title: Pseudomonas fluorescens-like bacteria from the stomach: a microbiological and molecular study.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi UP 221005, India.
authors: Saurabh Kumar
authors: SK
authors: Patel
authors: Chandra Bhan
authors: CB
authors: Pratap
authors: Ajay Kumar
authors: AK
authors: Verma
authors: Ashok Kumar
authors: AK
authors: Jain
authors: Vinod Kumar
authors: VK
authors: Dixit
authors: Gopal
authors: G
authors: Nath
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001706:Biopsy; D018834:Chaperonin 60 / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability; D010088:Oxidoreductases / Q000378:metabolism; D010802:Phylogeny; D011551:Pseudomonas fluorescens / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D011706:Pyloric Antrum / Q000382:microbiology*; D012329:RNA, Bacterial / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D021521:Ribotyping; D014510:Urease / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D010088:Oxidoreductases; D014510:Urease
keywords: Acid-tolerant bacteria; Antral biopsy; HSP60; Helicobacter pylori; Nested polymerase chain reaction; Pseudomonas fluorescens