Open access peer-reviewed chapter

Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases Related to the GIT

Written By

Luís Cláudio Lima de Jesus, Fernanda Alvarenga Lima, Nina Dias Coelho-Rocha, Tales Fernando da Silva, Júlia Paz, Vasco Azevedo, Pamela Mancha-Agresti and Mariana Martins Drumond

Submitted: 15 June 2019 Reviewed: 01 July 2019 Published: 13 December 2019

DOI: 10.5772/intechopen.88325

From the Edited Volume

The Health Benefits of Foods - Current Knowledge and Further Development

Edited by Liana Claudia Salanță

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Abstract

Many diseases that affect the gastrointestinal tract (GIT) have great influence on the quality of life of the majority of patients. Many probiotic strains are being highly studied as a promising candidate due to their beneficial effect reported in the GIT. With the purpose of increasing the beneficial characteristics of some probiotics strains and, consequently, to improve further the reported results, many probiotic strains expressing or encoding different proteins, with anti-inflammatory activities, have been developed. These recombinant strains have been reported as good candidates for the treatment of different pathological conditions, especially colitis and mucositis disease since they have been shown to have positive results and good perspectives for GIT inflammation. Thus, this chapter will first address the aspects of the gastrointestinal tract in humans as well as its microbiota. In a second moment, it will discuss about chronic diseases, mainly the intestinal ones. Finally, it will discuss about probiotics, especially concerning on lactic acid bacteria (LAB), and its action in the prevention and treatment of these diseases. At the final part, we will point out aspects on the development of recombinant strains and the results found in the literature on disease models.

Keywords

  • L. lactis
  • Lactobacillus
  • DNA vaccine
  • heterologous protein

1. The human gastrointestinal tract

The human gastrointestinal tract is formed by a complex ecosystem which includes the gastrointestinal epithelium, immune cells, and resident microbiota [1] and comprehends one of the biggest existent interfaces between the host, environmental factors, and antigens in the human body.

The intestine encompasses a broad variety of microorganisms (bacteria, archaea, eukarya, and viruses) [2] from more than 3500 different species [3, 4] that coevolved with the host in a mutually beneficial relationship [5, 6]. The composition and density of bacterial populations in adult individuals differ considerably over the GIT. The area of the GIT that has highest microorganism abundance is the colon (1014) followed by dental plaque (1012), ileum (1011), saliva (1011), and skin (1011) [7]. However, low concentrations (up to 102–107 cells/mL) and bacterial diversity are found in the upper GIT (stomach, duodenum, jejunum) [3, 4], since the presence of acid, bile salts, and pancreatic secretions hinders the bacterial colonization [8], so that there is no nutritional competition between the microbiota and the host [9]. Thus, both function and structure of microbial communities are significant and are closely related. However, function could be the more important measure of microbiome health, since bacterial ecology suggests that analogous ecosystems have similar function although they have moderately diverse composition [10, 11].

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2. Gut microbiota

The importance and the specific functions that gut microbiota has in human nutrition and health are well settled. The attributed functions can be classified in three classes: metabolic, protective, and trophic [12]. The gene diversity of the microbial community provides a variety of enzymes and biochemical pathways, specific to the host, able to contribute to short-chain fatty acid (SCFA) production by carbohydrate fermentation and production of some vitamins such as K, B12, biotin, folic acid, and pantothenate. These factors added to synthesis of amino acids from ammonia or urea contributing to the metabolic function of the microbiota [13, 14].

The gut microbiota’s protective function is related to barrier effect, once the resident bacteria generate a resistance line which avoid pathogens/opportunistic bacteria and maintain normal mucosal function. The activity of some bacteria to secrete antimicrobial substances, such as bacteriocins, is able to inhibit the growth of other bacteria and nutrient competition [15, 16].

Regarding trophic functions of gut microbiota, the interaction between resident microorganisms has influence in differentiation and proliferation of epithelial cells [17], as well as in the development and regulation of the immune system by numerous and varied interactions between microbes, epithelium, and gut lymphoid tissues [18].

It is important to highlight that the interactions between the gut microbiota and the host immune system are required to preserve the gut homeostasis [19, 20, 21]. When this relationship is affected, alterations in bacterial function and diversity lead to the imbalance in the composition of the resident microbiota, favoring either the growing of pathogenic bacteria or the decreasing in beneficial bacteria in a process known as dysbiosis [22], which appoint a great threat to gut integrity and is intrinsically related to the development and progression of several diseases, such as inflammatory bowel diseases.

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3. Chronic inflammatory diseases

One of the most well-characterized chronic inflammatory diseases that mainly affect the digestive tract is inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD). The exact etiology of IBD is still unclear, but the strict relation between genetic and the environmental factors, such as enteric immune dysregulation and alterations in the intestinal microbiome [23, 24], is broadly known. Besides, these diseases generate substantial morbidity and have a high prevalence in developed countries (5 in 1000 individual are affected) they remain to increase in developing nations [25].

Both diseases, UC and CD, present different pathogenesis, symptomatology, inflammatory profiles, and gut microbiota composition. CD is characterized by the irregular transmural inflammation (extending deeply into the submucosal regions) which can affect any portion of the GIT and often made difficult by strictures, abscesses, and fistulae. On the other hand, the inflammation presented in UC is restricted to the superficial layers of the intestinal mucosa characterized by mucosa erosion and/or ulcer, generally localized in the region of the gut most colonized by bacteria, the colon [26, 27]. In addition, regarding the immune response associated with these diseases, it is possible to relate CD with an increased IL-12, IL-23, IL-27, interferon γ (IFN-γ), and tumor necrosis factor-α (TNF-α) production, all associated with Th1 and Th17 immune responses, different from UC which is correlated with a Th2 immune response, with high levels of IL-5 and transforming growth factor-β (TGF-β) production [28].

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4. IBD complications and microbiota manipulation

It is important to highlight that the principal cause of death in IBD patients is colorectal cancer (CRC) [29]. Frequent episodes of inflammatory process in the intestinal mucosa are related to development of this disease, which is the second most frequently identified cancer in females and the third in males.

There are increased evidences that environmental factors such as lifestyle and diet alterations have effect in CRC incidence [30]. This effect has been documented because there is evidence showing an essential relationship between dietary antigens and antigens of commensal bacteria with the regulatory T cells (Tregs), which maintain the immune tolerance and, consequently, reduce the risk of tumorigenesis associated with inflammation [31].

In this context, it was reported that the higher consumption of diet rich in grains and vegetables decreases the incidence of CRC. This effect involves different mechanisms such as the diminution in the fecal transit time due to the increase in the stool bulk, and consequently, it reduces the contact of carcinogen with colon cells and the fermentation of these fibers of colonic components [14, 32]. In addition, significant reduction in concentration of acetate, propionate, and butyrate with increase in fecal pH [33] and the decrease in the number of obligate anaerobe microorganisms have been reported in individuals with colon cancer [34] when compared with healthy people. Thus, intestinal environmental alterations are the keys to evolution toward adenoma and afterward to CRC progression [35].

It has been also reported that up to 30% of patients with UC need surgical management such as the restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) [36]. This procedure removes the entire colon and rectum while preserving the anal sphincter and, hence, normal bowel function and fecal continence, therefore acting as an internal pelvic place for intestinal contents [37]. Around 50–60% of UC patients with following IPAA develop inflammation in the ileal pouch, generating the condition called “pouchitis.” The reported incidence of pouchitis is variable, generally because of the diagnostic criteria that have been used to define this syndrome [38, 39]. In addition, although its pathogenesis is uncertain, the main hypothesis for the mechanism by which the disease occurs is the break in the mucosal barrier generated by dysbiotic microbiome in susceptible patients, generating an unusual mucosal immune activation [40]; still the disease typically responds to antibiotics.

Corresponding to the increased attention given to the role of the intestinal microbiota in a variety of diseases, there has been an intense exploration of potential means to manipulate the intestinal microbiome either by probiotic administration or fecal microbiota transplant (FMT) for therapeutic effect [41].

In this context, a randomized clinical trial based on a 1-week treatment with anaerobically prepared donor FMT, compared with autologous FMT, resulted in a higher probability of remission in 8 weeks for patients with UC, revealing that stool administration from healthy donors to UC or CD patients is an intervention that seeks to restore a healthier balance of gut microbes and control IBD [42]. Data on FMT for Crohn’s disease is rather more limited than for UC, but it has been shown that single standardized FMT resulted in a clinical remission sustained for more than 9 months in CD patients [43]. However, the authors suggest that further studies are needed to enhance the knowledge about the use of stool transplantation for IBD treatment.

Alteration in the gut microbiome composition with increase in some groups of microorganisms, such as Clostridium and Fusobacterium, was also reported in patients with pouchitis [44, 45]. In this context, literature evidences indicate that the probiotic administration such as VSL#3 is effective in the chronic pouchitis prevention [46]. On the other hand, FMT to pouchitis treatment did not report the same beneficial results. Only three reports with this approach [47, 48, 49] exposed that neither clinical remission nor any adequate response was observed in the evaluated patients suggesting that the efficacy of FMT for pouchitis after proctocolectomy is limited [49]. The importance of standardization of this procedure needs to be highlighted to improve its efficacy, since frequency, route of administration (e.g., endoscopy, nasogastric tube, colonoscopy), and the criteria of choice of healthy donor are very important parameters to be considered.

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5. Intestinal mucositis

Different chemotherapy regimens such as FOLFOX (5-fluorouracil and oxaliplatin), FOLFIRI (5-fluorouracil and irinotecan), and triple FOLFOXIRI regimen (5-fluorouracil, oxaliplatin, and irinotecan) [50, 51] are adopted for different types of cancer but with a broad range of collateral effects.

Mucositis is the most common side effect in patients undergoing chemotherapy/radiotherapy treatments, which consist in an inflammation and/or ulcers in the gastrointestinal tract [52] with consequent loss of cells from the epithelial barrier of the GIT. Many symptoms are related to gastrointestinal mucositis, such as diarrhea, severe abdominal pain, bleeding, fatigue, malnutrition, dehydration, electrolyte imbalance, and infections, with potential fatal complications which can conduce to reduction or interruption of antitumor treatment [53] and consequently leads to longer hospitalization.

