance of the most represented bacterial taxa at the genus level for each sample. Amoxicillin-treatment in HSD-fed mice found a significant decrease in Lactobacillus, Streptococcus, Unc Clostridiaceae, Clostridium, [Ruminococcus], Unc Erysipelotrichaceae, Allobaculum, Desulfovibrio, Unc Coriobacteriaceae, Unc F16 and a significant increase in Bacteroides and Akkermansia. The color intensity in each sample is normalized to represent its relative ratio in the four groups. The color scale of higher (dark red) and lower (dark blue) shows the relative abundances of top 36 mean abundance of bacterial communities. CD: Standard—chow diet for 3 weeks; CD + Amox: Standard—chow diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week; HSD: High-salt diet for 3 weeks; HFD + Amox: High-salt diet for 3 weeks and subjected to Amoxicillin treatment in 3rd week.

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Table 8 Short term effect of high-salt (4% in chow) diet followed by Amoxicillin treatment on the mean relative abundance of major bacterial groups at the species level in mice.
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The HSD-fed mice did not show any alteration in the abundance of Bacteroides but increased the abundance of Akkermansia (p < 0.0001) compared to the CD- fed mice. However, Amoxicillin-treated HSD-fed mice caused a significant increase in Bacteroides (p < 0.0001) and Akkermansia (p < 0.0001). In this study, Amoxicillin treatment in HSD-fed mice also resulted in a significant increase in Bacteroides acidifaciens, Bacteroides uniformis (p < 0.0001), and Akkermansia muciniphila (p < 0.0001) linked to an increase in insulin sensitivity and gut epithelial integrity. Amoxicillin-treated CD-fed mice also showed a significant increase in Bacteroides acidifaciens and Akkermansia muciniphila (p < 0.0001) compared to the CD-fed mice.

There was a significant increase in the relative abundance of gut opportunistic pathogens such as Veillonella (p = 0.0007), Prevotella (p = 0.0002), Rothia (p < 0.0001) in HSD-fed mice compared to CD-fed mice. HSD-fed mice also showed a significant increase in Veillonella dispar (p = 0.0013), Rothia mucilaginosa (p < 0.0001), a) compared to the CD-fed mice. However, Amoxicillin treatment in HSD-fed mice caused a significant increase in Veillonella (p < 0.0001), Klebsiella (p < 0.0001), Prevotella (p = 0.0002), Rothia (p = 0.0184), and Enterococcus (p < 0.0001)). Amoxicillin treated HSD-fed mice also showed a significant increase in Veillonella dispar (p < 0.0001), Rothia mucilaginosa (p < 0.0184), and Enterococcus casseliflavus (p < 0.0001). Similarly, amoxicillin treatment in CD-fed mice also resulted in a significant increase in Veillonella, Klebsiella (p < 0.0001), Prevotella (p = 0.0002), and Rothia (p < 0.0001).

Taken together, our result indicates that HSD for a short period of 3 weeks in mice could significantly change the gut microbiota profile (phylum, family and genus level) known to develop the pathophysiological features of metabolic syndrome-related inflammation. Importantly, our investigation observed that Amoxicillin treatment in HSD-fed mice causes reshaping of specific gut bacteria associated with improvement in the pathophysiological attributes of metabolic disease related inflammation, during the consumption of HSD in mice.

We found that no significant histopathological damage occurs in CD-fed mice, HSD-fed mice and mice receiving amoxicillin treatment with these diets (Fig. 8). The photomicrograph of the heart tissue of HSD-fed mice showed mild cardiomyocyte hypertrophy and associated tissue degenerative changes (3a). Liver histopathology also indicated the changes in HSD-fed mice, including oddly shaped cells and decreased cell proliferation (3b). No significant changes were observed in the kidney tissue of HSD-fed mice (3c). However, these histopathological changes were insufficient to conclude that they were significant. No significant changes in the heart, liver, and kidney were observed in Amoxicillin treated HSD-fed mice (Fig. 4a, 4b, 4c). Our study indicates that HSD for a short period could not cause significant histopathological changes in the heart, liver and kidney of mice (Fig. 8).

