The Influence of Gut Microbial Species on Diabetes Mellitus

Diabetes mellitus (DM) is a metabolic disorder with an alarming incidence rate and a considerable burden on the patient’s life and health care providers. An increase in blood glucose level and insulin resistance characterizes it. Internal and external factors such as urbanization, obesity, and genetic mutations could increase the risk of DM. Microbes in the gut influence overall health through immunity and nutrition. Recently, more studies have been conducted to evaluate and estimate the role of the gut microbiome in diabetes development, progression, and management. This review summarizes the current knowledge addressing three main bacterial species: Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus and their influence on diabetes and its underlying molecular mechanisms. Most studies illustrate that using those bacterial species positively reduces blood glucose levels and activates inflammatory markers. Additionally, we reported the relationship between those bacterial species and metformin, one of the commonly used antidiabetic drugs. Overall, more research is needed to understand the influence of the gut microbiome on the development of diabetes. Furthermore, more efforts are required to standardize the model used, concentration ranges, and interpretation tools to advance the field further.


Diabetes Mellitus (DM)
Diabetes mellitus is one of the main leading causes of morbidity and mortality worldwide [1]. It is a chronic metabolic disease characterized by hyperglycemia, an elevation in the blood glucose level caused by a defect in insulin secretion and/or action [2][3][4]. Diabetes is classified into three main types based on its genetics, etiology, and diagnostic criteria: type 1, type 2, and gestational diabetes [5]. Their complications in several organs, such as the heart, eyes, and kidneys, profoundly affect the patient's quality of life [6]. Depending on the kind and duration of diabetes, the symptoms may include polyuria, polyphagia, polydipsia, and weight loss [7]. Currently, oral and injectable antidiabetic drugs, insulin therapy, and lifestyle management are the primary therapeutic modalities used to treat diabetes. However, the alarmingly high rate of diabetes worldwide shows the necessity to develop new and more effective therapeutic approaches to target the disease and its complications [8].

Gut Microbiome and Diabetes
The human gut microbiome comprises 100 trillion bacterial species in the intestinal tract [9]. It is regulated by internal and external factors such as genetics, diet, and medications [10]. The gut microbiome influences the overall health status of an individual through nutrition, physiology, and immunity [11]. Disruption in the diversity of the gut microbiome is linked to multiple pathological conditions, including diabetes [12,13]. Gut dysbiosis and increased gut permeability result in the translocation of lipopolysaccharide, which can activate the innate immune system [14]. In diabetic patients, the level of lipopolysaccharide

Bifidobacterium adolescentis
Bifidobacteria are Gram-positive, non-spore-forming, and non-motile bacteria known to be the first colonizer of the infant gut [38]. Their presence in the gut has been linked to several beneficial effects on the host as they prevent intestinal inflammation, colonic adenomas, and cancer [39]. Bifidobacterium adolescentis is a vital gut flora in adults [40,41]. In patients with type 2 diabetes, the abundance of B. adolescentis in the intestine is significantly reduced [42]. Using B. adolescentis (1 × 10 8 cfu/mL) daily on twenty volunteers aged 50 to 60 for thirty days as a supplementation alleviates gut microbiome disorder and reduces blood glucose [43].
Additionally, administering eight strains of B. adolescentis (2 × 10 8 cfu/mL) for 12 weeks in diabetic mice restored gut microbiome homeostasis, alleviated inflammation, and increased the abundance of short-chain fatty acid-producing microorganisms [44]. Moreover, supplementing B. adolescentis (5 × 10 8 cfu/mL) in mice fed a high-fat diet daily for twelve weeks improved insulin sensitivity and reduced visceral fat accumulation [45]. Unfortunately, the literature lacks more data that support or challenge the observed beneficial effects of B. adolescentis administration in diabetes. Furthermore, protocol standardization is required to ensure the safety and efficacy of using such an approach.