This pathology occurs due to cytotoxic effects of anticancer drugs/radiotherapy that cause damage at the DNA of stem cell (epithelial cell progenitors) with intense oxidative stress and consequent cell death. This apoptotic process is exacerbated affecting the absorption by shortening the villi structure of enterocytes and causing the loss of epithelial barrier with an invasion of inflammatory cells (neutrophils, eosinophils, and macrophages) leading to an increased production of inflammatory mediators at the mucosal area with consequent epithelial erosion and ulceration. The progressive destruction of mucosal integrity causes the rupture of the tight junctions proteins, leading to an increase in the intestinal permeability with subsequent penetration of commensal microbiota to the submucosal layer generating bacteria translocation which exacerbates the inflammatory process and intensifies the symptoms [53, 54, 55, 56, 57]. Besides, the intestinal microbiota composition is also modified by the chemotherapeutic drugs and radiotherapy action [54, 58, 59] resulting in dysbiosis. After the end of treatment, recovery and restoration of the GIT structure occur [60].

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6. Metabolic syndrome

Besides IBD and mucositis, it has been reported that intestinal microbiota has an intrinsic effect on metabolism, potentially contributing to several features of the pathophysiology of metabolic syndrome [61, 62]. The metabolic syndrome is an accumulation of various risk factors (glucose intolerance, hyperinsulinemia, hypertension, as well as dyslipidemia) which can often be associated with insulin resistance, hypertension with abdominal fat accumulation, and obesity [63, 64, 65].

The etiology of metabolic syndrome is not well-defined; however there are evident characteristics and life habits that could contribute to its development such as unbalanced diet, smoking, lack of physical activity, and the genetic predisposition [66]. These factors directly increase the risk of cardiovascular disease and chronic diseases as type 2 diabetes mellitus and obesity, and the interaction between components of both the clinical and biological phenotypes of the syndrome contributes to the development of a pro-inflammatory state [67].

The inflammatory process observed in MS is directly associated with increased oxidative stress. The reactive oxygen species (ROS) are capable of mediating symptoms of diabetes mellitus, such as insulin resistance and decrease in insulin secretion, and attend as precursors for the formation of LDLox (oxidized low-density lipoproteins), responsible for a large part of the development of atherosclerotic lesions, and the increase in circulating cholesterol fractions and glucose [68, 69]. In addition, chronic diseases are directly related to changes in the intestinal microbiome [70, 71], and they are also associated with elevated circulating levels of pro-inflammatory cytokines such as TNF and IL-6 [72].

The probiotic use in attenuating symptoms of different inflammatory diseases is widely reported in the literature. Among the commercial probiotics studied for treatment of these diseases, only a few products have been extensively tested in clinical trials in patients with MS, in order to demonstrate an effective result on weight loss, lipid metabolism, and reduction of inflammatory markers.

Studies performed with Lactobacillus strains have shown the ability of these probiotics in reducing the lipid accumulation in adipose tissues, as well as in inducing the subexpression of lipogenic genes [73, 74]. Animals that received diets with high concentrations of lipids and then treated with L. gasseri SBT2050 had shown lower intestinal permeability and bacterial translocation, as well as reduction of inflammatory parameters, suggesting that this strain improves the intestinal barrier function [75, 76, 77, 78]. In addition, L gasseri BRN17 was studied to treat animals with MS caused by the carbohydrate-rich diet consumption. This strain reduced the accumulation of adipose tissue in mice, and it has a beneficial effect on weight loss [79, 80, 81]. Another important approach with associated probiotics (Bifidobacterium, Lactobacillus, and S. thermophilus) for treatment of overweight patients has shown an improvement in lipid profile, as well as insulin sensitivity [82]. Besides, recently Hsieh e collaborators [83] demonstrated that administration of live Lactobacillus reuteri ADR-1 and killed Lactobacillus reuteri ADR-3 strain ameliorated type 2 diabetes mellitus in a clinical trial. The results indicated that the consumption of ADR-1 displayed a reduction effect on serum glycated hemoglobin (HbA1c), triglyceride, and cholesterol levels. On the other hand, the intake of ADR-3 showed a beneficial effect on blood pressure reduction. Besides, a reduction in the levels of pro-inflammatory cytokines (IL-1β), increase in antioxidant enzyme (superoxide dismutase), and the changes in intestinal microflora composition (increase in intestinal level of Lactobacillus spp. and Bifidobacterium spp. and decrease in Bacteroidetes) were observed. Thus, these strategies highlight the beneficial and potential effect of interventions targeting gut microbiota modulation by the use of probiotic strains to treat components or complications of metabolic syndrome.

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7. Functional foods

The human being for more than 4000 years has been consuming fermented products, by the fermentation process. At the beginning this practice was done to preserve foods from either physical, chemical, or microbial alterations. The microorganisms participating in this process are the lactic acid bacteria, extensively widespread in nature and also belong to the GIT communities, able to convert the sugar in lactic acid as well as produce other metabolites which contribute to food modifications, either sensorial or nutritional value. Thus, the terminology “functional food” was attributed to food with health benefits to the consumer including nutritional and physiological function [84, 85, 86].

During the fermentation, these bacteria can contribute to improving the digestion of nutrients (lactose, proteins, small peptides, and polysaccharides); providing essential micronutrients (vitamins) as well as bioactive compounds (metabolites) with potential health benefits to the host, such as prevention against enteric inflammation [87, 88]; providing antimicrobial, antihypertensive, hypocholesterolemic, immunomodulatory, antioxidant, and anticancer effects [46, 85, 89, 90, 91, 92]; showing ability to regulate the immunity; and, consequently, improving host quality of life [93].

Therefore, the gut communities and the microbial-derived molecules present in the gut lumen have been strongly influenced, either qualitatively or quantitatively, by consumption of dairy products [94] such as yogurts, cheeses, and fermented milk, among other fermented products using probiotic bacteria. Thus, the microbiota manipulation by functional food, probiotics, and prebiotics are evaluated as a beneficial option for treatment of GIT diseases [95].

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8. Lactic acid bacteria: the largest group of probiotic bacteria

There is a constant interaction between the host and the bowel commensal bacterial community in order to maintain the homeostasis [3, 96, 97, 98]. However, when this mutualist relationship is compromised, the intestinal microbiota may cause and/or contribute to either the establishment or the progression of inflammatory diseases [96, 97, 98, 99]. In this context, the search for therapeutic strategies that minimize the development and progression of pathologies caused directly and indirectly by the unbalance of the commensal microbiota has grown. The consumption of probiotic bacteria is one of these strategies, as they present several effects, such as ability to improve the intestinal barrier, stimulate the systemic and mucosal immune system, regulate the composition of the intestinal microbiota, and provide essential micronutrients (such as vitamins and SCFAs) and other bioactive compounds (metabolites) with potential health benefits for the host [100, 101, 102, 103].

Probiotics are defined as “live microorganisms that offer host health benefits when administered in adequate amounts” [104, 105]. The majority of the studied probiotics belongs to the group of lactic acid bacteria. However, other microorganisms with probiotic properties also deserve attention, such as yeasts (Saccharomyces spp.) and bacteria of the genus Bifidobacterium and Faecalibacterium, among others [106, 107, 108].

LAB, which include, mainly, species from the genus Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, and Streptococcus, constitute a group of Gram-positive, anaerobic or aerotolerant, nonspore-forming, nonmobile, and highly low pH-tolerant microorganisms. However, the main characteristic of this group is its ability to produce lactic acid as the final product of the fermentation of carbohydrates [109, 110, 111].

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9. Probiotic effects in gastrointestinal inflammation

LAB are often present in the human gut but also can be introduced by the ingestion of fermented foods, such as yogurt and other fermented milk products and fermented cured meat by-products [103], having the generally recognized as safe (GRAS) status by the Food and Drug Administration (FDA). Lactobacillus spp., Streptococcus spp., and Lactococcus spp. are the major LAB species with probiotic effects, and they have been used in therapeutic applications for treatment and prevention of various intestinal disorders [112, 113].

Scientific evidence reveals that the mechanisms by which probiotic bacteria ameliorate inflammatory bowel damage are heterogeneous, strain specific, and dependent on the number of available bacteria. Thus, administration of probiotic bacteria, specially LAB, improves intestinal inflammatory responses by (i) modulation and normalization of perturbed intestinal microbial communities; (ii) competitive exclusion of pathogens such as Staphylococcus aureus and Salmonella typhimurium, among others; (iii) bacteriocin and SCFA production; (iv) enzymatic activities related to metabolization of a number of carcinogens and other toxic substances; (v) adhesion to mucosal cells, cell antagonism, and mucin production; (vi) intestinal permeability reduction by tight junctions protein modulation (e.g., zonulin, claudin, occludin, junctional adhesion molecule); (vii) modulation of the immune system by stimulating Tregs cells, IgA production by B cells, and NF-kβ signaling pathway inhibition; and (viii) interaction with the brain-gut axis via the generation of bacterial metabolites ( Figure 1 ) [103, 114, 115, 116, 117, 118].

Figure 1.

A schematic diagram about potential action mechanisms of probiotic bacteria.

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10. Recombinant LAB probiotics

In order to potentialize the beneficial effects of probiotic strains, research has been conducted over the last decades, based on genetic engineering techniques, especially those related to DNA manipulation. Thus, modern methods of genetic engineering open the new opportunities to design and create genetically modified probiotic strains with the desired characteristics or to exclusively target a specific pathogen or toxin to be used either as a vaccine or for drug delivery [119, 120]. Since most of the probiotic strains are part of the LAB group, most of the genetic manipulation studies are carried out with species that belong to this group, such as Lactococcus and Lactobacillus genera. Consequently, recombinant probiotics have been created for mucosal delivery of therapeutic and/or prophylactic molecules comprising DNA, peptides, single-chain variable fragments, cytokines, enzymes, and allergens [121, 122], leading to the concept of “biodrug” for the prevention and treatment of various diseases [123]. Thus, researches have emphasized the use of species of these genera in two different approaches: the first as producers of heterologous protein and the second as vehicle for delivery of DNA vaccines [124].

10.1 LAB as producers of heterologous protein

Many studies are carried out with Lactococcus lactis due to its economic importance in the production of cheese and its easy growth and manipulation. In addition, it was the first species of LAB to have its genome completely sequenced, which allowed a greater understanding of its genetic and physiological mechanisms, aiding in the development of technological packages for its genetic manipulation in a laboratory environment [124, 125, 126, 127, 128].

