Helicobacter pylori and the gastric microbiota

Helicobacter pylori and the gastric microbiota

Best Practice & Research Clinical Gastroenterology 27 (2013) 39–45 Contents lists available at SciVerse ScienceDirect Best Practice & Research Clini...

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Best Practice & Research Clinical Gastroenterology 27 (2013) 39–45

Contents lists available at SciVerse ScienceDirect

Best Practice & Research Clinical Gastroenterology

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Helicobacter pylori and the gastric microbiota Lars Engstrand, MD, Professor *, Mathilda Lindberg, PhD, Reserach Fellow Department of Microbiology, Tumor & Cell Biology, Karolinska Institutet and Science for Life Laboratory, Stockholm, Sweden

a b s t r a c t Keywords: Stomach Gastric microbiota Helicobacter pylori Gastric cancer Atrophic gastritis Acid reducing medication

The human microbiota along the gastrointestinal tract is currently extensively studied and a number of studies focuses on elucidating the association between a more or less diverse intestinal microbial community and health and disease. The human stomach is considered to be exclusively inhabited by Helicobacter pylori and further lacks a colonizing non-H. pylori bacterial flora due to the acidic environment. However, recently a limited number of studies using molecular-based methods have provided a broader picture of the stomach microbiota. The question is whether changes in gastric pH or antibiotic treatment can lead to significant shifts in the stomach microbiota that may be involved in disease development such as gastric cancer. Ó 2013 Published by Elsevier Ltd.

Introduction It is well known that Helicobacter pylori is capable of colonizing the gastric mucosa and forms the main cause of chronic active gastritis [1]. This inflammation can progress to peptic ulcer disease and premalignant gastric lesions, e.g. atrophic gastritis [2]. On the other side, H. pylori also serves the useful function of regulating immune cells in the stomach. Also, the widespread use of antibiotics early in life may have profound effects over time since repeated high doses of antibiotics during childhood have led to that a shrinking share of adults is infected with H. pylori today [3]. Little is known about other members of the human gastric microbial ecosystem in health and disease. The harsh gastric environment have led to assumptions that the human stomach does not harbour a complex microbiota. Characterization of the gastric microbiota has traditionally relied on cultivation of * Corresponding author. Tel.: þ46 70 678 0318. E-mail addresses: [email protected], [email protected] (L. Engstrand). 1521-6918/$ – see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bpg.2013.03.016

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gastric juice or mucosal biopsies [4]. Most of the microbiota in the GI-tract is located in the intestinal lumen or in the loose mucus close to the lumen without direct contact to the epithelium [5,6]. The mucus layer in the stomach and the GI-tract is divided into the firmly attached mucus layer and the loose mucus layer. The mucus layer is a protective barrier that prevents epithelial colonization of microbes and prevents diffusion of gastric acid, to the epithelial surface. Thus, inflammation in the GI-tract is caused either by bacterial penetration of the mucus layer or by a disruption of the mucus layer [6]. Mobility and ability to get through the mucus layer is highly dependant on the bacterial motility, shape and by their mucus degrading ability [7]. In addition, the viscosity of the gastric mucus layer is pH dependant where an increase in pH results in a lower viscosity that is easier to penetrate for microorganisms [8]. When molecular based techniques were introduced, most PCR-based compositional studies of the gastric microbiota were focused on specific genera, e.g. Lactobacilli and Helicobacter [9]. Attempts to further characterize the bacterial profiles in gastric biopsy specimens obtained from patients with H. pylori-associated gastritis were taken by using temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments [10]. Genera including Enterococcus, Pseudomonas and Staphylococcus were detected and Helicobacter spp. appeared to be part of a complex, presumably indigenous, microbial flora in the stomach (Fig. 1).