Figure 8
figure 8

Short-term effect of high-salt diet (4%) followed by Amoxicillin treatment on the heart, liver, and kidney of mice. The figure represents histopathogical examination of heart, liver and kidney sections by hematoxylin and eosin staining. The photomicrograph of the heart tissue of HSD-fed mice showed mild cardiomyocyte hypertrophy and associated degenerative changes (3a). Liver histopathology also indicated the changes in HSD-fed mice, including oddly shaped cells and decreased cell proliferation (3b). No significant changes were observed in the kidney issue of HSD-fed mice (3c). However, these histopathological changes were insufficient to conclude as the significant observations. No significant changes were observed in the heart, liver and kidney of Amoxicillin treated HSD-fed mice (4a, 4b and 4c).

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Discussion

Excessive dietary salt intake is associated with metabolic abnormalities such as hypertension, cardiovascular complications, renal injury, and gut inflammation20,21. A metabolic syndrome is characterized by abnormal cholesterol levels, high fasting blood sugar level, high triglyceride level, and chronic inflammation, caused primarily by modulation of the gut microbiota22,23. Further, it is speculated that diabetes patients who take a certain class of antibiotics are more likely to have severe blood sugar fluctuations than those who take other types of the drugs. Nevertheless, determining how antibiotics treatment in the context of HSD diet influences gut microbiota, haemato-biochemical parameters, as well as histopathological changes in vital organs, is a topic of high interest to researchers. Earlier studies focused on the detrimental effect of consuming a high amount of salt for a long period on human health24,25,26,27,28,29. However, less has been known about the short-term impact of an HSD on gut microbiota and associated pathophysiology. In our study, we fed mice a short-term HSD diet for 3 weeks, followed by an Amoxicillin intervention in the 3rd week.

Previous investigations have shown that HSD for the period of 4 weeks or 8 weeks did not lead to any change in the faecal bacterial richness and diversity but displayed even variability or similarity in bacterial composition within groups7,30. Our findings are not consistent with previous research and showed that HSD treatment in mice causes an increase in taxonomic richness and diversity7,30, particularly of specific gut bacteria such as Streptococcus Clostridium, Unc Peptostreptococcaceae, Veillonella, Allobaculum, Neisseria, Prevotella, Haemophilus, Rothia, Bifidobacteria and Unc RF 39, which were exclusively detected in HSD-fed mice. However, Amoxicillin treatment resulted in a reverse effect in HSD-fed mice by depletion of Clostridium, Unc Clostridium Dehalobacterium Allobaculum, Bifidobacteria, and Unc RF 39. In addition, our study also identified the different compositions of gut microbiota in each group.

Here, we consistently found a significant increase in the F/B ratio in HSD-fed mice associated with the pathophysiology of metabolic syndrome7,31,32,33,34. Amoxicillin treatment in HSD-fed mice substantially reduced the F/B ratio with a significant increase in the abundance of Proteobacteria. To our knowledge, this is the first study to demonstrate that HSD intake causes a substantial rise in Desulfovibrionaceae, Erysipelotrichaceae, and Enterobacteriaceae, which induce the pathophysiology of obesity and metabolic syndrome-related inflammation35,36. Enterobacteriaceae usually cause acute infective process37,38. Furthermore, we were also observed a significant increase in Coriobacteriaceae in HSD-fed mice. Previous studies reported that the occurrence of Coriobacteriaceae has a positive correlation with obesity and type-2 diabetes39,40,41 Furthermore, HSD-fed mice also observed the significant increase of F16 known to be involved in the manifestation of inflammatory bowel diseases14. However, the treatment of Amoxicillin-in mice fed on HSD demonstrated a decrease in the abundance of Desulfovibrionaceae, Erysipelotrichaceae, Coriobacteriaceae, F16, and increased in the level of Enterobacteriaceae. Another key finding in this study was that Bacteroidaceae known to be negatively related to many metabolic phenotypes such as plasma insulin levels and weight gain, were exclusively increased in the Amoxicillin-treated both HSD-fed mice and CD-fed mice42. Unexpectedly, our study found significant increase in Verrucomicrobiaceae reported for a negative association with endotoxemia, adipose tissue inflammation, and insulin resistance in HSD-fed mice34. Amoxicillin treatment further significantly increased the abundance of Verrucomicrobiaceae.

