Original Article
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World J Gastroenterol. Feb 21, 2013; 19(7): 1056-1067
Published online Feb 21, 2013. doi: 10.3748/wjg.v19.i7.1056
Pseudomonas fluorescens-like bacteria from the stomach: A microbiological and molecular study
Saurabh Kumar Patel, Chandra Bhan Pratap, Ajay Kumar Verma, Ashok Kumar Jain, Vinod Kumar Dixit, Gopal Nath
Saurabh Kumar Patel, Chandra Bhan Pratap, Ajay Kumar Verma, Gopal Nath, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi UP 221005, India
Ashok Kumar Jain, Vinod Kumar Dixit, Department of Gastroenterology, Institute of Medical Sciences, Banaras Hindu University, Varanasi UP 221005, India
Author contributions: Patel SK performed the major part of the experimental work, interpreted the results and prepared the manuscript; Pratap CB and Verma AK assisted in performing experiments; Jain AK and Dixit VK coordinated and helped in collection of human material, and also helped in shaping the manuscript; Nath G was responsible for genesis of the hypothesis, designing of the experiment, analysis of data and shaping of the manuscript; all authors read and approved the final draft.
Supported by Department of Biotechnology, Government of India, No. 102/IFD/SAN/PR1310/2006-07; Council of Scientific and Industrial Research, New Delhi, India, in the form of Senior Research Fellowship (to Patel SK)
Correspondence to: Dr. Gopal Nath, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Pandit Madan Mohan Malviya Rd, Varanasi UP 221005, India. gopalnath@gmail.com
Telephone: +91-542-6703484 Fax: +91-542-2367568
Received: July 19, 2012
Revised: September 13, 2012
Accepted: September 22, 2012
Published online: February 21, 2013
Abstract

AIM: To characterize oxidase- and urease-producing bacterial isolates, grown aerobically, that originated from antral biopsies of patients suffering from acid peptic diseases.

METHODS: A total of 258 antral biopsy specimens were subjected to isolation of bacteria followed by tests for oxidase and urease production, acid tolerance and aerobic growth. The selected isolates were further characterized by molecular techniques viz. amplifications for 16S rRNA using universal eubacterial and HSP60 gene specific primers. The amplicons were subjected to restriction analysis and partial sequencing. A phylogenetic tree was generated using unweighted pair group method with arithmetic mean (UPGMA) from evolutionary distance computed with bootstrap test of phylogeny. Assessment of acidity tolerance of bacteria isolated from antrum was performed using hydrochloric acid from 10-7 mol/L to 10-1 mol/L.

RESULTS: Of the 258 antral biopsy specimens collected from patients, 179 (69.4%) were positive for urease production by rapid urease test and 31% (80/258) yielded typical Helicobacter pylori (H. pylori) after 5-7 d of incubation under a microaerophilic environment. A total of 240 (93%) antral biopsies yielded homogeneous semi-translucent and small colonies after overnight incubation. The partial 16S rRNA sequences revealed that the isolates had 99% similarity with Pseudomonas species. A phylogenetic tree on the basis of 16S rRNA sequences denoted that JQ927226 and JQ927227 were likely to be related to Pseudomonas fluorescens (P. fluorescens). On the basis of HSP60 sequences applied to the UPGMA phylogenetic tree, it was observed that isolated strains in an aerobic environment were likely to be P. fluorescens, and HSP60 sequences had more discriminatory potential rather than 16S rRNA sequences. Interestingly, this bacterium was acid tolerant for hours at low pH. Further, a total of 250 (96.9%) genomic DNA samples of 258 biopsy specimens and DNA from 240 bacterial isolates were positive for the 613 bp amplicons by targeting P. fluorescens-specific conserved putative outer membrane protein gene sequences.

CONCLUSION: This study indicates that bacterial isolates from antral biopsies grown aerobically were P. fluorescens, and thus acid-tolerant bacteria other than H. pylori can also colonize the stomach and may be implicated in pathogenesis/protection.

Keywords: Antral biopsy, Helicobacter pylori, Pseudomonas fluorescens, HSP60, Nested polymerase chain reaction, Acid-tolerant bacteria