Fermented Foods: Are They Tasty Medicines for Helicobacter pylori Associated Peptic Ulcer and Gastric Cancer?

More than a million people die every year due to gastric cancer and peptic ulcer. Helicobacter pylori infection in stomach is the most important reason for these diseases. Interestingly, only 10–20% of the H. pylori infected individuals suffer from these gastric diseases and rest of the infected individuals remain asymptomatic. The genotypes of H. pylori, host genetic background, lifestyle including smoking and diet may determine clinical outcomes. People from different geographical regions have different food habits, which also include several unique fermented products of plant and animal origins. When consumed raw, the fermented foods bring in fresh inocula of microbes to gastrointestinal tract and several strains of these microbes, like Lactobacillus and Saccharomyces are known probiotics. In vitro and in vivo experiments as well as clinical trials suggest that several probiotics have anti-H. pylori effects. Here we discuss the possibility of using natural probiotics present in traditional fermented food and beverages to obtain protection against H. pylori induced gastric diseases.


INTRODUCTION
"Let food be thy medicine and medicine be thy food." Hippocrates  Every year peptic ulcer and gastric cancer takes approximately 301,000 and 740,000 lives, respectively (Piazuelo and Correa, 2013;Naghavi et al., 2015). Although both diseases have multiple etiologies like stress, diet, smoking and host genetic background, Helicobacter pylori infection is perhaps the most critical among them (Malfertheiner et al., 2014). However, every H. pylori infected individual does not develop peptic ulcer or gastric cancer. More than half of the world population is infected by H. pylori, but 10-20% of the infected people suffer from these diseases (Dorer et al., 2009). Why ∼80% of the H. pylori infected people in any given population never suffer from gastric disorders is unknown at present. Also, the clinical outcomes among the H. pylori infected population suffering from gastric disorders vary tremendously with geography (Covacci et al., 1999). For example, gastric cancer is fairly common in East-Asian countries like Japan and Korea, but in most African countries and India, the incidence of gastric cancer is low in spite of having high prevalence of H. pylori infection (Holcombe, 1992;Singh and Ghoshal, 2006;Shiota et al., 2013). Variations in bacterial and human genetic factors have been linked to explain the differences in clinical outcome, but our understanding of H. pylori infection and related diseases are really incomplete. Microbiota is the ecological community of commensal, symbiotic and pathogenic microorganisms that literally share our body space. Microbiome is the combined genomes of the microbiota (Lederberg, 2001). Recent metagenomic analyses of DNA isolated from gastric tissue specimens show that human stomach is the niche of many bacterial species (Maldonado-Contreras et al., 2011). While the exact significance of the microbes that co-exist in highly acidic gastric milieu is not understood till date, it seems apparent that H. pylori infection can alter the dynamics of gastric microbiota (Andersson et al., 2008). However, microbiota can also be modulated by several other factors like alteration in immunity due to other infections and change in lifestyle including food and beverage consumptions (De Filippo et al., 2010). Interestingly, almost every geographical location has unique tradition of consuming fermented foods and beverages (Campbell-Platt, 1987). These fermented foods are rich source of bacteria, yeasts and molds and many of these microbes provide benefits to hosts and act as probiotics (Tamang et al., 2016a,b). More intriguingly, adding purified probiotics to therapy against H. pylori gives better eradication rate and reduces the side effects of antibiotics (Zhang et al., 2015). Unfortunately, however, the significances of the natural probiotics in traditional fermented foods and beverages are less studied in the context of H. pylori associated diseases. In this mini-review, we will discuss how probiotics present in different fermented foods and beverages may have a role in preventing H. pylori related gastric diseases.