Fig. 1. Upper: Heatmap of six stomach biopsies. High abundance of H. pylori is illustrated by red colour, low abundance is illustrated by blue colour. Each band represents a different genera in the stomach. Biopsy 1 ¼ H. pylori-associated gastritis with normal acid production and non-atrophic gastric mucosa. Biopsy 6 ¼ Severe atrophic gastritis with low acid production and no H. pylori present. Biopsy 2–5 ¼ gradually scoring of atrophic gastritis where 2 is low and 5 is high. When H. pylori abundance decreases the number of non-H. pylori genera increase in the stomach e.g. a more diverse microbiota develops. Lower: Proposed development of gastric cancer. Factors that influence the microbiota composition in the stomach are here antibiotic and PPI treatment. The invasion of nonH. pylori microbiota in the stomach over the years is illustrated by the blue arrow.

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Introduction of sequence based molecular methods showed that H. pylori was the most abundant phylotype in the human stomach in H. pylori positive subjects [9,11]. Whether the stomach is home to distinct bacterial communities that interacts with the host remains however to be verified. A better understanding of the resident microbial communities in health and disease should shed light on the pathogenesis, diagnosis, and treatment of gastric illnesses. By exploring the structure and dynamics of microbial communities, the relationships between their members, the interaction with the host, and differences between communities in health and disease, we will hopefully achieve deeper understanding on microbial ecology and the involvement of the microbiota in disease development [12]. Culture of gastric biopsies and/or gastric juice has been the method of choice before the introduction of molecular techniques. Classical microbiological characterization of the gastric microbiota underestimated the biodiversity but provided phenotypic data on the species level. It is generally accepted that approximately 80% of the bacteria found by molecular tools in the human gut cannot be cultured [13]. Consequently, the major population easily isolated from stools is composed of bacteria that grow quickly in classical high-nutrient growth media. Examples where culture-based techniques have been used are investigation of anti-H. pylori treatment to study the ecological effect on the gastric microbiota [14] and studies of co-existens rates of Lactobacillus spp. and H. pylori in the stomach [15,16]. The gastric samples were inoculated on selective and non-selective agar media and incubated both aerobically and anaerobically [17]. Different colony types could then be identified to genus and species levels by morphological and biochemical analysis. Mass spectrometry for identification of pathogenic bacteria obtained after culture are currently widely used in clinical microbiology laboratories as a complement to standard identification procedures [18]. Culture-independent investigation of the human microbiota is one of the most studied areas of microbiology today. It also has a clear potential to benefit clinical practice. Most analysis of community structure focuses on sequencing of targeted regions e.g. the 16S rRNA gene using different high-throughput sequencing platforms [19]. The methodology is becoming cheap, more sophisticated and advances are made every year [20]. Short run-time instruments and reductions in sample size will also help to introduce microbiota analysis in clinical practice. Physicians will most likely within a couple of years request information obtained from microbiota analyses to direct treatment and apply these methods as diagnostic and preventive tools in daily work with patients [21]. One problem associated with genomic data is that it does not address whether an organism is alive or not. Antibiotic treatment will kill the bacteria but not remove the DNA from the sample. This problem can however be complemented with transcriptome analysis, or proteomic, and metabolomic data sets, which analyse gene expression and metabolic data that are more likely to be derived specifically from living cells. Also, the simultaneous advances in human genetics and genomics offer opportunities for combining studies of host genotype with microbiota phenotype. Methods for viewing the microbiota as a quantitative trait and relating this to host genotype are being developed [22].

Practice points  New DNA-based diagnostic tools to get information about the gastric microbiota are available.  The structures of microbial communities in the GI-tract are associated with health and disease  Acid reducing medication such as PPI are affecting the gastric environment and gastric microbiota composition.