There are several ways to make LAB produce heterologous proteins, and the most used form is through the insertion of a plasmid into its cytoplasm. Plasmids are elements of extrachromosomal DNA that are naturally found in prokaryotes. With the advent of the recombinant DNA technique, these elements have been manipulated to act as molecular vehicles that allow the production of proteins of interest by the bacterium [129].

The first heterologous protein production system based on plasmid insertion in LAB was developed for L. lactis. These systems included both inducible and constitutive promoters, which ensure efficient expression of the antigen of interest under different conditions [130, 131]. Although it is possible to choose the type of promoter to be used in the vector, the vast majority of expression vectors present inducible promoters that allow controlled expression of the protein of interest by protecting against aggregation and protein degradation in the bacterial cytoplasm. On the other hand, these vectors present safety issues that need to be analyzed since it is necessary to introduce chemical compounds into the culture medium to induce protein expression prior to animal administration [132, 133, 134].

With the improvement of cloning and expression techniques, several production systems were developed, specifically for LAB, allowing the production of different molecules of interest, including pathogen antigens, by a large number of LAB species [135, 136, 137, 138, 139]. The most commonly used regulation systems in LAB are the following:

10.1.1 Nisin-controlled gene expression (NICE)

Among the heterologous production systems, the most widely studied is the nisin-controlled gene expression system. This system is based on the expression of three genes (nisA, nisF, and nisR) that are involved in the production and regulation of the antimicrobial peptide nisin, which is naturally secreted by different strains of L. lactis. In this system the membrane-located histidine kinase NisK senses the signal inducer nisin and autophosphorylates and then transfers the phosphorous group to the intracellular response regulator protein NisR which acts as a transcription activator of nisA/nisF and induces gene expression under pNis promoter. Depending on the presence or absence of the corresponding targeting signals, the protein is either expressed into the cytoplasm or the cell envelope or secreted into the external medium [140]. Thus, it has already been successfully used for the expression of different proteins of medical and biotechnological interest [141, 142].

10.1.2 Xylose-inducible expression system (XIES)

In 2004, Miyoshi and colleagues [143] developed the xylose-inducible expression system whose promoter is the xylose permease gene (pxylT) found in L. lactis NCDO2118. This system produces either cytoplasmic or secreted proteins being activated in the presence of xylose and strongly repressed in the presence of glucose, fructose, or mannose [143].

10.1.3 Stress-inducible controlled expression system (SICE)

More recently, the stress-inducible controlled expression system was developed using the L. lactis groESL promoter [134]. This system induces expression of proteins of interest via stress stimuli such as those found in the GIT (e.g., bile salt, acid pH, antimicrobial peptide, and heat shock proteins) [134, 144]. This system does not require the induction of bacterial culture or the presence of regulatory genes, being a good alternative in the delivery and production of therapeutic proteins at mucosal surfaces.

10.2 LAB as a live vehicle to deliver DNA vaccine plasmids to eukaryotic cells

Among the available approaches to stimulate efficient mucosal responses, the use of bacterial system for DNA delivery and its expression using the eukaryotic cell machinery have been extensively explored. Unlike the production of heterologous protein, in which the bacterium is responsible for the synthesis of the protein of interest, in the DNA vaccine platform, the bacteria only act as a delivery vehicle for prophylactic and therapeutic purposes [109, 145].

New vectors had been developed to approach the DNA vaccine using LAB as live delivery vehicles [146, 147, 148, 149, 150]. These vectors present a series of common characteristics such as the presence of a eukaryotic promoter, which allows protein expression by eukaryotic cells; a prokaryotic region, which has a selection marker (usually antibiotic resistance); a multiple cloning site, where the open reading frame (ORF) of interest will be inserted; and a prokaryotic origin of replication, which ensures that the plasmid replicates only in prokaryotic cells [151]. Some molecules (IL-10, IL-4, and HSP65) have been cloned in these vectors to evaluate their effect, especially as a treatment approach in diseases related to the bowel [152, 153], as well as reporters (GFP and Cherry) which allowed the understanding of this platform in the mammalian body [148, 154]. Although further studies need to be conducted in order to elucidate whether the cloning of ORFs of interest in these vectors is really effective pointing to disease prevention and treatment, this approach is undoubtedly an important tool for the development of new techniques with potential in the medical clinic.

11. Next-generation recombinants: using CRISPR-Cas system

Among the different techniques used to construct recombinant LAB strains, the most recent is associated with the use of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, based on the use of a system present in several bacterial strains that works as part of the adaptive immune system of bacteria and archaea against the presence of external DNA, such as plasmids and bacteriophages [155, 156, 157, 158, 159].

Although this system has been studied for more than 30 years [160], it was only in 2013 that the first experiments were carried out emphasizing its use as a tool for genome editing [161, 162]. Evaluating the CRISPR databases, it is possible to observe that about 46% of all bacterial genomes presents the CRISPR-Cas system, and this percentage reaches approximately 63% of the sequenced Lactobacillus genomes [163]. The natural presence of this system in most of the LAB strains expands the possibilities of genetic manipulation of microorganisms of this group, including probiotic ones [164].

The first gene editing experiment in LAB based on the CRISPR-Cas system was conducted by Oh and van Pijkeren [165] where they were able to edit three different regions of the genome, with efficiency up to 100% in the selected clones. After this pioneering work, few others were published focusing on LAB gene editing [166, 167, 168].

Therefore, the use of this technology is presented as a widely viable strategy to be applied in LAB, enabling the development of food-grade recombinant strains in order to allow their future use in the clinic [169].

12. Use of recombinant LAB to treat GIT-related disorders

The use of recombinant L. lactis strains, as well as others recombinant LAB strains, using different systems has shown promising results in many studies as an alternative therapy to treat, especially, GIT inflammation and other diseases ( Table 1 ).

Microorganism Gene Expression System Inflamation Condition Anti-Inflamatory Properties References
L. lactis MG1363 Mouse IL-10 SICE Mouse model of DNBS-induced colitis Restoration of intestinal architecture; IgA production and IL-6 reduction; Reduced tissue damage [134]
L. lactis MG1363 Mouse IL-10 and IL-4 pValac vector Mouse model of DSS/TNBS-induced colitis Decreased IL-6, IL-12 and MPO activity
Reduced tissue damage
[152, 153]
L. lactis NZ9000 Mouse TGF-β1; IL-10
and leukocyte protease inhibitor
Human Elafin
NICE Mouse model of DSS-induced colitis Reduced tissue damage
Decreased pro-inflammatory cytokines
[174]
L. lactis NCDO 2118 Human 15-lipoxygenase-1 XIES Mouse model of DSS-induced colitis Reduced tissue damage [175]
L. lactis NCDO 2118 M. leprae Hsp65 protein XIES Mouse model of DSS-induced colitis Restoration of intestinal architecture
CD4+Foxp3+ and CD4+LAP+ regulatory T cells production
[176]
B. bifidum BS42 Mouse IL-10 BEST Mouse model of DNBS-induced colitis Reduced tissue damage [177]
L. casei BL23 Superoxide dismutase A from L.lactis MG1363
Catalase from L.plantarum ATCC
pLEM415 vector Mouse model of TNBS-induced Crohn’s disease Reduced tissue damage
Reduced microbial translocation
Increase IL-10/INF-γ reduction
[180]
S. thermophilus CLR807 Superoxide dismutase A from L.lactis MG1363
Catalase from L.plantarum ATCC
pIL253 vector Mouse model of TNBS-induced colitis Reduced tissue damage
Reduced microbial translocation
IL-17 reduction
[181]
L.lactis AG013 Human Trefoil Factor 1 (Htff-1) ThyA native promoter of L.lactis Hamster model of radiation-induced oral mucositis Reduced clicnical scores of oral mucositis [186]
L. lactis NZ9000 Human pancreatitis associated protein (Reg3A) NICE Mouse model of 5-FU-induced intestinal mucositis Microbiota Regulation
Villus architecture preservation
Increased Paneth cells activity
[185, 187]
L.lactis NCDO2118 M. leprae Hsp65 protein XIES Mice model of experimental encephalomyelitis Increased CD4+Foxp3+ regulatory T cells
Reduced encephalytogenic CD4+ T cells
[184]
L.lactis MG1363 Mouse IL-17 SICE Mice model HPV-induced cancer Reduced tumor size
Induced IL-6 and IL-17 secretion
[182]
L.lactis NZ9000 M. leprae Hsp65 protein and peptide derived of human Hsp60 protein NICE Mice model of diabetes type 1 Reduction of insulitis
Inhibition of T cell proliferation
[183]

Table 1.

Protein with anti-inflammatory properties produced in different strains of bacteria.

To arrive at mucosa in sufficient quantities to exert their therapeutic effects, many LAB strains must survive, during their passage through the GIT, stressor factors such as pH, temperature, bile salt concentration, and the presence of antimicrobial peptides [170, 171, 172]. In this context, an interest approach was recently developed by Coelho-Rocha and colleagues [154] using an encapsulated recombinant strain (L. lactis pExu:mcherry) and tested it through the GIT at different times post-administration. They have shown that the microencapsulation process is an effective method to improve DNA delivery, guaranteeing a greater number of viable bacteria able to reach different sections of the bowel [154].

The use of recombinant probiotics to improve therapeutic approaches has been widely studied using different systems with different molecules. As IBDs are a serious clinical topic, many strategies have been tested trying to improve previous results found with wild type strains.

L. lactis MG1363 strain carrying the pTREX1 vector expressing the mouse IL-27 protected mice against the inflammatory effects of dextran sulfate sodium (DSS)-induced colitis. This recombinant strain was able to reduce disease activity scores and pathology features of the large and small bowels and also led to reduced levels of inflammatory cytokines IL-1β, TNF-α, and IFN-γ in colonic tissue. In addition, reduction in the number of CD4+ and IL-17+ T cells in gut-associated lymphoid tissue and increase in IL-10 production were observed [173].

Besides, it was also demonstrated in a DSS-induced colitis mouse model that the oral administration of L. lactis NZ900 strain harboring the NICE system expressing either the anti-inflammatory cytokine IL-10, TGF-β1, secretory leukocyte protease inhibitor (SLPI), or elafin was able to ameliorate some clinical parameters in inflamed mice. Even though it was possible to observe the reduction of weight loss and diarrhea, microscopic colonic damage scores, colon thickness, and myeloperoxidase (MPO) activity, the authors reported that treatments with recombinant L. lactis strain delivering either SLPI or elafin were more efficient to reduce signs of colitis than treatments with anti-inflammatory cytokines. Altogether these recombinant strains display anti-inflammatory effects in inflamed mice [174].

