Microbiome insights into the perforated duodenal ulcer via metagenomic sequencing

Background: As a part of the digestive duct, duodenum, populates abundant commensal microbes, of which Helicobacter pylori was considered as the most widely and only recognized causative agent, especially in perforated duodenal ulcer patients. Although the most researches have been performed between duodenal ulcer diseases and the invader H. pylori , the relationship between perforated duodenal ulcer and the associated microbiome remained to be explored. Results: Metagenomic sequencing were used to profile the microbes underlying duodenal ulcer. The metagenome results of 6 duodenal ulcer samples (3 ulcer foci and3 sites around the ulcer foci) were obtained and compared. Ulcer foci and (healthy control (3 sites around the ulcer foci) were found to exist distinct microbial distributions. And interestingly, Streptococcus mitis was highly enriched in duodenal ulcer foci. Conclusion: The dysbiosis of microbiomes might underlie duodenal ulcer diseases, while the increased abundance of S. mitis is a novel finding. Whether there is causal link between S. mitis and duodenal ulcer remains to be determined. This represents the first report on the gut microbiome of duodenum or duodenal ulcer, that revealed that gut microbiota dysbiosis, especially S. mitis , might play an unexpected role in the disease. The S. mitis and microbiome dysbiosis can be new direction in the duodenal ulcer study. the collected for and part data


Background
Intestinal tract is one of the most important digestive apparatus closely relevant to human health [1,2], of which digestive tract is populated by a large number of 4 microbes [2], with cell numbers (10 14 ) exceed that of body cells (10 13 ) by a factor of ten [3]. These "aboriginal inhabitants" protect the host through communication with immune system. Activation of Toll-like receptors (TLRs) by commensal microflora is critical for the protection against gut injury and associated mortality, for these bacteria are recognized by TLRs under physiological status [4]. Beyond that, gut microbiota monitor multiple aspects of host, for instance energy harvest from the diet and energy storage [5], intestinal angiogenesis [6] and host immunity [7].
The gut microbes contribute to resist numerous invading pathogens [8][9][10]. Bacillus bifidus can protect or resist the ulcerative inflammation in gut [11], while the Escherichia coli often causes diarrhea [12].. The immunologic response and endogenous or exogenous compounds metabolism in the liver are interweaved with the gut microbes [10]. E. coli evolution abilityis associated with the gut microbiota homeostasis [13]. Inflammatory bowel diseases, Crohn's disease and ulcerative colitis, are chronic idiopathic disorders causing inflammation of the gastro-intestinal tract [14]. It's reported that more than 60 percent population was carrier of an opportunistic pathogen H. pylori [15,16], the culprit of superficial gastritis. The duodenum, one of the most important digestive apparatus, is located between stomach and jejunum. Although H. pylori was widely considered as a culprit of these gastroduodenal disorders [17,18],few direct evidences can be found for its role in duodenal ulcer, which was considered as the result of convulsion in stomach and duodenum. Thus, the further analysis between duodenal ulcer and its microbiome required to be performed.
In this study, metagenome sequencing was performed for 3 duodenal ulcer patients, 6 samples were collected including 3 ulcer foci and 3 sites around the ulcer foci.
Each patient contributed two samples, one from the ulcerate foci, the sites around the ulcer foci as control. Metagenomic data showed unexpected enrichment of Streptococcus mitis in the ulcer sites.

DNA Extraction and library construction
Samples separated from 3 patients were harvested and sent to Shanghai Biotechnology Corporation for metagenomic sequencing. Briefly, DNA was extracted according to conventional protocol. For the quality monitoring, the degradation and potential contamination were determined on agarose gels. DNA concentration was measured. The contents above 1µg are used to construct library. DNA sample was fragmented by sonication to a size of 350bp, then DNA fragments were endpolished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. At last, PCR products were purified (AMPure XP system) and libraries were analyzed for size distribution by Bioanalyzer and quantified using real-time PCR.