The oral and upper GI tract microbiota The oral cavity has a high abundance of microorganisms with 109 bacteria per mL saliva and 1011 bacteria per gram dental plaque [23]. The microbiota in the healthy oral cavity is site specific but some species, such as Streptococcus mitis, Gemella adiacens and several Prevotella spp. are detected in most sites. The mouth has both soft and hard tissue and different species seem to preferentially colonize

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either of the tissue types. Great similarities have been found in the oral microbiota in different individuals and these species and genera have been identified as the core microbiome. The most prevalent genera and families in the oral core microbiome are Streptococcus, Corynebacterium, Neisseria, Rothia, Veillonellaceae, Heamophilus, Actinomyces, Granulicatella and Prevotella [24]. This is also consistent with findings in throat swabs [25]. Bik et al found similar results in the oral cavity when ten healthy individuals were analysed and at least 15 bacterial genera were conserved among the individuals [26]. Probably these genera follow the swallowed saliva further down in the GI tract and it is unclear if they colonize this niche or just pass. In biopsies obtained from oesophagus a similar but more sparse biota has been found, with Streptococcus, Rothia, Veillonellaceae, Granulicatella and Prevotella as the most prevalent genera [27]. It was also found that the oral microbiota was more stable after antibiotic treatment and over long periods compared to the intestinal microbiota [25]. The core gastric microbiota in a healthy stomach In the normal acidic stomach a sparse cultivable non-Helicobacter microbiota has been found dominated by Veillonella spp., Lactobacillus spp., and Clostridium spp. [28]. A more diverse microbiota has been seen when using 16S rRNA based methods and the main genera found in stomachs have except Helicobacter been, Streptococcus, Prevotella, Veillonella and Rothia [9,11,29]. The gastric microbiota is well adapted to the gastric environment and also to environmental changes in their specific stomach. Both lactobacilli and streptococci are among the species that have been cultured from stomach samples [15,16,28,29]. Using molecular based methods the most commonly found genus in the stomach is streptococci [9,11,29]. By sequencing we determined that, among 13 individuals with normal pathology and without dominance of Helicobacter sequences, the majority of the phyla/genera were represented in all individuals (our own unpublished data). The five most abundant genera from two other studies [9,29] were all among the ten most abundant in our study. Thus, a core microbiota with and without H. pylori can be defined even though different approaches for DNA extraction, primer design and sequencing were used in each study. Two specific streptococci in a majority of individuals (unpublished data) were identified representing the Streptococcus mitis group i.e. streptococci that are aciduric, can survive in a pH around four and produce hydrogen peroxide [30,31] and are considered to belong to the commensal oral microbiota [32]. In addition, the Streptococcus mitis group was the most frequently isolated streptococci by culturing in a previous study [29] and was the most common sequence match within the streptococci grouping in other 16S rRNA gene based studies [9]. When analysing the cultivable lactic acid bacteria (LAB) community on Rogosa agar plates Streptococcus salivarius was the most commonly isolated streptococci and present in 7% of the individuals (our own unpublished data). Lactobacillus as well as Streptococcus belong to the LAB group. Although lactobacilli have been isolated from the human stomach they do not seem to be as common as streptococci. Of the different lactobacilli species that has been isolated from the human stomach Lactobacillus gasseri, Lactobacillus fermentum and Lactobacillus rhamnosus are most dominant [33]. It is not known if lactobacilli isolated from the stomach are true colonizers or derived from food or the oral cavity but we have previously been able to isolate clonally related Lactobacillus strains from two sampling occasions, four years apart, in four individuals. These observations indicate that there are Lactobacillus strains capable of colonizing the stomach (our own unpublished data). The gastric microbiota in corpus predominant atrophic gastritis Persistent infection of the gastric mucosa by H. pylori initiates an inflammatory cascade that progresses into atrophic gastritis, a condition associated with reduced capacity for secretion of gastric acid and an increased risk of developing gastric cancer. A reduced capacity for gastric acid secretion allows survival and proliferation of other microbes that normally are killed by the acidic environment. There are no published studies on characterization of the gastric microbiota in pre-cancer lesions i.e. exclusively in severe atrophic gastritis. A shift was however found in the most prevalent genera from Prevotella to Streptococcus in the atrophic stomach and this shift further confirms that the gastric