Approaches using the invasive L. lactis MG1363 FnBPA+, by expressing the FnBpA protein at their surface and carrying the pValac eukaryotic expression vector coding either the IL-10 cytokine [rL. lactis FnPBA+ (pValac:il-10)] or the IL-4 cytokine [rL. lactis FnPBA+ (pValac:il-4)] in DSS or trinitrobenzenesulfonic acid (TNBS)-induced acute model of colitis, respectively, were also investigated. The administration of L. lactis FnPBA+ (pValac:il-10) recombinant strain was capable to reduce the intestinal inflammation by increasing IL-10 levels and sIgA production, accompanied by decreasing IL-6, as well as the restoration of intestinal architecture of mice colon [153]. Besides, the engineered L. lactis FnPBA+ (pValac:il-4) was able to slump the level of pro-inflammatory cytokine (IL-12, IL-6) and myeloperoxidase activity and increase levels of IL-4 and IL-10, consequently decreasing the colitis harshness [153].

The human 15-lipoxygenase-1-producing L. lactis NCDO2118 harboring the xylose-inducible expression system (pXylt:CYT:15-LOX-1) was also effective in attenuating the symptoms of DSS-induced colitis in a murine model [175]. Its oral administration improved the body weight, decreased pro-inflammatory cytokines (IFN-γ and IL-4) while increasing the anti-inflammatory cytokine IL-10, and, consequently, ameliorated the macroscopic damage scores associated with the inflammation.

The oral pretreatment with genetically modified L. lactis NCDO2118 able to secrete HSP65 protein from Mycobacterium leprae, using XIES system (pXylt:SEC:hsp65), prevented DSS-induced colitis in C57BL/6 mice [176]. This protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; it also increased IL-10 production in colonic tissue and expansion of CD4+FoxP3+ and CD4+ latency-associated peptide (LAP+) regulatory T cells in the spleen and mesenteric lymph nodes. Besides, the authors showed that this effect was dependent on IL-10 and toll-like receptor 2 (TLR-2) [176].

Although L. lactis represents an excellent candidate for a live mucosal vector delivery system, other bacteria have also been explored as promising live vehicles for molecule expression with therapeutic properties, such as Lactobacillus, Bifidobacterium, and Streptococcus. In this context, Mauras et al. [177] using the new Bifidobacteria Expression SysTem (BEST) allowing the production of IL-10 in Bifidobacterium bifidum BS42(pBESTExp4:il-10 and pBESTBL1181:il-10) demonstrated that the use of these recombinant strains in a DNBS-induced colitis model showed its ability to decrease local inflammation and confirmed therefore its potential for delivery of therapeutic molecules in the colon.

It is well known that IBD is associated with oxidative stress by the increase in concentration of reactive oxygen species in the GIT and impaired antioxidant defenses [178, 179]. In this context, it has been shown that some probiotic LAB strains may play a protective role in IBD by expressing antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) [180, 181].

LeBlanc et al. and Del Carmen et al. [180, 181] showed, respectively, that L. casei BL23 and S. thermophilus CRL807 transformed with two different plasmids (pLEM415:mnkat; pLEM415:sodA) (pIL253:sodA and pIL253:mnkat) harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes exhibited anti-inflammatory activities in a mouse model of Crohn’s and colitis disease induced by trinitrobenzenesulfonic acid (TNBS). The authors observed a reduction in weight loss, fewer liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the IFN-γ/IL-10 [180] and IL-10/IL-17 [181] cytokine production in the large intestines of mice treated with either CAT- or SOD-producing lactobacilli/streptococci.

The stress-inducible controlled expression (SICE) system represented by L. lactis MG1363 strain harboring the pLB333 plasmid was developed to avoid the external induction of culture before the host administration [134]. Several interesting molecules were cloned in this system such as IL-10 [134] and IL-17 [182], and the effect of L. lactis secreting them was evaluated in mice models. L. lactis (pSICE:il-10) was tested in a DNBS-induced colitis mice model, resulting in a significant reduction in colitis parameters with improvement in weight loss and a decrease in macroscopic scores [134]. The intranasal administration with L. lactis secreting IL-17A (pSICE:il-17), in a mice model of human papilloma virus (HPV)-induced cancer, was able to reduce tumor size and induce IL-6 and IL-17 secretion in reactivated splenocytes from mice challenged with the tumoral cell line [182]. Both works confirmed the potential use of L. lactis harboring the SICE system to deliver interesting molecules either to colitis or colon cancer patients [134, 182].

Although many studies have focused on the use of recombinant bacteria for the treatment of IBDs, as was previously discussed, the use of recombinant probiotic strains expressing/delivering therapeutic molecules has been explored for treatment or prevention of other diseases such as mucositis, cancer, obesity, multiple sclerosis, and diabetes [182, 183, 184, 185].

An in vivo study reported by Caluwaerts et al. [186] showed that recombinant L. lactis AG013 secreting human trefoil factor 1(hTFF-1) was able to reduce the severity and course of radiation-induced oral mucositis. Carvalho et al. [187] also demonstrated that a recombinant strain of L. lactis NZ9000 using the inducible NICE system to express the human pancreatitis-associated protein (PAP) was able to prevent 5-FU-induced intestinal mucositis in a murine model. It was observed that this protein preserved villous architecture, increased Paneth cell activity [187], and suppressed the growth of Enterobacteriaceae during inflammation [185].

It also has been shown that oral administration of a recombinant L. lactis NCDO2118 strain (pXylT:SEC:hsp65) prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice [184]. Mice fed daily with this recombinant strain increased the number of natural and inducible CD4+FoxP3+ and CD4+ latency-associated peptide (LAP+) regulatory T cells in the spleen, inguinal and mesenteric lymph nodes, as well as in the spinal cord. In addition, a reduction in the recruitment of encephalitogenic CD4+ T cells to the spinal cord was observed, which decreased IgG response against HSP65 and induced an anti-inflammatory cytokine profile (IL-17 reduction and IL-10 increase) during EAE development.

The oral administration of recombinant L. lactis expressing HSP65 and tandemly repeated P277 (pCYT:HSP65-6P277) was also analyzed in a model of type 1 diabetes mellitus (DM1) [183]. The authors observed that oral administration of recombinant L. Lactis resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis, and induced HSP65- and P277-specific T-cell immunotolerance, as well as antigen-specific proliferation of splenocytes, demonstrating to be an effective therapeutic approach in preventing DM1 [183].

Another study using the E. coli Nissle 1917 strain engineered to secrete N-acylphosphatidylethanolamines (NAPEs) (pDEST-At1g78690 expression plasmid) demonstrated that this strain was able to reduce the obesity of mice fed with a high-fat diet when added to drinking water. N-acyl phosphatidylethanolamines are precursors to the N-acylethanolamine (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reducing food intake and obesity. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis than mice receiving standard water or control bacteria [188]. In addition, it was observed that changes on intestinal microbiota significantly decreased the abundance of Firmicutes and increased the abundance of Proteobacteria. Thus, these results provide evidence of the potential efficacy of this approach to inhibit the development of metabolic disorders and related diseases.

13. Conclusion

Currently the association between disease progression, especially chronic inflammatory diseases, and intestinal dysbiosis has been more frequently observed. As a clinical strategy, the use of probiotic bacteria, which naturally benefit the host, has been increasingly used on the treatment of diseases related to the GIT. In view of the good results obtained with this approach, researchers have sought through bacterial genetic modification to increase the beneficial potential of probiotics, either through their use for heterologous protein production or as a vehicle for vaccinal plasmid delivery, by developing recombinant bacterial strains and by testing their action in different disease models. And while there are still a number of questions that need to be answered about the use of genetically modified organisms for health care, especially in human, the use of these strains has proven to be a potentially effective therapeutic alternative, so much so that clinical trials using recombinant lineages have already been authorized and conducted in humans.