Our genus-level results confirmed the abundance of gut microbiota, including Lactobacillus, Streptococcus, Ruminococcus gnavus, Allobaculum, Desulfovibrio, Neisseria. subflava, Clostridium and Haemophilus in HSD-fed mice. Most importantly, our study showed a surprise increase in the abundance of Lactobacillus that is not in line with the previous studies, which reported their suppression in HSD-fed mice7,30,44. Although the Lactobacillus genus is potentially probiotic bacteria, however its effect is species and even strain specific. Lactobacillus abundance is known to have a positive correlation with fasting blood glucose and HbA1c45. However, in our study, the representative species, Lactobacillus reuteri, which is well known to play an active role in the maintenance of the gut epithelial barrier by secreting antibiotic substances such as reuteri, and is also reported to have a beneficial impact on the pathophysiology of T2D, was observed for a decrease in their proportion in HSD-fed mice46,47. A previous study has reported a positive correlation between Streptococcus and metabolic disease development48. There is an agreement with the recent publication article by Wang, C. et al., which showed the increase in the abundance of Ruminococcus in HSD-fed mice7. Additionally, this study observed the increase of Ruminococcus gnavus linked to production of a polysaccharide that stimulates the release of inflammatory cytokines and induces inflammatory bowel disease49. Congruently, our investigation also found a higher level of Allobaculum in HSD-fed mice44. An earlier study has shown the positive association between the Allobaculum and the expression of angiogenin-like protein 4 (ANGPTL4), which has a crucial role in lipid deposition by repressing the lipoprotein lipase (LPL) enzyme and developing the pathophysiology of the obesity-related metabolic disorder50. In this investigation, we also observed a higher proportion of Desulfovibrio known to provoke intestinal barrier dysfunction and insulin resistance51,52. The previous study has shown a positive correlation between Neisseria and plasma cholesterol53. Further, the increase in the abundance of Neisseria subflava is considered an indicator of a change in microbial community composition to become acid-secreting54. Most strains of Clostridium and Haemophilus are potentially pathogenic to humans55. In our study Amoxicillin treatment to HSD-fed mice caused a decrease in the abundance of these specific gut bacteria.

Furthermore, a previous study has shown that Bacteroides are capable of preventing salt induced effects due to having salt tolerant genes56. Similarly, this study did not show any change in the abundance of Bacteroides in HSD-fed mice. Interestingly, we found an increase in the abundance of Bacteroides in Amoxicillin treated HSD-fed mice. Moreover, this study observed an exclusive increase in the proportion of Bacteroides acidifaciens and Bacteroides uniformis in Amoxicillin treated HSD-fed mice. A previous study has shown that these gut bacteria species improve insulin sensitivity and prevent obesity by activating Glucagon-like peptide-1 (GLP-1)23. Most unexpectedly, our study showed an increase in the abundance of Akkermansia muciniphila known for its decrease in glucose intolerance57,58. Along with beneficial impact of Amoxicillin on HSD-fed mice, we also observed the abundance of some opportunistic gut pathogen bacteria such as Klebsiella, Prevotella melanogeni, Veillonella dispar, and Enterococcus casseliflavus in Amoxicillin treated HSD-fed mice. These are the opportunistic pathogens that cause the disruption in the gut59,60,61,62,63,64,65, Collectively, these results suggest that Amoxicillin treatment in mice fed HSD causes a change in specific gut microbiota associated with improving the pathophysiological attributes of metabolic disorder related inflammation however, it also puts some at risk for opportunistic pathogens.

Earlier studies have shown that the gut microbiota is a key regulator of energy homeostasis66,67,68. and their composition is related to insulin sensitivity and host glycemic regulation47,69. The previous researchers lend support to the view that a long-term intake of a high-salt diet is associated with the pathophysiology of metabolic syndrome71,72,73. However, some studies have demonstrated that HSD for a short period also reduced insulin sensitivity in human and rat models74,75. Our study found that HSD increases the blood glucose level with abundance of specific gut microbiota such as Lactobacillus, Streptococcus, Ruminococcus gnavus, Allobaculum, and Desulfovibrio. These microbiotas are known for having a positive correlation with high plasma glucose level45,48,49,50,51. Contrary to our findings, Pitynski-Miller, D et al., reported that high-salt diets might block high–fat–diet-induced obesity either by impairing fat absorption or by unknown mechanisms76. Most strikingly, our data showed that Amoxicillin treatment in HSD-fed mice significantly reduced the glucose level, suggesting its hypoglycemic effect. In fact, previous studies have shown that antibiotics deplete or even reshape specific gut microbial communities involved in glucose metabolism77,78. Our study elucidates a significant suppression of Streptococcus, Lactobacillus, Ruminococcus gnavus, Allobaculum, Desulfovibrio, and enrichment of Bacteroides acidifaciens, Bacteroides uniformis, and Akkermansia muciniphila in Amoxicillin-treated HSD-fed mice. Incongruent to Seck et al., our result revealed the exceptionally enrichment of Akkermansia muciniphila reported for improving glucose tolerance in HSD-fed mice79,80. These findings suggest that Amoxicillin-treatment in HSD-fed mice results in the restoration of glucose levels near to true values by substantially changing the composition of these gut bacteria. Although, the conclusion of the study opens the door for future research to find out which specific gut microbiota’s metabolites involve in the restoration of blood glucose levels towards the normal range by improving the insulin signaling in the mice.