Data Analysis
The clustering of the index-coded samples was performed on a cBot Cluster Generation System according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform and paired-end reads were generated. The Clean Data is assembled and analyst [19] by SOAPdenovo software [20,21]. All samples' Clean Data are compared to each Scaffolds respectively by SoapAligner software (soap 2.21) to acquire the PE reads Gene prediction and abundance analysis 6 The Scaftigs (≥ 500 bp) assembled from both single and mixed are all predicted for the ORF by MetaGeneMark (V2.10, http://topaz.gatech.edu/GeneMark/) software, and the length information shorter than 100nt were filtered. For ORF predicted, CD-HIT software is adopted to redundancy and obtain the unique initial gene catalogue. The Clean Data of each sample is mapped to initial gene catalogue using SoapAlignerand. Genes with reads ≤ 2 in each sample was filtered and the gene catalogue

Results
The duodenal ulcer microbiota was significantly altered by metagenomic study.
To explore the metagenomic change of duodenal ulcer patients, 6 samples (3 ulcer foci and 3 healthy controls) were subjected to metagenomic sequencing. Consistent 8 with the role of gut microbiota change in metabolic disorders [8], multiple central metabolic pathways were underrepresented in duodenal ulcer patients, such as ABC transporters, amino acids biosynthesis, carbon metabolism and pyrimidine metabolism ( Figure 1A). These underrepresented pathways were highlighted ( Figure   1B). D-glutamine and D-glutamate metabolism, sphingolipid metabolism and nitrotoluene degradation were significantly decreased. Tetracycline biosynthesis pathway was enriched 3000 folds. Bacteria population density was auto-regulated [22]. The enrichment of tetracycline biosynthesis pathway and antibiotics resistance genes (with abundance over 0.03%) indicated the specifically proliferating of microbes bearing these genes ( Figure 1C). The percentage of duodenum microbes is similar between ulcer foci and the healthy control (Figure 2A), except the virus.

Discussion
Gut microbiota is increasingly recognized as key player in host healthy [10,20], largely due to close physical contact [23] and modulating the differentiation of immune cell subsets [24], the production of cytokines and chemokines to influence the T cell repertoire of the intestine and surrounding tissue, and the production of soluble immune mediator IgA [25,26]. Bifidobacterium abundance was changed by gene-diet interaction [27]. However, to our knowledge, there is no reports on the gut microbiota of duodenum or duodenal ulcer.
In this study, metagenomic results showed that bacterium instead of virus or fungi dominated the duodenum (Figure 2A). Though both health control and ulcer sites are populated with bacteria, significant difference exists. S. salivarius, S. parasanguinis, Megasphaera micronuciformis, Porphyromonas endodontails are abundant in health control ( Figure 2B), G. sanguinis, Fusobacterium periodonticum, G. haemolysans, S. mitis [28], bacteria frequently found in oral cavity, were found also dominant in ulcer foci ( Figure 2B). In general, the top 10 species of the ulcer foci are more abundant than TOP 10 in health control ( Figure 2B). The duodenal ulcer was accompanied by abundance change of dominant bacteria. Some hemolytic bacteria were found in duodenal ulcer, while absent in health control. G. haemolysans, G. sanguinis, S. mitis might underlie the duodenal ulcer. Gut type is a recently well recognized conception in gut microbiota field. Prevotella, Bacteroides and Ruminococcaceae were core for gut types identification [29]. In our results, Prevotella are predominant both in patients (6.9%) and health (6.9%), Bacteroides is presentin patients (0.2%) and absent in health. Ruminococcaceae (0. 0008%) was found in patients only. The result suggested that ulcer patients and healthy control have different gut type.
S. mitis is one of mitis group Streptococci, which are abundant members of the microbiota on all surfaces in the oral cavity and pharynx birth and through lifespan [28]. As an oral parasite, Streptococcus mitis is opportunistic pathogens implicated in dental caries [30], subacute bacterial endocarditis [31], brain abscesses [32]. The role of S. mitis in duodenal ulcer warrants further study. The copies of some bacterial genes involved in metabolic pathways are low in ulcer patients. The underrepresented KEGG [33] pathways are ABC transporters [34], amino acids biosynthesis [35] and carbon metabolism [36] (   The microbiota difference between the duodenal ulcer foci and site around the ulcer foci. A.

Figure 3
Microbes ratio of 353 duodenal ulcer patients cultured on agar.