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microbiota is not only a reflection of the oral microbiota (our own unpublished data). The greatest increase in the atrophy group represent the mitis group within the Streptococcus spp. The gastric microbiota in gastric cancer The pH is increased in gastric cancer as in severe atrophic gastritis and consequently both conditions can lead to an increased number of bacteria in the gastric environment. In gastric cancer the number of bifidobacteria/lactobacilli, Veillonella and streptococci are increased as determined by culture [17,34]. We investigated the gastric microbiota from ten patients with gastric cancer and compared with the gastric microbiota from five dyspeptic controls [35]. The analysis revealed a complex bacterial community in the cancer patients that was not significantly different from that in the controls. Sequencing revealed 102 phylotypes, with representatives from five bacterial phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria). The gastric cancer microbiota was dominated by different species of the genera Streptococcus, Lactobacillus, Veillonella and Prevotella. The respective role of these species in development of gastric cancer remains to be determined. Among the Streptococcus a group including S. mitis and Streptococcus parasanguinis was shown to dominate [35]. The changes in the microbiota in individuals with gastric cancer resemble those seen in the atrophic stomach (our own unpubished data). The importance of H. pylori and non-H. pylori bacteria in the pathogenesis of atrophic gastritis and gastric cancer needs further studies. The gastric microbiota in individuals treated with acid reducing drugs Treatment with proton pump inhibitors (PPI) has been shown to alter the gastric microbiota by providing bacterial overgrowth in the stomach. The non-H. pylori flora colonizes the gastric mucosa of a large proportion of patients treated long-term with acid inhibition [36]. This overgrowth consists mainly of oral bacteria that instead of being killed in the normally acidic stomach survives. It has also been speculated that acid suppressive drugs may lead to gastric cancer because of the bacterial overgrowth. Suppression of gastric acid will alter the bacterial flora of the upper gastrointestinal tract and studies have confirmed that PPIs do alter the bacterial population in the stomach [37] Further research to more fully quantify the risk associated with PPI therapy is required. However, there is a possibility that bacteria can enhance the production of carcinogenic nitrosamines. It was found that the increased risk of atrophy development is only seen when acid suppressive drugs are used in H. pylori positive subjects [38,39]. No increased risk has been observed in H. pylori negative individuals or after H. pylori eradication. This suggests that the microbiota acquired cannot cause atrophy on its own but could enhance atrophy development together with H. pylori [40]. It has also been shown that a previously antrum dominated H. pylori infection after treatment with acid inhibitors shifted to a more corpus predominant infection [36]. The less acidic pH in corpus promotes H. pylori to penetrate deeper in the gastric pits and increase the inflammation and provide a faster progression to atrophy [41]. As discussed above treatment with acid inhibitory drugs has by cultivation methods been shown to effect survival of bacteria in the stomach. However, no significant differences has been found regarding diversity and composition of the microbiota by using 16S rRNA gene based methods [9]

Research agenda:  The possible impact of the non-H. pylori microbiota in the stomach needs to be defined.  Options to manipulate the gastric microbiota to prevent gastro-duodenal diseases should be studied.  H. pylori as part of the commensal flora is controversial. The eventual protective effect of H. pylori colonization against certain diseases should be further evaluated in well-designed prospective epidemiological studies.

