Distal esophagus microbiota in gastroesophageal reflux disease

The purpose of this study is to compare persons with and without reflux in terms of microbial load and presence of selected microorganism species. Within the scope of this study, patients diagnosed with reflux due to regurgitation of sour liquid two or more times a week and a burning sensation in the chest, and control group patients investigated for anemia underwent upper gastrointestinal tract endoscopy. Fifty patients who presented to İzmir Katip Çelebi University Atatürk Education Research Hospital between February 2015 and June 2015 were included in the study. Of them, 30 were assigned to the reflux group and 20 to the control group. Informed consent was obtained from all those involved in the study. During the endoscopic procedure performed under anesthesia, two biopsy specimens were taken from the distal 5 cm of esophagus gastroesophageal junction using forceps. One of the biopsies was sent to the pathology laboratory in formaldehyde for histological examination. The other biopsy material was stored in glycerol in Eppendorf tubes at -80°C to use in planned molecular studies. After all the biopsy specimens were collected, total DNA isolation was performed. The 16s rDNA region, which is universally found in bacteria, was amplified with specific primers and the ampfied target gene cloned into the Promega T Easy vector, because the cloning method is the most suitable method for detecting the bacterial load differences between the groups. Subsequently, recombinant plasmid was transferred to the E.coli JM109 component cell and then plasmid isolation was performed. In order to generate a standard curve in real time PCR, the number of copies of the target gene was calculated and found to be 8.27 x 109 gene copy number/µl. A standard curve was obtained as a result of the real time PCR performed by taking five concentrations after the dilutions. The number of the copies of the target gene observed after cloning was approximately 104- 105copy number in the gastroesophageal reflux patients and 104- 105copy number in the control group patients. The difference was not statistically significant (p <0,005). In the study, the presence of bacterial Prevotella intermedia, Streptococcus pyogenes, Veillonella parvula, Enterococcus faecalis, Fusobacterium nucleatum, Neisseria meningitidis, Bacteroides fragilis, Lactobacillus acidophilus and fungal Candida albicans species was investigated using the real time PCR method in which the species-specific primers were used. In the 30 reflux patients included in the study, the percentages of the species determined conducted with the real time PCR were as follows: Prevotella intermedia (23%), Veillonella parvula (17%), Fusobacterium nucleatum (17%), Neisseria meningitidis (17%), Bacteroides fragilis (10%), Lactobacillus acidophilus (3%) and Streptococcus pyogenes (27%). In the 20 control group patients the percentages were as follows: Prevotella intermedia (20%), Veillonella parvula (15%), Fusobacterium nucleatum (10%), Neisseria meningitidis (10%), Bacteroides fragilis (20%), Lactobacillus acidophilus (15%) and Streptococcus pyogenes (35%). A informed volunteer form was developed by asking all the participants various questions. The responses given to the questions in the form revealed that as the gastroesophageal reflux patients' Body Mass Index increased so did the number of Veillonella parvula, Fusobacterium nucleatum, Neisseria meningitidis and Helicobacter pylori significantly (p <0.005). The Helicobacter pylori species was statistically significantly higher in the reflux patients who smoked or took alcohol than in the control group patients (p <0.005). According to the responses, Veillonella parvula, Fusobacterium nucleatum and Neisseria meningitidis species were dominant in the reflux group patients having undergone cholecystectomy (gallbladder surgery) while Fusobacterium nucleatum, Neisseria meningitidis and Bacteroides fragilis species were statistically significantly high in the control group patients (p <0.005). No significant data were obtained in the patients using the proton pump inhibitor in both groups in terms of the species detected. The amounts of Helicobacter pylori, Veillonella parvula, Fusobacterium nucleatum, Neisseria meningitidis and Streptococcus pyogenes species were statistically significantly high in the reflux patients taking pain relievers for a long time (p <0.005). The amounts of Veillonella parvula, Fusobacterium nucleatum, Neisseria meningitidis and Streptococcus pyogenes were significantly high in reflux patients with Grade A esophagitis (p <0.005). Veilonella parvula, Fusobacterium nucleatum and Neisseria meningitidis species were higher in the Helicobacter pylori-positive reflux patients (p <0.005). According to the results, these microorganism most of which are Gr (-) and reside in the mouth are present in the distal esophagus in patients with gastroesophageal reflux disease. There was no difference between the number of total bacteria in the two groups with distal esophageal reflux disease, but the types of bacterial varied. In addition to medical treatments, provision of probiotic medicines and giving appropriate nutritional advice can help these patients. Key words: Gastroesophageal reflux disease, Microbiota, Real time PCR, Cloning