High salt consumption increases cardiovascular diseases (CVDs) risk by impairing endothelial function, most notably by reducing nitric oxide (NO) availability81,82. The increase in total cholesterol levels in short term is associated with significant decreases in endothelium-dependent vasodilation83,84. Metabolic factors, such as high glucose and triglyceride levels, are considered as major risk factors in contributing to endothelial dysfunction by decreasing nitric oxide absorption85,86. In our study HSD-fed mice showed increased levels of serum triglycerides and significant increases in glucose levels. The increase in these metabolites is likely to cause vascular endothelial dysfunction and may account for the short-term increase in total cholesterol in mice fed HSD. Importantly, our data consistently showed that even short-term HSD exposure to mice could result in increase the cholesterol level significantly along, with significant change in microbiota composition87. There are further evidences that the gut microbiota has the capacity to alter the blood lipid composition, particularly cholesterol, through gut bacteria related metabolites68,88,89,90. In fact, it is well known that the intestinal gut bacteria determine the circulating cholesterol level. Previous studies have shown that Erysipelotrichaceae and Coriobacteriaceae have a positive correlation with high plasma cholesterol in human and animal models89,91,92,93,94. The gut bacteria are directly involved in the pathogenesis of cardiovascular disease (CVD) through the alteration of circulating cholesterol levels. We also found the higher abundance of specific gut microbiota such as Lactobacillus, Enterobacteriaceae, Streptococcus and increased F/B ratio reported with cardiovascular diseases (CVD)95. Moreover, one study has shown a positive correlation between Neisseria and plasma cholesterol53. Interestingly, we were also able to detect similar modulation in the composition of gut microbiota and we believe that this may be the likely cause of increasing the plasma cholesterol in HSD-fed mice. However, the investigation to find out which gut bacteria metabolites are involved in the increase in the level of cholesterol is the topic of future research interest. We further believe that Amoxicillin treatment in HSD-fed mice could improve endothelial function by lowering serum triglyceride and glucose levels, which could lead to a reduction in cholesterol levels. Importantly, the treatment with Amoxicillin resulted in almost the suppression of these specific gut bacteria except for the enrichment of Enterobacteriaceae in HSD-fed mice. Amoxicillin treated HSD-fed mice however showed a slight but not significant reduction in cholesterol levels.

Our research consistently revealed the increase in creatinine levels in HSD-fed mice96. Many studies have indicated that high salt intake induces the pathophysiology of progressive impairment in renal function either through BP-dependent or independent mechanisms97,98,99. A high sodium intake impairs vascular function primarily by decreasing nitric oxide bioavailability. However, prostaglandins, adenosine, pH, PO2, and myogenic factors have been reported as major regulators of this response100,101. In addition, the metabolic factors, such as glucose and triglycerides at high level can also contribute to endothelial dysfunction by increasing oxidative stress and decreasing nitric oxide bioavailability96. Furthermore, our study found an increase in serum triglycerides and a significant increase in glucose levels in HSD-fed mice, which may result in endothelial remodelling causing adverse effects on renal hemodynamics by reducing glomerulus filtration efficiency, which in turn could increase blood creatinine levels. In our study we found that Amoxicillin treatment in HSD-fed mice caused a significant decrease in serum triglycerides and glucose toward the control values that could improve vascular endothelial function and result in an increase in the glomerular filtration rate and decrease in serum creatinine levels in mice. Here, we provide an insightful clinical finding that Amoxicillin treatment in HSD-fed mice significantly reduced plasma creatinine levels to a control range.