H. pylori INFECTION AND GASTRIC DISEASES
Presence of spiral bacilli in stomach have been reported several times during the past century, but the culture of this slow growing species remained unsuccessful until a serendipitous prolonged incubation of human gastric specimens in microaerophillic conditions during Easter holidays by Barry James Marshall and John Robin Warren (Doenges, 1938;Freedberg and Barron, 1940;Warren and Marshall, 1983;Marshall and Warren, 1984). To prove Koch's postulate Barry Marshall drank pure culture of H. pylori, which resulted in hypochlorhydric vomiting and gastritis before he was treated with antibiotics (Marshall et al., 1985). Subsequently, a huge number of studies confirmed the role of H. pylori virulence factors in peptic ulcer and gastric cancer and H. pylori was classified as a type I carcinogen by WHO (Malfertheiner et al., 2014).
H. pylori expresses many virulence factors, but two multitasking proteins, the vacuolating cytotoxin (VacA) and the cytotoxin-associated gene A (CagA), seem to play the most crucial role in developing the gastro-duodenal diseases. The VacA is a secreted toxin, which forms large cytoplasmic vacuoles inside the host cells (Leunk et al., 1988). The VacA is also involved in reducing mitochondrial transmembrane potential, releasing cytochrome c, inducing cell death, activating MAPkinases and inhibiting T-cell activation (Galmiche et al., 2000;Gebert et al., 2003;Willhite and Blanke, 2004;Yamasaki et al., 2006;Torres et al., 2007). The vacA gene has mosaic structures viz. s1/s2 alleles (encoding signal peptides), m1/m2 alleles (encoding mid-regions) and i1/i2/i3 (encoding intermediate regions) (Cover et al., 1994;Atherton et al., 1995Atherton et al., , 1999Rhead et al., 2007). The s1 and the i1 alleles of vacA are associated with aggressive clinical outcomes (Rhead et al., 2007;Yamaoka, 2010). The H. pylori strains carrying vacA s1 usually carry cagA gene, which is located in the cag-pathogenicity island Xiang et al., 1995;Yamaoka, 2010). The cagA + strains are associated with more severe diseases in most regions . The cagA gene shows length polymorphism at the 3 end and this variable region encodes EPIYA motifs that undergo phosphorylation once the CagA protein is translocated into the host cells (Yamaoka et al., 1998;Higashi et al., 2002). The phospho-CagA interacts and deregulates the SHP-2 protein, which leads to cancer, but the CagA can hijack cellular pathways also by phosphorylation independent manner (Higashi et al., 2002;Hatakeyama, 2014).
Polymorphisms in host immune genes also contribute to determine the clinical status of the host (Datta De and Roychoudhury, 2015). For example, polymorphisms in interleukin-1 (IL1) and tumour necrosis factor (TNF) genes have been shown to play important roles in progression of gastric diseases among Scottish, Japanese, American, and Indian populations (El-Omar, 2001;Datta De and Roychoudhury, 2015). Moreover, every geographic region has unique lifestyle including food and beverage intakes, which are known to have effects on gut microbiota (De Filippo et al., 2010).
It is now appreciated that human stomach microbiota consists of 44 bacterial phyla, dominated by four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (Maldonado-Contreras et al., 2011). A study using Swedish patients showed that the presence of H. pylori in stomach may significantly alter the relative abundance of other bacteria (Andersson et al., 2008). Colonization by specific groups of bacteria seems to correlate with H. pylori infection status. H. pylori colonization dramatically reduced the diversity and increased the colonization of Proteobacteria. Positive H. pylori status in America is also associated with increased abundance of Proteobacteria, Spirochetes and Acidobacteria, and with decreased abundance of Actinobacteria, Bacteroidetes and Firmicutes (Maldonado-Contreras et al., 2011). Recently, in mouse, it has been shown that H. pylori colonization can influence both gastric and intestinal microbiota (Kienesberger et al., 2016). While it appears that the stomach and intestinal microbiota in the presence and in the absence of H. pylori infection may have a role in gastric diseases the mechanism is not known.
Treatment for all H. pylori infections has been recommended for several geographical locations (Shiota et al., 2013;Malnick et al., 2014). The usual treatment regimen for H. pylori is a short course of two antibiotics (mostly clarithromycin and amoxicillin) along with proton pump inhibitors (e.g., omeprazole or lansoprazole). The treatment, however, is complicated by several factors like bacterial resistance to antibiotics, re-infection, side effects (bloating, diarrhea and taste disturbances) and alteration of healthy gut microbiota (Malnick et al., 2014;Zhang et al., 2015). The destruction of the commensal flora may lead to increased prevalence of opportunistic pathobionts, like Clostridium difficile (Malnick et al., 2014). Hence, the treatment of H. pylori using antibiotics has the risk for microbiota imbalance or dysbiosis, which may lead to other diseases. Also, eradication of H. pylori may lead to esophageal cancer (Blaser, 2008;Blaser and Falkow, 2009). Therefore, alternative approach that can eradicate or prevent H. pylori infection without affecting gut microbiota is needed.
Lactobacillus is normally present in human intestinal tract including stomach and it is tolerant to acid and bile (Ruiz et al., 2013). Therefore, Lactobacillus is an attractive candidate for probiotic for the treatment of H. pylori related gastric diseases. Bhatia et al. (1989) showed that the culture supernatant of Lactobacillus acidophilus inhibits H. pylori in vitro due to an extracellular secretory product. Direct application of L. acidophilus on blood agar plate can also inhibit H. pylori (Vilaichone et al., 2002). Subsequently, it was found that both L. acidophilus and L. casei subsp. rhamnosus can inhibit H. pylori due to the production of lactic acid (Midolo et al., 1995;Enany and Abdalla, 2015). The lactic acid produced by L. casei strain Shirota inhibits 70% of urease activity in vitro and significantly reduces the levels of H. pylori colonization in mouse model (Sgouras et al., 2004). Lorca et al. (2001) studied antibacterial activity of 17 Lactobacillus strains on 10 H. pylori strains and concluded that the inhibition was due to acid production. They also found that autolysis of L. acidophilus after 24 h of culture releases a proteinaceous compound and this event is related to the bactericidal effect (Lorca et al., 2001). Furthermore, H. pylori colonized mice when treated with a commercial mixture of live probiotics (L. rhamnosus, strain R0011, and L. acidophilus, strain R0052) they suppressed colonization of H. pylori strain SS1 (Johnson-Henry et al., 2004).
H. pylori infected MKN45 cells showed increased expression of Smad7 and NFkB, and induced pro-inflammatory cytokines IL-8 and TNF-α in vitro. Probiotic L. acidophilus pre-treatment, however, inactivate the Smad7 and NFkB pathways and reduces the H. pylori induced inflammation . Using gnotobiotic murine model, it was shown that L. salivarius infection also inhibits the colonization of H. pylori and associated inflammatory responses like IL-8 release (Kabir et al., 1997;Avía et al., 1998).
Since in vitro experiments and in vivo mouse studies showed promising results, a significant number of clinical trials have been performed in the recent past (Table 1). Several metaanalyses published in 2013 revealed that addition of probiotics in triple therapy against H. pylori improves overall efficacy and reduces the side effects of therapy like nausea, diarrhea metallic taste, abdominal/epigastric pain (Ruggiero, 2014). However, it needs further improvement since the benefits conferred by the probiotics are often not too remarkable. For example, a meta-analysis based on literature search strategy suggest that use of probiotics (mostly Lactobacillus, Bifidobacterium and Streptococcus and in few trials Enterococcus, Clostridium, Saccharomyces etc) in triple therapy improve eradication rate of H. pylori by ∼10% and reduce adverse effects of therapy by ∼15% (Zhang et al., 2015).