References

  1. 1. Thursby E, Juge N. Introduction to the human gut microbiota. Biochemical Journal. 2017;474(11):1823-1836
  2. 2. Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Research [Internet]. 2009;19(12):2317-2323. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19819907
  3. 3. Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nature Reviews. Immunology. 2014;14(10):667-685
  4. 4. Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Nageshwar Reddy D. Role of the normal gut microbiota. World Journal of Gastroenterology [Internet]. 2015;21(29):8787-8803. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26269668
  5. 5. Neish AS. Microbes in gastrointestinal health and disease. Gastroenterology [Internet]. 2009;136(1):65-80. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19026645
  6. 6. Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science [Internet]. 2005;307(5717):1915-1920. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15790844
  7. 7. Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biology [Internet]. 2016;14(8):1-14. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27541692
  8. 8. Guarner F. Enteric flora in health and disease. Digestion [Internet]. 2006;73(1):5-12. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16498248
  9. 9. Walter J, Ley R. The human gut microbiome: Ecology and recent evolutionary changes. Annual Review of Microbiology [Internet]. 2011;65(1):411-429. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21682646
  10. 10. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature [Internet]. 2012;486(7402):207-214. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22699609
  11. 11. Bäckhed F, Fraser CM, Ringel Y, Sanders ME, Sartor RB, Sherman PM, et al. Defining a healthy human gut microbiome: Current concepts, future directions, and clinical applications. Cell Host & Microbe [Internet]. 2012;12(5):611-622. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23159051
  12. 12. Guarner F, Malagelada J-R. Gut flora in health and disease. The Lancet (London, England) [Internet]. 2003;361(9356):512-519. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12583961
  13. 13. Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annual Review of Nutrition [Internet]. 2002;22(1):283-307. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12055347
  14. 14. den Besten G, van Eunen K, Groen AK, Venema K, Reijngoud D-J, Bakker BM. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. Journal of Lipid Research [Internet]. 2013;54(9):2325-2340. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23821742
  15. 15. Hooper LV, Xu J, Falk PG, Midtvedt T, Gordon JI. A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 1999;96(17):9833-9838. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10449780
  16. 16. Liévin V, Peiffer I, Hudault S, Rochat F, Brassart D, Neeser JR, et al. Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity. Gut [Internet]. 2000;47(5):646-652. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11034580
  17. 17. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science [Internet]. 2001;291(5505):881-884. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11157169
  18. 18. Gensollen T, Iyer SS, Kasper DL, Blumberg RS. How colonization by microbiota in early life shapes the immune system. Science [Internet]. 2016;352(6285):539-544. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27126036
  19. 19. Littman DR, Pamer EG. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell Host & Microbe [Internet]. 2011;10(4):311-323. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22018232
  20. 20. Renz H, Brandtzaeg P, Hornef M. The impact of perinatal immune development on mucosal homeostasis and chronic inflammation. Nature Reviews. Immunology [Internet]. 2011;12(1):9-23. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22158411
  21. 21. Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI. Human nutrition, the gut microbiome and the immune system. Nature [Internet]. 2011;474(7351):327-336. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21677749
  22. 22. Honda K, Littman DR. The microbiome in infectious disease and inflammation. Annual Review of Immunology [Internet]. 2012;30(1):759-795. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22224764
  23. 23. Rogler G, Vavricka S. Exposome in IBD: Recent insights in environmental factors that influence the onset and course of IBD. Inflammatory Bowel Diseases [Internet]. 2015;21(2):400-408. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25358064
  24. 24. Singh UP, Singh NP, Murphy EA, Price RL, Fayad R, Nagarkatti M, et al. Chemokine and cytokine levels in inflammatory bowel disease patients. Cytokine [Internet]. 2016;77:44-49. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26520877
  25. 25. Benchimol EI, Manuel DG, Guttmann A, Nguyen GC, Mojaverian N, Quach P, et al. Changing age demographics of inflammatory bowel disease in Ontario, Canada: A population-based cohort study of epidemiology trends. Inflammatory Bowel Diseases [Internet]. 2014;20(10):1761-1769. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25159453
  26. 26. Cho JH. The genetics and immunopathogenesis of inflammatory bowel disease. Nature Reviews. Immunology [Internet]. 2008;8(6):458-466. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18500230
  27. 27. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annual Review of Immunology [Internet]. 2010;28(1):573-621. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20192811
  28. 28. Sartor RB. Mechanisms of disease: Pathogenesis of Crohn’s disease and ulcerative colitis. Nature Clinical Practice. Gastroenterology & Hepatology [Internet]. 2006;3(7):390-407. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16819502
  29. 29. Wanders LK, Dekker E, Pullens B, Bassett P, Travis SPL, East JE. Cancer risk after resection of polypoid dysplasia in patients with longstanding ulcerative colitis: A meta-analysis. Clinical Gastroenterology and Hepatology [Internet]. 2014;12(5):756-764. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23920032
  30. 30. Rattray NJW, Charkoftaki G, Rattray Z, Hansen JE, Vasiliou V, Johnson CH. Environmental influences in the etiology of colorectal cancer: The premise of metabolomics. Current Pharmacology Reports [Internet]. 2017;3(3):114-125. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28642837
  31. 31. Erdman SE, Poutahidis T. Roles for inflammation and regulatory T cells in colon cancer. Toxicologic Pathology [Internet]. 2010;38(1):76-87. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20019355
  32. 32. Brownlee I, Dettmar P, Strugala V, Pearson J. The interaction of dietary fibres with the colon. Current Nutrition & Food Science [Internet]. 2006;2(3):243-264. Available from: http://www.eurekaselect.com/openurl/content.php?genre=article&issn=1573-4013&volume=2&issue=3&spage=243
  33. 33. Ohigashi S, Sudo K, Kobayashi D, Takahashi O, Takahashi T, Asahara T, et al. Changes of the intestinal microbiota, short chain fatty acids, and fecal pH in patients with colorectal cancer. Digestive Diseases and Sciences [Internet]. 2013;58(6):1717-1726. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23306850
  34. 34. McGarr SE, Ridlon JM, Hylemon PB. Diet, anaerobic bacterial metabolism, and colon cancer: A review of the literature. Journal of Clinical Gastroenterology [Internet]. 2005;39(2):98-109. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15681903
  35. 35. Bonnet M, Buc E, Sauvanet P, Darcha C, Dubois D, Pereira B, et al. Colonization of the human gut by E. coli and colorectal cancer risk. Clinical Cancer Research [Internet]. 2014;20(4):859-867. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24334760
  36. 36. Steinhart AH, Ben-Bassat O. Pouchitis: A practical guide. Frontline Gastroenterology [Internet]. 2013;4(3):198-204. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28839726
  37. 37. Sagar PM, Pemberton JH. Intraoperative, postoperative and reoperative problems with ileoanal pouches. The British Journal of Surgery [Internet]. 2012;99(4):454-468. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22307828
  38. 38. Svaninger G, Nordgren S, Oresland T, Hultén L. Incidence and characteristics of pouchitis in the Kock continent ileostomy and the pelvic pouch. Scandinavian Journal of Gastroenterology [Internet]. 1993;28(8):695-700. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8210985
  39. 39. Simchuk EJ, Thirlby RC. Risk factors and true incidence of pouchitis in patients after ileal pouch-anal anastomoses. World Journal of Surgery [Internet]. 2000;24(7):851-856. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10833254
  40. 40. Schieffer KM, Williams ED, Yochum GS, Koltun WA. Review article: The pathogenesis of pouchitis. Alimentary Pharmacology & Therapeutics [Internet]. 2016;44(8):817-835. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27554912
  41. 41. Cao Y, Zhang B, Wu Y, Wang Q, Wang J, Shen F. The value of fecal microbiota transplantation in the treatment of ulcerative colitis patients: A systematic review and meta-analysis. Gastroenterology Research and Practice. 2018;2018(1):1-12
  42. 42. Costello SP, Hughes PA, Waters O, Bryant RV, Vincent AD, Blatchford P, et al. Effect of fecal microbiota transplantation on 8-week remission in patients with ulcerative colitis: A randomized clinical trial. JAMA: The Journal of the American Medical Association. 2019;321(2):156-164
  43. 43. Zhang FM, Wang HG, Wang M, Cui BT, Fan ZN, Ji GZ. Fecal microbiota transplantation for severe enterocolonic fistulizing Crohn’s disease. World Journal of Gastroenterology. 2013;19(41):7213-7236
  44. 44. Zella GC, Hait EJ, Glavan T, Gevers D, Ward DV, Kitts CL, et al. Distinct microbiome in pouchitis compared to healthy pouches in ulcerative colitis and familial adenomatous polyposis. Inflammatory Bowel Diseases [Internet]. 2011;17(5):1092-1100. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20845425
  45. 45. Reshef L, Kovacs A, Ofer A, Yahav L, Maharshak N, Keren N, et al. Pouch inflammation is associated with a decrease in specific bacterial taxa. Gastroenterology. 2015;149(3):718-727. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26026389
  46. 46. Nguyen N, Zhang B, Holubar SD, Pardi DS, Singh S. Treatment and prevention of pouchitis after ileal pouch-anal anastomosis for chronic ulcerative colitis. Cochrane Database of Systematic Reviews [Internet]. 2019;5(1):1-10. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26593456
  47. 47. Landy J, Walker AW, Li JV, Al-Hassi HO, Ronde E, English NR, et al. Variable alterations of the microbiota, without metabolic or immunological change, following faecal microbiota transplantation in patients with chronic pouchitis. Scientific Reports. 2015;5(1):12955
  48. 48. Stallmach A, Lange K, Buening J, Sina C, Vital M, Pieper DH. Fecal microbiota transfer in patients with chronic antibiotic-refractory pouchitis. The American Journal of Gastroenterology [Internet]. 2016;111(3):441-443. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27018122
  49. 49. Nishida A, Imaeda H, Inatomi O, Bamba S, Sugimoto M, Andoh A. The efficacy of fecal microbiota transplantation for patients with chronic pouchitis: A case series. Clinical Case Reports [Internet]. 2019;7(4):782-788. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30997086
  50. 50. Keefe DM, Schubert MM, Elting LS, Sonis ST, Epstein JB, Raber-Durlacher JE, et al. Updated clinical practice guidelines for the prevention and treatment of mucositis. Cancer [Internet]. 2007;109(5):820-831. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17236223