Consistently, our data clearly showed a decrease in plasma urea in HSD-fed mice102. According to a previous study, ammonia in the gut lumen is not the result of host urease activity, but rather of proteolytic and microbial urease activity103. Indeed, HSD caused decreased digestion of protein as well as reduced transit time in the digestive tract by modulation of specific gut bacteria7. Further, the increase of protein in urine excretion was also associated with the intake of high salt intake104. We believe the poor absorption of protein in the intestine and loss through urine could be the probable reason for a low level of blood urea in HSD-fed mice. Notably, Amoxicillin treatment in HSD-fed mice restored blood urea concentration to the control range. Similarly, Amoxicillin treatment in CD-fed mice also decreased plasma urea levels. A previous study observed that oral antibiotic administration affected urea kinetics by increasing urine excretion and increasing blood concentration of urea N by affecting N metabolism105. In the intestine, Bacteroides spp. secrete proteases at the brush border of absorptive cells, leading to higher levels of ammonia105. Our study also found an increase in the abundance of Bacteroides spp. for both HSD-fed mice and CD-fed mice that could be the probable cause of the increase in serum urea. The present study also consistently observed the effect of HSD on blood cells especially the increase in the concentration of thrombocytes in mice106. However, Amoxicillin treatment in CD-fed mice did not alter haematological parameters substantially. One study found that HSD significantly increased IL-6 production and induced the platelet production107,108. Interestingly, Amoxicillin treatment brought the elevated levels of the thrombocytes in HSD-fed mice closer to the true levels. Based on these observations, we could suggest that Amoxicillin treatment restores thrombocytes value by decreasing the level of specific cytokines such as IL-6. Accordingly Amoxicillin appears to be able to reduce the risk of clotting, stroke or heart attack to the individual on HSD. However, more advanced studies are needed to pinpoint the underlying mechanism by which Amoxicillin treatment in HSD-fed mice reduced the level of the thrombocytes to control levels.

Several studies have found that long term intake of HSD leads to obesity, and the pathophysiology of metabolic syndrome71,72,73. According to a study conducted by Dobrian, et al. on the laboratory rats by providing an HSD for a long duration caused obesity and an increasing adipocyte size109. However, we conducted a short-term study by providing HSD for a period of 3 weeks and did not find any significant gain in the weight of mice. Our results are consistent with the established observations in the field which demonstrate that a high-salt diet has been reported to cause hyperphagia in mice and humans, but body weights remained similar acutely (over 4 wks.) under ad libitum dietary conditions due to a hypercatabolic state110,111.

Many studies have provided extensive evidence that HSD consumption promotes the development of hypertrophy as well as fibrosis in the heart and kidney by overexpressing cytokines, namely transforming growth factor-1 (TGF-1)112,114,114. Also another study found that HSD cause the excess production of reactive oxygen species (ROS) which causes liver fibrosis115. This study observed only mild cardiomyocyte hypertrophy and associated tissue degenerative changes, oddly shaped cells, and a decrease in cell proliferation in the heart of the HSD-fed mice. However, these small pathological findings did not lead to any significant conclusions. Further, mice fed HSD did not develop any significant pathological changes in the kidney. Our study suggests that short term intake of HSD did not cause any significant pathological progression in the heart, liver and kidney of mice. Any significant pathological changes were not observed in the heart, liver and kidney of Amoxicillin treated HSD-fed mice. We believe that Amoxicillin treatment showed beneficial impact by reducing the minimal pathological changes observed in the heart of HSD-fed mice by inhibiting the upregulation of transforming growth factor as also reported by Melhus et al.116.

In summary, the present study showed that Amoxicillin treatment has a positive impact on a high salt diet as shown by haematology and serum biochemistry findings. There was a significant reshaping of the gut microbiota ascribed to their beneficial impact by improving the biochemical and physiological outcomes. However, this study has some limitations like the use of mouse models. Therefore, the data obtained from this study may not be directly extrapolated to humans and need to be validated. Further, we decreased the faecal samples per group and examined their relative proportions in the total bacterial population for cost-effectiveness. Taken together, our study findings provide proof of concept that Amoxicillin has a positive impact on the pathophysiological attributes of metabolic disease related inflammation collectively by improving biochemical health and gut health. We believe that long term exposure to HSD is necessary to induce the complete pathophysiology of metabolic disorders in the host. Therefore, further studies would be required to evaluate the long-term effects of antibiotic administration in mouse models of metabolic syndrome, especially in the context of diabetics.