ROLE OF FERMENTED FOODS AND BEVERAGES AGAINST H. pylori ASSOCIATED DISEASES
Fermentation of food dates back to the early ages of human evolution and provides an effective way of preserving food for longer durations (McGovern et al., 2004). Many of the bacteria, yeasts and molds that are present in fermented foods and beverages are known probiotics and probably provide health benefits when consumed raw (Stanton et al., 2005). The significance of the microbes present in fermented food in maintaining human health was first noticed by Elie Metchnikoff (Mackowiak, 2013). He hypothesized that the long and healthy lives of Bulgarian peasants were due to the regular consumption of sour milk and yogurt containing the necessary beneficial microbes (Mackowiak, 2013).
Many of the probiotic that are isolated directly from the fermented foods, particularly fermented dairy products, have anti H. pylori effects. Based on dietary interviews it was found that yogurt, but not unfermented dairy products, when consumed one serving per week or more has protective effect against H. pylori infection in Mexican population (Ornelas et al., 2007). Several strains of Lactobacilli and two strains of yeast directly isolated from yogurt were found to have inhibitory effect on H. pylori (Oh et al., 2002). A meta-analysis of randomized controlled trials shows that there is ∼10% improvement in eradication rates when using fermented milk based probiotics, which seems to be better than capsule/sachet-based bacteriaonly preparations (Sachdeva and Nagpal, 2009). Similarly, 4-week treatment with L. gasseri-containing yogurt improved the efficacy of triple therapy in patients with H. pylori infection (Deguchi et al., 2012). Another study showed that H. pylori infected children have a lower number of Bifidobacterium in their gut, but intake of probiotics-containing yogurt had multiple effects like, restoration of Bifidobacterium, reduction of H. pylori load, increase in IgA and decrease in IL-6 (Yang and Sheu, 2012). Three Single center, double-blind, prospective, randomized, placebo-controlled trial