  51. 51. Falcone A, Ricci S, Brunetti I, Pfanner E, Allegrini G, Barbara C, et al. Phase III trial of infusional fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) compared with infusional fluorouracil, leucovorin, and irinotecan (FOLFIRI) as first-line treatment for metastatic colorectal cancer: The Gruppo Oncologico Nor. Journal of Clinical Oncology [Internet]. 2007;25(13):1670-1676. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17470860
  52. 52. Peterson DE, Bensadoun R-J, Roila F, ESMO Guidelines Working Group. Management of oral and gastrointestinal mucositis: ESMO Clinical Practice Guidelines. Annals of Oncology, The Journal of the European Society for Medical Oncology [Internet]. 2011;22(6):78-84. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21908510
  53. 53. Sonis ST. The pathobiology of mucositis. Nature Reviews. Cancer [Internet]. 2004;4(4):277-284. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15057287
  54. 54. Stringer A, Gibson R, Bowen J, Keefe D. Chemotherapy-induced modifications to gastrointestinal microflora: Evidence and implications of change. Current Drug Metabolism. 2009;10(1):79-83
  55. 55. Stringer AM. Interaction between host cells and microbes in chemotherapy-induced mucositis. Nutrients. 2013;5(5):1488-1499
  56. 56. Song M-K, Park M-Y, Sung M-K. 5-Fluorouracil-induced changes of intestinal integrity biomarkers in BALB/c mice. Journal of Cancer Prevention [Internet]. 2013;18(4):322-329. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25337561
  57. 57. Li H-L, Lu L, Wang X-S, Qin L-Y, Wang P, Qiu S-P, et al. Alteration of gut microbiota and inflammatory cytokine/chemokine profiles in 5-fluorouracil induced intestinal mucositis. Frontiers in Cellular and Infection Microbiology. 2017;7(1):455
  58. 58. van Vliet MJ, Harmsen HJM, de Bont ESJM, Tissing WJE. The role of intestinal microbiota in the development and severity of chemotherapy-induced mucositis. PLoS Pathogens. 2010;6(5):e1000879
  59. 59. Ribeiro RA, Wanderley CWS, Wong DVT, Mota JMSC, Leite CAVG, Souza MHLP, et al. Irinotecan- and 5-fluorouracil-induced intestinal mucositis: Insights into pathogenesis and therapeutic perspectives. Cancer Chemotherapy and Pharmacology. 2016;78(5):881-893
  60. 60. Duncan M, Grant G. Oral and intestinal mucositis—Causes and possible treatments. Alimentary Pharmacology & Therapeutics. 2003;18(9):853-874
  61. 61. Mazidi M, Rezaie P, Kengne AP, Mobarhan MG, Ferns GA. Gut microbiome and metabolic syndrome. Diabetes and Metabolic Syndrome: Clinical Research and Reviews [Internet]. 2016;10(2):150-157. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26916014
  62. 62. He M, Shi B. Gut microbiota as a potential target of metabolic syndrome: The role of probiotics and prebiotics. Cell & Bioscience [Internet]. 2017;7(1):54. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29090088
  63. 63. Morgen CS, Sorensen TIA. Obesity: Global trends in the prevalence of overweight and obesity. Nature Reviews. Endocrinology. 2014;10(9):503-504
  64. 64. Alfano M, Canducci F, Nebuloni M, Clementi M, Montorsi F, Salonia A. The interplay of extracellular matrix and microbiome in urothelial bladder cancer. Nature Reviews. Urology. 2016;13(2):77-90
  65. 65. Dolpady J, Sorini C, Di Pietro C, Cosorich I, Ferrarese R, Saita D, et al. Oral probiotic VSL#3 prevents autoimmune diabetes by modulating microbiota and promoting indoleamine 2,3-dioxygenase-enriched tolerogenic intestinal environment. Journal Diabetes Research. 2016;2016(1):7569431
  66. 66. Eckel RH, Alberti K, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet. 2010;375(9710):181-183
  67. 67. Vona R, Gambardella L, Cittadini C, Straface E, Pietraforte D. Biomarkers of oxidative stress in metabolic syndrome and associated diseases. Oxidative Medicine and Cellular Longevity [Internet]. 2019;2019(1):1-19. Available from: https://www.hindawi.com/journals/omcl/2019/8267234/
  68. 68. Sonnenberg GE, Krakower GR, Kissebah AH. A novel pathway to the manifestations of metabolic syndrome. Obesity Research. 2004;12(2):180-186
  69. 69. Roberts CK, Sindhu KK. Oxidative stress and metabolic syndrome. Life Sciences. 2009;84(21-22):705-712
  70. 70. Clavel T, Haller D. Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: Implications for chronic inflammation. Inflammatory Bowel Diseases. 2007;13(9):1153-1164
  71. 71. Clavel T, Desmarchelier C, Haller D, Gérard P, Rohn S, Lepage P, et al. Intestinal microbiota in metabolic diseases: From bacterial community structure and functions to species of pathophysiological relevance. Gut Microbes. 2014;5(4):544-551
  72. 72. Brestoff JR, Artis D. Immune regulation of metabolic homeostasis in health and disease. Cell. 2015;161(1):146-160
  73. 73. Hamad EM, Sato M, Uzu K, Yoshida T, Higashi S, Kawakami H, et al. Milk fermented by Lactobacillus gasseri SBT2055 influences adipocyte size via inhibition of dietary fat absorption in Zucker rats. The British Journal of Nutrition. 2009;101(5):716-724
  74. 74. Kawano M, Miyoshi M, Ogawa A, Sakai F, Kadooka Y. Lactobacillus gasseri SBT2055 inhibits adipose tissue inflammation and intestinal permeability in mice fed a high-fat diet. Journal of Nutritional Science. 2016;5(1):23
  75. 75. Lopetuso LR, Scaldaferri F, Bruno G, Petito V, Franceschi F, Gasbarrini A. The therapeutic management of gut barrier leaking: The emerging role for mucosal barrier protectors. European Review for Medical and Pharmacological Sciences. 2015;19(6):1068-1076
  76. 76. Miyoshi M, Ogawa A, Higurashi S, Kadooka Y. Anti-obesity effect of Lactobacillus gasseri SBT2055 accompanied by inhibition of pro-inflammatory gene expression in the visceral adipose tissue in diet-induced obese mice. European Journal of Nutrition. 2014;53(2):599-606
  77. 77. Kadooka Y, Sato M, Ogawa A, Miyoshi M, Uenishi H, Ogawa H, et al. Effect of Lactobacillus gasseri SBT2055 in fermented milk on abdominal adiposity in adults in a randomised controlled trial. The British Journal of Nutrition. 2013;110(9):1696-1703
  78. 78. Yun SI, Park HO, Kang JH. Effect of Lactobacillus gasseri BNR17 on blood glucose levels and body weight in a mouse model of type 2 diabetes. Journal of Applied Microbiology. 2009;107(5):1681-1686
  79. 79. Kang JH, Yun S II, Park HO. Effects of Lactobacillus gasseri BNR17 on body weight and adipose tissue mass in diet-induced overweight rats. Journal of Microbiology. 2010;48(5):712-714
  80. 80. Jung SP, Lee KM, Kang JH, Yun S II, Park HO, Moon Y, et al. Effect of Lactobacillus gasseri BNR17 on overweight and obese adults: A randomized, double-blind clinical trial. Korean Journal of Family Medicine. 2013;34(2):80-89
  81. 81. Rajkumar H, Mahmood N, Kumar M, Varikuti SR, Challa HR, Myakala SP. Effect of probiotic (VSL#3) and omega-3 on lipid profile, insulin sensitivity, inflammatory markers, and gut colonization in overweight adults: A randomized, controlled trial. Mediators of Inflammation. 2014;2014:348959
  82. 82. Shirouchi B, Nagao K, Umegatani M, Shiraishi A, Morita Y, Kai S, et al. Probiotic Lactobacillus gasseri SBT2055 improves glucose tolerance and reduces body weight gain in rats by stimulating energy expenditure. The British Journal of Nutrition. 2016;116(3):451-458
  83. 83. Hsieh M-C, Tsai W-H, Jheng Y-P, Su S-L, Wang S-Y, Lin C-C, et al. The beneficial effects of Lactobacillus reuteri ADR-1 or ADR-3 consumption on type 2 diabetes mellitus: A randomized, double-blinded, placebo-controlled trial. Scientific Reports [Internet]. 2018;8(1):16791. Available from: http://www.nature.com/articles/s41598-018-35014-1
  84. 84. Silva HLA, Balthazar CF, Esmerino EA, Neto RPC, Rocha RS, Moraes J, et al. Partial substitution of NaCl by KCl and addition of flavor enhancers on probiotic Prato cheese: A study covering manufacturing, ripening and storage time. Food Chemistry. 2018;248(1):192-200
  85. 85. Moura CS, Lollo PCB, Morato PN, Esmerino EA, Margalho LP, Santos-Junior VA, et al. Assessment of antioxidant activity, lipid profile, general biochemical and immune system responses of Wistar rats fed with dairy dessert containing Lactobacillus acidophilus La-5. Food Research International. 2016;90(1):275-280
  86. 86. Lollo PCB, De Moura CS, Morato PN, Cruz AG, de Castro WF, Betim CB, et al. Probiotic yogurt offers higher immune-protection than probiotic whey beverage. Food Research International. 2013;54(1):118-124
  87. 87. Corsello G, Carta M, Marinello R, Picca M, De Marco G, Micillo M, et al. Preventive effect of cow’s milk fermented with lactobacillus paracasei CBA L74 on common infectious diseases in children: A multicenter randomized controlled trial. Nutrients. 2017;9(7):e669
  88. 88. Acurcio LB, Sandes SHC, Bastos RW, Sant’anna FM, Pedroso SHSP, Reis DC, et al. Milk fermented by Lactobacillus species from Brazilian artisanal cheese protect germ-free-mice against Salmonella typhimurium infection. Beneficial Microbes. 2017;8(4):579-588
  89. 89. Agerholm-Larsen L, Raben A, Haulrik N, Hansen AS, Manders M, Astrup A. Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. European Journal of Clinical Nutrition. 2000;54(4):288-297
  90. 90. de Moreno de LeBlanc A, Matar C, LeBlanc N, Perdigón G. Effects of milk fermented by Lactobacillus helveticus R389 on a murine breast cancer model. Breast Cancer Research. 2005;7(4):R477-R486
  91. 91. Lollo PCB, Morato PN, Moura CS, Almada CN, Felicio TL, Esmerino EA, et al. Hypertension parameters are attenuated by the continuous consumption of probiotic Minas cheese. Food Research International. 2015;76(1):611-617
  92. 92. Rodrigues R, Guerra G, Soares J, Santos K, Rolim F, Assis P, et al. Lactobacillus rhamnosus EM1107 in goat milk matrix modulates intestinal inflammation involving NF-κB p65 and SOCs-1 in an acid-induced colitis model. Journal of Functional Foods. 2018;50(1):78-92
  93. 93. Tojo Sierra L, Leis TR, Tojo Gonzalez R. Prebiotics and probiotics in childhood helath and disease. Journal of Gastroenterology and Hepatology. 2003;26(1):37-49
  94. 94. Vinolo MAR, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by short chain fatty acids. Nutrients [Internet]. 2011;3(10):858-876. Available from: http://www.mdpi.com/2072-6643/3/10/858
  95. 95. Kanauchi O, Andoh A, Mitsuyama K. Effects of the modulation of microbiota on the gastrointestinal immune system and bowel function. Journal of Agricultural and Food Chemistry [Internet]. 2013;61(42):9977-9983. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24070265
  96. 96. Carvalho RDDO, do Carmo FLR, de Oliveira Junior A, Langella P, Chatel J-M, Bermúdez-Humarán LG, et al. Use of wild type or recombinant lactic acid bacteria as an alternative treatment for gastrointestinal inflammatory diseases: A focus on inflammatory bowel diseases and mucositis. Frontiers in Microbiology [Internet]. 2017;8(1):800. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28536562
  97. 97. Ni J, Wu GD, Albenberg L, Tomov VT. Gut microbiota and IBD: Causation or correlation? Nature Reviews. Gastroenterology & Hepatology [Internet]. 2017;14(10):573-584. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28743984
  98. 98. Kim D, Zeng MY, Núñez G. The interplay between host immune cells and gut microbiota in chronic inflammatory diseases. Experimental & Molecular Medicine [Internet]. 2017;49(5):e339. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28546562