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Summary The discovery of H. pylori and the introduction of molecular-based methods to determine the microbiota have increased the interest in studies of the gastric microbiota in health and disease. Most microbiota studies in the stomach have so far been limited to descriptive results with speculations about a possible role of the gastric microbiota in disease development. Consequently, there are still many questions to be answered. We do not know whether a majority of the non-H. pylori microbiota in the stomach colonizes or just pass the gastric niche even though some species of lactobacilli seem to colonize the stomach. We need more studies to clarify the gastrointestinal effects of acid suppressive therapy and the consequences of intragastric bacterial overgrowth in the presence or absence of H. pylori. The most important area of research today is to design longitudinal prospective studies in humans to clarify the role of the non-H. pylori microbiota. If we can perform such studies and narrow in on the true colonizers of the stomach e.g. the bacteria that interacts and may be involved in pathogenesis, we will hopefully in the near future provide therapeutic alternatives e.g. probiotics to restore a healthy gastric microbiota. Conflict of interest None. Acknowledgement We thank the Söderbergs foundation for support. References [1] Sibony M, Jones NL. Recent advances in Helicobacter pylori pathogenesis. Current Opinion in Gastroenterology 2012 Jan; 28(1):30–5. [2] Conteduca V, Sansonno D, Lauletta G, Russi S, Ingravallo G, Dammacco FH. Pylori infection and gastric cancer: state of the art (Review). International Journal of Oncology 2013 Jan;42(1):5–18. [3] Blaser MJ. Equilibria of humans and our indigenous microbiota affecting asthma. Proceedings of the American Thoracic Society 2012 May;9(2):69–71. [4] Lagier J-C, Million M, Hugon P, Armougom F, Raoult D. Human gut microbiota: repertoire and variations. Frontiers in Cellular and Infection Microbiology 2012 Jan;2:136. [5] Van der Waaij LA, Harmsen HJM, Madjipour M, Kroese FGM, Zwiers M, Van Dullemen HM, et al. Bacterial population analysis of human colon and terminal ileum biopsies with 16S rRNA-based fluorescent probes: commensal bacteria live in suspension and have no direct contact with epithelial cells. Inflammatory Bowel Diseases 2005 Oct;11(10):865–71. [6] Swidsinski A, Loening-Baucke V, Theissig F, Engelhardt H, Bengmark S, Koch S, et al. Comparative study of the intestinal mucus barrier in normal and inflamed colon. Gut 2007 Mar;56(3):343–50. [7] Swidsinski A, Sydora BC, Doerffel Y, Loening-Baucke V, Vaneechoutte M, Lupicki M, et al. Viscosity gradient within the mucus layer determines the mucosal barrier function and the spatial organization of the intestinal microbiota. Inflammatory Bowel Diseases 2007 Aug;13(8):963–70. [8] Goddard AF, Spiller RC. The effect of omeprazole on gastric juice viscosity, pH and bacterial counts. Alimentary Pharmacology & Therapeutics 1996 Feb;10(1):105–9. [9] Bik EM, Eckburg PB, Gill SR, Nelson KE, Purdom EA, Francois F, et al. Molecular analysis of the bacterial microbiota in the human stomach. Proceedings of the National Academy of Sciences of the United States of America 2006 Jan 17;103(3): 732–7. [10] Monstein HJ, Tiveljung A, Kraft CH, Borch K, Jonasson J. Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis. Journal of Medical Microbiology 2000 Sep;49(9):817–22. [11] Andersson AF, Lindberg M, Jakobsson H, Bäckhed F, Nyrén P, Engstrand L. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PloS One 2008 Jan;3(7). e2836. [12] De Vos WM, De Vos EAJ. Role of the intestinal microbiome in health and disease: from correlation to causation. Nutrition Reviews 2012 Aug;70(Suppl. 1):S45–56. [13] Vartoukian SR, Palmer RM, Wade WG. Strategies for culture of “unculturable” bacteria. FEMS Microbiology Letters 2010 Aug 1;309(1):1–7. [14] Adamsson I, Nord CE, Lundquist P, Sjöstedt S, Edlund C. Comparative effects of omeprazole, amoxycillin plus metronidazole versus omeprazole, clarithromycin plus metronidazole on the oral, gastric and intestinal microflora in Helicobacter pylori-infected patients. The Journal of Antimicrobial Chemotherapy 1999 Nov;44(5):629–40. [15] Roos S, Engstrand L, Jonsson H. Lactobacillus gastricus sp. nov., Lactobacillus antri sp. nov., Lactobacillus kalixensis sp. nov. and Lactobacillus ultunensis sp. nov., isolated from human stomach mucosa. International Journal of Systematic and Evolutionary Microbiology 2005 Jan;55(Pt 1):77–82.

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