Bifidobacterium adolescentis
Bifidobacteria are Gram-positive, non-spore-forming, and non-motile bacteria known to be the first colonizer of the infant gut [38]. Their presence in the gut has been linked to several beneficial effects on the host as they prevent intestinal inflammation, colonic adenomas, and cancer [39]. Bifidobacterium adolescentis is a vital gut flora in adults [40,41]. In patients with type 2 diabetes, the abundance of B. adolescentis in the intestine is significantly reduced [42]. Using B. adolescentis (1 × 10 8 cfu/mL) daily on twenty volunteers aged 50 to 60 for thirty days as a supplementation alleviates gut microbiome disorder and reduces blood glucose [43].
Additionally, administering eight strains of B. adolescentis (2 × 10 8 cfu/mL) for 12 weeks in diabetic mice restored gut microbiome homeostasis, alleviated inflammation, and increased the abundance of short-chain fatty acid-producing microorganisms [44]. Moreover, supplementing B. adolescentis (5 × 10 8 cfu/mL) in mice fed a high-fat diet daily for twelve weeks improved insulin sensitivity and reduced visceral fat accumulation [45]. Unfortunately, the literature lacks more data that support or challenge the observed beneficial effects of B. adolescentis administration in diabetes. Furthermore, protocol standardization is required to ensure the safety and efficacy of using such an approach. Figure 2 highlights the main pathways affected by B. adolescentis administration in diabetes.

Figure 2.
Overview illustration of Bifidobacterium adolescentis on diabetes. The figure shows the three main pathways: inflammation, insulin response, and the production of short-chain fatty acids by microorganisms. Created with BioRender.com.

Bifidobacterium bifidum
Bifidobacterium bifidum is one species of naturally occurring microbiota detected in breastfed infants [46]. It is considered a dominant resident of the gut population [47]. B. bifidum consists of 3000 genes that encode carbohydrate enzymes such as glycosyl transferases (GTs), glycosyl hydrolases (GHs), and carbohydrate esterases (CEs) [48]. This showed the ability of B. bifidum to metabolize host-derived glycans such as human milk oligosaccharides and mucin [49]. Using B. bifidum in diabetes management has started to gain more scientific attention recently. A single administration dosage of 1 × 10 7 cfu/mL daily for 28 days reduced fasting blood glucose, glycosylated hemoglobin, triglycerides (TG), and total cholesterol in Wistar rats [50]. Additionally, diabetic patients treated with a collection of probiotics, including B. bifidum (2 × 10 9 cfu/mL) daily for 12 weeks,

Bifidobacterium bifidum
Bifidobacterium bifidum is one species of naturally occurring microbiota detected in breastfed infants [46]. It is considered a dominant resident of the gut population [47]. B. bifidum consists of 3000 genes that encode carbohydrate enzymes such as glycosyl transferases (GTs), glycosyl hydrolases (GHs), and carbohydrate esterases (CEs) [48]. This showed the ability of B. bifidum to metabolize host-derived glycans such as human milk oligosaccharides and mucin [49]. Using B. bifidum in diabetes management has started to gain more scientific attention recently. A single administration dosage of 1 × 10 7 cfu/mL daily for 28 days reduced fasting blood glucose, glycosylated hemoglobin, triglycerides (TG), and total cholesterol in Wistar rats [50]. Additionally, diabetic patients treated with a collection of probiotics, including B. bifidum (2 × 10 9 cfu/mL) daily for 12 weeks, significantly decreased insulin resistance, fasting blood glucose, and increased insulin sensitivity and HDL cholesterol level. It also improved the total antioxidant capacity and reduced the C-reactive protein level [51]. The combination treatment of different Bifidobacterium spp., including B. bifidum and excluding B. adolescentis, ameliorated insulin resistance and reduced blood glucose levels in mice [52]. More studies are needed to evaluate how B. bifidum manages diabetes. Additionally, studies that address the influence of B. bifidum and B. adolescentis may be essential for better treatment outcomes. Figure 3 highlights the main pathways affected by B. bifidum administration in diabetes.
significantly decreased insulin resistance, fasting blood glucose, and increased insulin sensitivity and HDL cholesterol level. It also improved the total antioxidant capacity and reduced the C-reactive protein level [51]. The combination treatment of different Bifidobacterium spp., including B. bifidum and excluding B. adolescentis, ameliorated insulin resistance and reduced blood glucose levels in mice [52]. More studies are needed to evaluate how B. bifidum manages diabetes. Additionally, studies that address the influence of B. bifidum and B. adolescentis may be essential for better treatment outcomes. Figure 3 highlights the main pathways affected by B. bifidum administration in diabetes.