  99. 99. Opazo MC, Ortega-Rocha EM, Coronado-Arrázola I, Bonifaz LC, Boudin H, Neunlist M, et al. Intestinal microbiota influences non-intestinal related autoimmune diseases. Frontiers in Microbiology [Internet]. 2018;9(1):432. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29593681
  100. 100. Huebner ES, Surawicz CM. Probiotics in the prevention and treatment of gastrointestinal infections. Gastroenterology Clinics of North America [Internet]. 2006;35(2):355-365. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16880070
  101. 101. Eom T, Kim YS, Choi CH, Sadowsky MJ, Unno T. Current understanding of microbiota- and dietary-therapies for treating inflammatory bowel disease. Journal of Microbiology [Internet]. 2018;56(3):189-198. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29492876
  102. 102. Weingarden AR, Vaughn BP. Intestinal microbiota, fecal microbiota transplantation, and inflammatory bowel disease. Gut Microbes [Internet]. 2017;8(3):238-252. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28609251
  103. 103. Plaza-Diaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Advances in Nutrition [Internet]. 2019;10(1):S49-S66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30721959
  104. 104. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews. Gastroenterology & Hepatology [Internet]. 2014;11(8):506-514. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24912386
  105. 105. Reid G, Gadir AA, Dhir R. Probiotics: Reiterating what they are and what they are not. Frontiers in Microbiology [Internet]. 2019;10(1):424. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30930863
  106. 106. Foligné B, Dewulf J, Vandekerckove P, Pignède G, Pot B. Probiotic yeasts: Anti-inflammatory potential of various non-pathogenic strains in experimental colitis in mice. World Journal of Gastroenterology [Internet]. 2010;16(17):2134-2145. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20440854
  107. 107. Sugahara H, Yao R, Odamaki T, Xiao JZ. Differences between live and heat-killed bifidobacteria in the regulation of immune function and the intestinal environment. Beneficial Microbes [Internet]. 2017;8(3):463-472. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28441886
  108. 108. Chang C-J, Lin T-L, Tsai Y-L, Wu T-R, Lai W-F, Lu C-C, et al. Next generation probiotics in disease amelioration. Journal of Food and Drug Analysis [Internet]. 2019;27(3):615-622. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1021949819300110
  109. 109. Wang M, Gao Z, Zhang Y, Pan L. Lactic acid bacteria as mucosal delivery vehicles: A realistic therapeutic option. Applied Microbiology and Biotechnology [Internet]. 2016;100(13):5691-5701. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27154346
  110. 110. Mokoena MP. Lactic acid bacteria and their bacteriocins: Classification, biosynthesis and applications against uropathogens: A mini-review. Molecules [Internet]. 2017;22(8):1-13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28933759
  111. 111. Makarova KS, Koonin EV. Evolutionary genomics of lactic acid bacteria. Journal of Bacteriology [Internet]. 2007;189(4):1199-1208. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17085562
  112. 112. Quinto EJ, Jiménez P, Caro I, Tejero J, Mateo J, Girbés T. Probiotic lactic acid bacteria: A review. Food and Nutrition Sciences [Internet]. 2014;05(18):1765-1775. Available from: http://www.scirp.org/journal/doi.aspx?DOI=10.4236/fns.2014.518190
  113. 113. Bron PA, Kleerebezem M. Lactic acid bacteria for delivery of endogenous or engineered therapeutic molecules. Frontiers in Microbiology [Internet]. 2018;9(1):1821. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30123213
  114. 114. Shi Y, Zhai Q, Li D, Mao B, Liu X, Zhao J, et al. Restoration of cefixime-induced gut microbiota changes by Lactobacillus cocktails and fructooligosaccharides in a mouse model. Microbiological Research [Internet]. 2017;200(1):14-24. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28527760
  115. 115. Halder D, Mandal M, Chatterjee SS, Pal NK, Mandal S. Indigenous probiotic Lactobacillus isolates presenting antibiotic like activity against human pathogenic bacteria. Biomedicine [Internet]. 2017;5(2):1-11. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28621711
  116. 116. Hemarajata P, Versalovic J. Effects of probiotics on gut microbiota: Mechanisms of intestinal immunomodulation and neuromodulation. Therapeutic Advances in Gastroenterology [Internet]. 2013;6(1):39-51. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23320049
  117. 117. Aliakbarpour HR, Chamani M, Rahimi G, Sadeghi AA, Qujeq D. The Bacillus subtilis and lactic acid bacteria probiotics influences intestinal mucin gene expression, histomorphology and growth performance in broilers. Asian-Australasian Journal of Animal Sciences [Internet]. 2012;25(9):1285-1293. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25049692
  118. 118. Gao K, Wang C, Liu L, Dou X, Liu J, Yuan L, et al. Immunomodulation and signaling mechanism of Lactobacillus rhamnosus GG and its components on porcine intestinal epithelial cells stimulated by lipopolysaccharide. Journal of Microbiology, Immunology, and Infection [Internet]. 2017;50(5):700-713. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26055689
  119. 119. Culligan EP, Hill C, Sleator RD. Probiotics and gastrointestinal disease: Successes, problems and future prospects. Gut Pathogens [Internet]. 2009;1(1):19. Available from: http://gutpathogens.biomedcentral.com/articles/10.1186/1757-4749-1-19
  120. 120. Amalaradjou MAR, Bhunia AK. Bioengineered probiotics, a strategic approach to control enteric infections. Bioengineered [Internet]. 2013;4(6):379-387. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23327986
  121. 121. Sleator RD, Hill C. Battle of the bugs. Science [Internet]. 2008;321(5894):1294-1295. Available from: http://www.sciencemag.org/cgi/doi/10.1126/science.321.5894.1294b
  122. 122. Wells J. Mucosal vaccination and therapy with genetically modified lactic acid bacteria. Annual Review of Food Science and Technology [Internet]. 2011;2(1):423-445. Available from: http://www.annualreviews.org/doi/10.1146/annurev-food-022510-133640
  123. 123. D’Silva I. Recombinant technology and probiotics. International Journal of Engineering & Technology. 2011;3(4):288-293
  124. 124. Wells JM, Mercenier A. Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nature Reviews. Microbiology [Internet]. 2008;6(5):349-362. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18345021
  125. 125. de Vos WM. Gene expression systems for lactic acid bacteria. Current Opinion in Microbiology [Internet]. 1999;2(3):289-295. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10383867
  126. 126. Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, et al. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Research [Internet]. 2001;11(5):731-753. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11337471
  127. 127. Felis GE, Dellaglio F. Taxonomy of Lactobacilli and Bifidobacteria. Current Issues in Intestinal Microbiology [Internet]. 2007;8(2):44-61. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17542335
  128. 128. Bermúdez-Humarán LG, Kharrat P, Chatel J-M, Langella P. Lactococci and Lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Microbial Cell Factories [Internet]. 2011;10(1):4. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21995317
  129. 129. Margaritis A, Bassi AS. Plasmid stability of recombinant DNA microorganisms. In: Prokop A, Bajpai R, Ho C, editors. Recombinant DNA Technology and Applications. New York: New York Academy of Sciences; 1991. pp. 316-332
  130. 130. Kleerebezem M, Beerthuyzen MM, Vaughan EE, de Vos WM, Kuipers OP. Controlled gene expression systems for lactic acid bacteria: Transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Applied and Environmental Microbiology [Internet]. 1997;63(11):4581-4584. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9361443
  131. 131. Pavan S, Hols P, Delcour J, Geoffroy MC, Grangette C, Kleerebezem M, et al. Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: A tool to study in vivo biological effects. Applied and Environmental Microbiology [Internet]. 2000;66(10):4427-4432. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11010894
  132. 132. Derré I, Rapoport G, Devine K, Rose M, Msadek T. ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis. Molecular Microbiology [Internet]. 1999;32(3):581-593. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10320580
  133. 133. Ruiz L, O’Connell-Motherway M, Zomer A, de los Reyes-Gavilán CG, Margolles A, van Sinderen D. A bile-inducible membrane protein mediates bifidobacterial bile resistance. Microbial Biotechnology [Internet]. 2012;5(4):523-535. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22296641
  134. 134. Benbouziane B, Ribelles P, Aubry C, Martin R, Kharrat P, Riazi A, et al. Development of a stress-inducible controlled expression (SICE) system in Lactococcus lactis for the production and delivery of therapeutic molecules at mucosal surfaces. Journal of Biotechnology [Internet]. 2013;168(2):120-129. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23664884
  135. 135. Wells JM, Robinson K, Chamberlain LM, Schofield KM, Le Page RW. Lactic acid bacteria as vaccine delivery vehicles. Antonie Van Leeuwenhoek [Internet]. 1996;70(2-4):317-330. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8879413
  136. 136. Mercenier A, Müller-Alouf H, Grangette C. Lactic acid bacteria as live vaccines. Current Issues in Molecular Biology [Internet]. 2000;2(1):17-25. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11464916
  137. 137. Thole JE, van Dalen PJ, Havenith CE, Pouwels PH, Seegers JF, Tielen FD, et al. Live bacterial delivery systems for development of mucosal vaccines. Current Opinion in Molecular Therapeutics [Internet]. 2000;2(1):94-99. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11249657
  138. 138. Seegers JFML. Lactobacilli as live vaccine delivery vectors: Progress and prospects. Trends in Biotechnology [Internet]. 2002;20(12):508-515. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12443872
  139. 139. Wells J, Mercenier A. Lactic acid bacteria as mucosal delivery system. In: Genetics of Lactic Acid Bacteria. New York: Kluwer Academic/Plenum Publishers; 2003. pp. 261-290
  140. 140. Mierau I, Kleerebezem M. 10 Years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Applied Microbiology and Biotechnology. 2005;68(6):705-717
  141. 141. Nouaille S, Ribeiro LA, Miyoshi A, Pontes D, Le Loir Y, Oliveira SC, et al. Heterologous protein production and delivery systems for Lactococcus lactis. Genetics and Molecular Research [Internet]. 2003;2(1):102-111. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12917806
  142. 142. Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermúdez-Humarán LG, et al. Protein secretion in Lactococcus lactis: An efficient way to increase the overall heterologous protein production. Microbial Cell Factories. 2005;4(1):2
  143. 143. Miyoshi A, Jamet E, Commissaire J, Renault P, Langella P, Azevedo V. A xylose-inducible expression system for Lactococcus lactis. FEMS Microbiology Letters [Internet]. 2004;239(2):205-212. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15476967
  144. 144. Desmond C, Fitzgerald GF, Stanton C, Ross RP. Improved stress tolerance of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338. Applied and Environmental Microbiology [Internet]. 2004;70(10):5929-5936. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15466535