Lactobacillus GG
Reduced side effects and overall treatment tolerability Armuzzi et al., 2001 strains of lactic acid bacteria, LY1, LY5 AND IF22, which are from the spent culture supernatant of fermented milk, showed anti-H. pylori effect (Lin et al., 2011). In china, several probiotics from traditional fermented foods were isolated and two strains of Lactobacillus-L. plantarum 18 and L. gasseri showed potential anti-H. pylori activity (Chen et al., 2010). Kefir, a fermented milk product was found to be effective in eradication and reducing side effects when used along with triple therapy (Bekar et al., 2011). An in vitro study proved that L. plantarum (MLBPL1) isolated from sauerkraut (fermented cabbage) had an anti-Helicobacter activity (Rokka et al., 2006). Interestingly, the main inhibitory activity is mostly associated with cell wall. Unfortunately, however, anti-H. pylori activity alone does not ensure protection from gastric diseases and gastric cancer may sometimes develop even after eradication of H. pylori since some of the H. pylori proteins like CagA may act by 'hit and run' mechanism (Shiota et al., 2013;Hatakeyama, 2014). More interestingly, prevalence of H. pylori and incidence of gastric diseases does not match in some countries. In Africa and India, the prevalence of H. pylori infection and associated gastritis is high, but the incidence of gastric cancer is very low (Holcombe, 1992;Singh and Ghoshal, 2006). On the other hand, East-Asian countries like Japan and Korea have high rates of gastric cancer (Singh and Ghoshal, 2006). Genotype alone cannot be responsible to explain the clinical outcome since nearly all H. pylori strains isolated from East-Asia are virulent (Shiota et al., 2013). Therefore, it is intriguing to compare the microbes that are present in traditional fermented foods and beverages of Japan or Korea and African countries ( Table 2). Apparently, fermented foods in African countries are based on milk, beans, grains and roots. They are dominated by Lactobacillus and other lactic acid bacteria. Conversely, Japanese fermented foods are primarily based on rice, soy and fish and these foods have varieties of bacteria and fungi. Interestingly, the soy foods may reduce the risk for gastric cancer, while high salt containing foods might be a risk factor in Japan and Korea (Hirayama, 1981;Woo et al., 2013).
A similar comparison would be exiting between the fermented foods of ethnic populations in North-Eastern India (e.g., ethnic populations in Sikkim state like Bhutias) and Mid-Eastern India (e.g., ethnic population of Jharkhand and West Bengal states like Santhals). North-Eastern states have highest incidence of gastric cancer in India (Pradhan et al., 2003(Pradhan et al., -2004. This high prevalence has been thought to be due to smoking and high salt consumption that possibly come from fermented and pickled foods including fish and meat (Phukan et al., 2005;Verma et al., 2012). Recent analyses of some of the fermented foods showed presence of huge microbial variety but their significances in gastric diseases have not been studied (Tamang and Sarkar, 1996;Tamang et al., 2016a,b). Unfortunately, not much is known about the microbes that are present in fermented foods consumed by the Santhals. But interestingly, they regularly consume intoxicating alcoholic beverages like Handia and Mahua fermented in traditional way and these beverages are not common elsewhere (Kumar and Rao, 2007). Among Santhals, infections with virulent H. pylori strains are extremely common without any manifestation of gastric diseases (Datta et al., 2003).
Our current understandings of microbes present in the ethnic fermented foods are incomplete at present, but with modern methodologies like metagenomic analysis using Nextgeneration sequencing the microbial species are now easy to identify (Mozzi et al., 2013). However, to prove or disprove the hypothesis-whether or not microbes present in the ethnic fermented food can protect certain population from peptic ulcer or gastric cancer is very tricky, particularly when H. pylori infection is not the only determinant in precipitating the gastric diseases (Parekh et al., 2014;De and Roychoudhury, 2015). How the microbes present in the ethnic fermented food can alter the pathogenicity of H. pylori in combination with gastric and duodenal microbiome as well as host immunity for different population is perhaps the key question at present.

CONCLUSION
H. pylori infection is the major risk factor for peptic ulcer and gastric cancer and the eradication of this bacterium using antibiotics is often unsuccessful. Several microbes with known probiotic activities are shown to have inhibitory effects against H. pylori in vitro and in vivo. Inclusion of probiotics in triple therapy leads to improved efficacy and reduced side effects. Most traditional fermented foods and beverages are natural sources of probiotic microbes. Microbes directly isolated from the fermented products are shown to have anti-H. pylori activity. Few studies showed that consumption of probiotics containing yogurt and kefir are somewhat beneficial in the context of H. pylori infection. Many ethnic populations have significantly low incidences of peptic ulcer and gastric cancer in spite of having very high prevalence of H. pylori infection. Incidentally, each ethnic population also has unique tradition of consuming fermented food and beverages that contain probiotics. It is intriguing to hypothesize that regular consumptions of these probiotics may have protective effect against peptic ulcer and gastric cancer for some populations. Analyzing these traditional fermented foods and beverages using modern techniques is needed to understand these microbes and their significances.

AUTHOR CONTRIBUTIONS
MN and DC equally contributed 60% of the mini-review works. SG contributed 15% and SC contributed 25% in the mini-review works.

FUNDING
This work was supported by Rajiv Gandhi Centre for Biotechnology (an autonomous institute, sponsored by Department of Biotechnology, Govt. of India).