Lactobacillus rhamnosus
Lactobacillus rhamnosus was first isolated in 1983 and is known for its ability to resist stomach acidity and strong avidity for intestinal cells. It has been widely used in targeting multiple pathological conditions, such as cancer, as an effective probiotic [53]. Administering L. rhamnosus daily (1 × 10 8 cfu/mL) in rodents for four weeks improved glucose tolerance by reducing endoplasmic reticulum stress [54]. Additionally, in mice fed a highfat diet, treating 10 9 cfu/mL of L. rhamnosus daily significantly reduced the insulin level and fasting blood glucose. It also reduced proinflammatory cytokines such as IL-6 and TNF-a [55].
Furthermore, oral administration of L. rhamnosus improved glucose tolerance in diabetic rats by downregulating the expression of glucose 6 phosphatase [56]. The administration of L. rhamnosus to diabetic mice reduced insulin, glycosylated hemoglobin, and fasting blood glucose levels and increased glucagon-like peptide 1 levels in serum [57]. Similar results were obtained when 3 month old male Zebrafish were used [58]. These observations show the urgent need for protocol standardization and model specification to estimate the beneficial effect of L. rhamnosus in diabetes. Figure 4 highlights the main pathways affected by L. rhamnosus administration in diabetes. Table 1 summarizes the data available in the literature that address the influence of the species Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus on diabetes mellitus. The table includes essential data about the targeted pathway tested, the mode of administration, the effects on diabetes, the follow-up period, and the method and model used in each study.

Lactobacillus rhamnosus
Lactobacillus rhamnosus was first isolated in 1983 and is known for its ability to resist stomach acidity and strong avidity for intestinal cells. It has been widely used in targeting multiple pathological conditions, such as cancer, as an effective probiotic [53]. Administering L. rhamnosus daily (1 × 10 8 cfu/mL) in rodents for four weeks improved glucose tolerance by reducing endoplasmic reticulum stress [54]. Additionally, in mice fed a high-fat diet, treating 10 9 cfu/mL of L. rhamnosus daily significantly reduced the insulin level and fasting blood glucose. It also reduced proinflammatory cytokines such as IL-6 and TNF-a [55].
Furthermore, oral administration of L. rhamnosus improved glucose tolerance in diabetic rats by downregulating the expression of glucose 6 phosphatase [56]. The administration of L. rhamnosus to diabetic mice reduced insulin, glycosylated hemoglobin, and fasting blood glucose levels and increased glucagon-like peptide 1 levels in serum [57]. Similar results were obtained when 3 month old male Zebrafish were used [58]. These observations show the urgent need for protocol standardization and model specification to estimate the beneficial effect of L. rhamnosus in diabetes. Figure 4 highlights the main pathways affected by L. rhamnosus administration in diabetes. Table 1 summarizes the data available in the literature that address the influence of the species Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus on diabetes mellitus. The table includes essential data about the targeted pathway tested, the mode of administration, the effects on diabetes, the follow-up period, and the method and model used in each study.

Discussion
Diabetes is a global metabolic condition with a high incidence rate worldwide. Developing new and improved therapeutic approaches to target the disease and its complications is necessary. The gut microbiota has been linked recently to diabetes. Here, we searched the literature and reported the role played by the three commonly addressed microbial species on diabetes: Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus. Both animal and human studies reported the influence of Bifidobacterium adolescentis administration on blood glucose level, an abundance of short-chain fatty acids, and inflammatory response in a dose dependent manner that ranges from 1 × 10 8 to 5 × 10 8 CFU/mL. Moreover, administering Bifidobacterium bifidum (1 × 10 7 -2 × 10 9 CFU/mL) reduced fasting blood glucose, insulin resistance, and improved sensitivity in human participants with diabetes and animal models. Unfortunately, this is not the case with Lactobacillus rhamnosus as most of the available studies reported the role of this species on diabetes in animal models only. Despite that, the data support the positive influence of this species on insulin resistance and lipid profile.
Throughout the literature, we observed the lack of standardization regarding the protocol followed, the model used, the diet used to induce diabetes in animal models, and the mode of administration, as most studies followed oral or intraperitoneal administration. Establishing standardized protocols that specify specific guidelines will help further advance the field. Additionally, the literature shows that many studies investigate a single microbial species. The gut microbiome is a community of microorganisms interacting with each other and the host. Isolating and investigating a single organism only might not be of great interest. As a starting point, a study may investigate the influence of the three bacterial species mentioned in this paper on diabetes in human and animal models and report the challenges and limitations. By doing so, we can then, step by step, look at the gut microbiome as a community in the context of health and diseases. The following sections highlight some essential topics that need further discussion and research for better treatment outcomes.