  145. 145. Cano-Garrido O, Seras-Franzoso J, Garcia-Fruitós E. Lactic acid bacteria: Reviewing the potential of a promising delivery live vector for biomedical purposes. Microbial Cell Factories. 2015;14(137):1-12
  146. 146. Guimarães V, Innocentin S, Chatel J-M, Lefèvre F, Langella P, Azevedo V, et al. A new plasmid vector for DNA delivery using Lactococci. Genetic Vaccines and Therapy [Internet]. 2009;7(1):4. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19208231
  147. 147. Tao L, Pavlova SI, Ji X, Jin L, Spear G. A novel plasmid for delivering genes into mammalian cells with noninvasive food and commensal lactic acid bacteria. Plasmid [Internet]. 2011;65(1):8-14. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20832422
  148. 148. Mancha-Agresti P, Drumond MM, do Carmo FLR, Santos MM, Dos Santos JSC, Venanzi F, et al. A new broad range plasmid for DNA delivery in eukaryotic cells using lactic acid bacteria: In vitro and in vivo assays. Molecular Therapy: Methods & Clinical Development [Internet]. 2017;4(1):83-91. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28344994
  149. 149. Yagnik B, Padh H, Desai P. Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis. Microbes and Infection [Internet]. 2016;18(4):237-244. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26655884
  150. 150. Yagnik B, Sharma D, Padh H, Desai P. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY. Microbiology and Immunology [Internet]. 2017;61(3-4):123-129. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28258689
  151. 151. Kutzler MA, Weiner DB. DNA vaccines: Ready for prime time? Nature Reviews. Genetics [Internet]. 2008;9(10):776-788. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18781156
  152. 152. Zurita-Turk M, Del Carmen S, Santos ACG, Pereira VB, Cara DC, Leclercq SY, et al. Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis. BMC Biotechnology [Internet]. 2014;14(1souz):73. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25106058
  153. 153. Souza BM, Preisser TM, Pereira VB, Zurita-Turk M, de Castro CP, da Cunha VP, et al. Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells. Microbial Cell Factories [Internet]. 2016;15(1):150. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27576902
  154. 154. Coelho-Rocha ND, de Castro CP, de Jesus LCL, Leclercq SY, de Cicco Sandes SH, Nunes AC, et al. Microencapsulation of lactic acid bacteria improves the gastrointestinal delivery and in situ expression of recombinant fluorescent protein. Frontiers in Microbiology [Internet]. 2018;9(1):2398. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30344518
  155. 155. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology [Internet]. 2005;151(8):2551-2561. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16079334
  156. 156. Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics [Internet]. 2007;8(1):172. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17521438
  157. 157. Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology [Internet]. 2008;190(4):1390-1400. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18065545
  158. 158. Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nature Biotechnology [Internet]. 2016;34(9):933-941. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27606440
  159. 159. Price VJ, Huo W, Sharifi A, Palmer KL. CRISPR-Cas and restriction-modification act additively against conjugative antibiotic resistance plasmid transfer in Enterococcus faecalis. mSphere [Internet]. 2016;1(3):1-13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27303749
  160. 160. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology [Internet]. 1987;169(12):5429-5433. Available from: http://www.ncbi.nlm.nih.gov/pubmed/3316184
  161. 161. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science [Internet]. 2013;339(6121):819-823. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23287718
  162. 162. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science [Internet]. 2013;339(6121):823-826. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23287722
  163. 163. Millen AM, Horvath P, Boyaval P, Romero DA. Mobile CRISPR/Cas-mediated bacteriophage resistance in Lactococcus lactis. PLoS One [Internet]. 2012;7(12):e51663. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23240053
  164. 164. Hidalgo-Cantabrana C, O’Flaherty S, Barrangou R. CRISPR-based engineering of next-generation lactic acid bacteria. Current Opinion in Microbiology [Internet]. 2017;37(1):79-87. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28622636
  165. 165. Oh J-H, van Pijkeren J-P. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Research [Internet]. 2014;42(17):e131. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25074379
  166. 166. Sanozky-Dawes R, Selle K, O’Flaherty S, Klaenhammer T, Barrangou R. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri. Microbiology [Internet]. 2015;161(9):1752-1761. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26297561
  167. 167. Song X, Huang H, Xiong Z, Ai L, Yang S. CRISPR-Cas9D10A nickase-assisted genome editing in Lactobacillus casei. Applied and Environmental Microbiology [Internet]. 2017;83(22):1-13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28864652
  168. 168. Berlec A, Škrlec K, Kocjan J, Olenic M, Štrukelj B. Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis. Scientific Reports [Internet]. 2018;8(1):1009. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29343791
  169. 169. de Castro CP, Drumond MM, Batista VL, Nunes A, Mancha-Agresti P, Azevedo V. Vector development timeline for mucosal vaccination and treatment of disease using Lactococcus lactis and design approaches of next generation food grade plasmids. Frontiers in Microbiology [Internet]. 2018;9(1):1805. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30154762
  170. 170. Bezkorovainy A. Probiotics: Determinants of survival and growth in the gut. The American Journal of Clinical Nutrition [Internet]. 2001;73(2):399S-405S. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11157348
  171. 171. Lo Curto A, Pitino I, Mandalari G, Dainty JR, Faulks RM, John Wickham MS. Survival of probiotic Lactobacilli in the upper gastrointestinal tract using an in vitro gastric model of digestion. Food Microbiology [Internet]. 2011;28(7):1359-1366. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21839386
  172. 172. Karl JP, Hatch AM, Arcidiacono SM, Pearce SC, Pantoja-Feliciano IG, Doherty LA, et al. Effects of psychological, environmental and physical stressors on the gut microbiota. Frontiers in Microbiology [Internet]. 2018;9(1):2013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30258412
  173. 173. Hanson ML, Hixon JA, Li W, Felber BK, Anver MR, Stewart CA, et al. Oral delivery of IL-27 recombinant bacteria attenuates immune colitis in mice. Gastroenterology [Internet]. 2014;146(1):210-221. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24120477
  174. 174. Bermúdez-Humarán LG, Motta J-P, Aubry C, Kharrat P, Rous-Martin L, Sallenave J-M, et al. Serine protease inhibitors protect better than IL-10 and TGF-β anti-inflammatory cytokines against mouse colitis when delivered by recombinant Lactococci. Microbial Cell Factories [Internet]. 2015;14(1):26. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25889561
  175. 175. dias CR, Morais K, Pereira VB, Gomes-Santos AC, Luerce TD, de Azevedo MS, et al. Oral administration of Lactococcus lactis expressing recombinant 15-lipoxygenase-1 (15 LOX-1) modulates chemically induced colitis in mice. Medical Research Archives [Internet]. 2016;4(7):1-13. Available from: http://journals.ke-i.org/index.php/mra/article/view/612
  176. 176. Gomes-Santos AC, de Oliveira RP, Moreira TG, Castro-Junior AB, Horta BC, Lemos L, et al. Hsp65-producing Lactococcus lactis prevents inflammatory intestinal disease in mice by IL-10- and TLR2-dependent pathways. Frontiers in Immunology [Internet]. 2017;8(1):30. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28194152
  177. 177. Mauras A, Chain F, Faucheux A, Ruffié P, Gontier S, Ryffel B, et al. A new bifidobacteria expression system (BEST) to produce and deliver interleukin-10 in Bifidobacterium bifidum. Frontiers in Microbiology [Internet]. 2018;9(1):3075. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30622516
  178. 178. Tian T, Wang Z, Zhang J. Pathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies. Oxidative Medicine and Cellular Longevity [Internet]. 2017;2017(1):4535194. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28744337
  179. 179. Bourgonje AR, von Martels JZH, Bulthuis MLC, van Londen M, Faber KN, Dijkstra G, et al. Crohn’s disease in clinical remission is marked by systemic oxidative stress. Frontiers in Physiology [Internet]. 2019;10(1):499. Available from: http://www.ncbi.nlm.nih.gov/pubmed/31080419
  180. 180. LeBlanc JG, del Carmen S, Miyoshi A, Azevedo V, Sesma F, Langella P, et al. Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn’s disease in mice. Journal of Biotechnology [Internet]. 2011;151(3):287-293. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21167883
  181. 181. Del Carmen S, de Moreno de LeBlanc A, Martin R, Chain F, Langella P, Bermúdez-Humarán LG, et al. Genetically engineered immunomodulatory Streptococcus thermophilus strains producing antioxidant enzymes exhibit enhanced anti-inflammatory activities. Applied and Environmental Microbiology [Internet]. 2014;80(3):869-877. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24242245
  182. 182. Jacouton E, Torres Maravilla E, Boucard A-S, Pouderous N, Pessoa Vilela AP, Naas I, et al. Anti-tumoral effects of recombinant Lactococcus lactis strain secreting IL-17A cytokine. Frontiers in Microbiology [Internet]. 2018;9(1):3355. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30728820
  183. 183. Ma Y, Liu J, Hou J, Dong Y, Lu Y, Jin L, et al. Oral administration of recombinant Lactococcus lactis expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice. PLoS One [Internet]. 2014;9(8):e105701. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25157497
  184. 184. Rezende RM, Oliveira RP, Medeiros SR, Gomes-Santos AC, Alves AC, Loli FG, et al. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells. Journal of Autoimmunity [Internet]. 2013;40(1):45-57. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22939403
  185. 185. Carvalho R, Vaz A, Pereira FL, Dorella F, Aguiar E, Chatel J-M, et al. Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP. Scientific Reports [Internet]. 2018;8(1):15072. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30305667
  186. 186. Caluwaerts S, Vandenbroucke K, Steidler L, Neirynck S, Vanhoenacker P, Corveleyn S, et al. AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis. Oral Oncology [Internet]. 2010;46(7):564-570. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20542722
  187. 187. Carvalho RD, Breyner N, Menezes-Garcia Z, Rodrigues NM, Lemos L, Maioli TU, et al. Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis. Microbial Cell Factories [Internet]. 2017;16(1):27. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28193209
  188. 188. Chen Z, Guo L, Zhang Y, Walzem RL, Pendergast JS, Printz RL, et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. The Journal of Clinical Investigation [Internet]. 2014;124(8):3391-3406. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24960158

Written By

Luís Cláudio Lima de Jesus, Fernanda Alvarenga Lima, Nina Dias Coelho-Rocha, Tales Fernando da Silva, Júlia Paz, Vasco Azevedo, Pamela Mancha-Agresti and Mariana Martins Drumond

Submitted: 15 June 2019 Reviewed: 01 July 2019 Published: 13 December 2019