Discussion
Diabetes is a global metabolic condition with a high incidence rate worldwide. Developing new and improved therapeutic approaches to target the disease and its complications is necessary. The gut microbiota has been linked recently to diabetes. Here, we searched the literature and reported the role played by the three commonly addressed microbial species on diabetes: Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus. Both animal and human studies reported the influence of Bifidobacterium adolescentis administration on blood glucose level, an abundance of short-chain fatty acids, and inflammatory response in a dose dependent manner that ranges from 1 × 10 8 to 5 × 10 8 CFU/mL. Moreover, administering Bifidobacterium bifidum (1 × 10 7 -2 × 10 9 CFU/mL) reduced fasting blood glucose, insulin resistance, and improved sensitivity in human participants with diabetes and animal models. Unfortunately, this is not the case with Lactobacillus rhamnosus as most of the available studies reported the role of this species on diabetes in animal models only. Despite that, the data support the positive influence of this species on insulin resistance and lipid profile.
Throughout the literature, we observed the lack of standardization regarding the protocol followed, the model used, the diet used to induce diabetes in animal models, and the mode of administration, as most studies followed oral or intraperitoneal administration. Establishing standardized protocols that specify specific guidelines will help further advance the field. Additionally, the literature shows that many studies investigate a single microbial species. The gut microbiome is a community of microorganisms interacting with each other and the host. Isolating and investigating a single organism only might not be of great interest. As a starting point, a study may investigate the influence of the three bacterial species mentioned in this paper on diabetes in human and animal models and report the challenges and limitations. By doing so, we can then, step by step, look at the gut microbiome as a community in the context of health and diseases. The following sections highlight some essential topics that need further discussion and research for better treatment outcomes.

The Influence of Combination Therapy on Diabetes
Diabetes is managed mainly by antidiabetic drugs such as metformin [61]. Its administration augments glucose uptake in tissues and reduces glucose output [62]. Due to its high efficacy and safety level, metformin is used as the first line of treatment in patients with type 2 diabetes [63]. Various research supports the influence of metformin on the gut microbiome [64]. In a randomized study of patients with type 2 diabetes, the administration of metformin altered the composition and function of the gut microbiome. The results also showed how metformin prompted the growth of B. adolescentis, which was associated with reduced blood glucose levels [65].
Additionally, metformin treatment altered the composition of the gut microbiome by enhancing the growth of Lactobacillus, Bifidobacterium, and Escherichia and reducing the abundance of Intestinibacter bartlettii [66]. Those reports prompted more research in the field of combination therapy and diabetes. The co-administration of metformin and B. bifidum in rats suppressed the metformin effect on feces while maintaining the antihyperglycemic effect of metformin [67]. Furthermore, the combination treatment of metformin and B. bifidum in 40 patients with diabetes for ten weeks significantly improved the gastrointestinal symptoms associated with metformin without altering the glucose control effect of the medication [68]. More studies are required to assess and evaluate those results on other bacterial species, such as B. adolescentis and L. rhamnosus. More research is needed to evaluate this approach with other antidiabetic drugs, such as sulfonylureas and meglitinides. Figure 5 highlights the influence of metformin on the three discussed bacterial species.

The Influence of Combination Therapy on Diabetes
Diabetes is managed mainly by antidiabetic drugs such as metformin [61]. Its administration augments glucose uptake in tissues and reduces glucose output [62]. Due to its high efficacy and safety level, metformin is used as the first line of treatment in patients with type 2 diabetes [63]. Various research supports the influence of metformin on the gut microbiome [64]. In a randomized study of patients with type 2 diabetes, the administration of metformin altered the composition and function of the gut microbiome. The results also showed how metformin prompted the growth of B. adolescentis, which was associated with reduced blood glucose levels [65].
Additionally, metformin treatment altered the composition of the gut microbiome by enhancing the growth of Lactobacillus, Bifidobacterium, and Escherichia and reducing the abundance of Intestinibacter bartlettii [66]. Those reports prompted more research in the field of combination therapy and diabetes. The co-administration of metformin and B. bifidum in rats suppressed the metformin effect on feces while maintaining the antihyperglycemic effect of metformin [67]. Furthermore, the combination treatment of metformin and B. bifidum in 40 patients with diabetes for ten weeks significantly improved the gastrointestinal symptoms associated with metformin without altering the glucose control effect of the medication [68]. More studies are required to assess and evaluate those results on other bacterial species, such as B. adolescentis and L. rhamnosus. More research is needed to evaluate this approach with other antidiabetic drugs, such as sulfonylureas and meglitinides. Figure 5 highlights the influence of metformin on the three discussed bacterial species.

The Influence of Flavonoids on Species Abundance
Flavonoids are natural compounds present abundantly in fruits and vegetables and exert several biological benefits, such as anticancer and anti-inflammatory properties. Our previous work extensively covers the influence of flavonoids and phytochemicals consumption on metabolic conditions such as diabetes and cancer. We also reported the relationship between the gut microbiome and flavonoid metabolism in the context of health and disease [69][70][71][72][73]. In this section, we report the influence of flavonoid consumption on the abundance of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus. An in vitro stimulated fermentation method was used to evaluate the influence of nine flavonoids-hesperidin, hesperetin-7-O-glucoside, hesperetin, naringin, prunin, naringenin, rutin, isoquercitrin, and quercetin-in 10 healthy Chinese volunteers. The results showed that the administration of hesperetin-7-O-glucoside, prunin, and isoquercitrin significantly enhanced the abundance of Bifidobacterium spp. [74]. Additionally, adding quercetin significantly increased the abundance of Bifidobacterium adolescentis in particular [75]. Interestingly, and in another report, the administration of quercetin enhanced the quantity of Lactobacillus rhamnosus while inhibiting the growth of pathogenic bacteria [76,77]. This may support the synergistic effect of the same flavonoids on the abundance of different bacterial species in the gut. Research that supports those findings in the context of diabetes is lacking in the literature. We think conducting more research in that area can provide insight into a potential new treatment/management for diabetes. Moreover, studies that evaluate the efficacy and safety of using flavonoids in combination with other antidiabetic drugs are necessary. Furthermore, the bioavailability challenge accompanying flavonoid administration may be improved if we better understand the role of the gut microbiome.

Fecal Microbiota Transplantation and Diabetes
Fecal microbiota transplantation (FMT) transfers the stool sample of a healthy participant into the colon of a patient suffering from a medical condition to restore the typical abundance and function of the gut microbiota [78,79]. The procedure is considered welltolerated and safe, with minor side effects such as abdominal cramps and diarrhea [80]. FMT has been used to treat metabolic syndrome, inflammatory bowel disease, and diabetes [81,82]. A 24-year-old patient with type 1 diabetes treated with FMT showed a graduate improvement in blood glucose level, glycosylated hemoglobin, and nutritional status. Additionally, the abundance of gut bacterial species changed after the treatment [83].
Furthermore, an open-labeled controlled trial of 13 patients with type 2 diabetes revealed that the treatment with FMT improved blood glucose levels, glycosylated hemoglobin, and the abundance of Bifidobacterium [84]. Furthermore, mice with type 2 diabetes were treated with FMT and reported an improvement in the level of insulin resistance while the level of inflammatory response was reduced. Additionally, Western blots and flow cytometry results reported inhibition of the apoptotic pathway after the FMT treatment [85]. Although none of the studies reported adverse side effects of the FMT treatment, more studies are required to assess and standardize the mode of administration, the concentration, and the safety of the procedure. Unfortunately, the literature still lacks more information that links the effect of FMT on restoring the abundance of the three bacterial species-Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus-in diabetic patients/models. Despite that, reporting the available data that support the positive influence of FMT on diabetes and gut microbiome profile, in general, may provide a roadmap for structured research in linking FMT to the three bacterial species and diabetes.

Conclusions
Diabetes mellitus is a chronic condition with a massive burden on patients worldwide. Developing new targets and management plans which can be used with the currently used treatment is essential. The gut microbiome has been recently used in diabetic research. Throughout our study, we observed a lack in the literature of data that addresses specific bacterial species and their correlation with diabetes. Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus rhamnosus are the most commonly addressed bacterial species with diabetes in the literature. Those bacterial species were reported to reduce the biochemical parameters of diabetes and improve its complications.
Unfortunately, the field still lacks standardization in the protocol followed, the models used, and the interpretations. Furthermore, more efforts are required to address the available online Atlases that discuss gut microbiome causality without solid evidence. Generally, the gut microbiome field will be essential in futuristic treatments, primarily when combined with other therapeutic options. However, more research is needed to evaluate the safety and efficacy of this proposed approach.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the article.