A protocol for isolating putative Helicobacter pylori from fecal specimens and genotyping using vacA alleles.
BACKGROUND
This study outlines steps for isolating and culturing Helicobacter pylori from freshly voided fecal specimens and genotyping isolates for vacA alleles.


METHODS
A family with four H. pylori-infected members participated in this pilot study. Criterion for participation was a positive test for H. pylori by the urea breath test. Fecal specimens from children were taken from a freshly soiled diaper, placed in cold buffer, and prepared for culture in less than 2 hours. Culturing of H. pylori utilized selective culture media and isolates were screened for negative Gram stain, positive catalase and oxidase tests, and positive H. pylori 16S ribosomal RNA polymerase chain reaction (PCR). Strain types were determined by vacA genotyping.


RESULTS
The isolation procedure is relatively simple, although 5-7 days are required for H. pylori culturing. Isolation and purification of DNA eliminated PCR inhibitors and resulted in reliable analyses. All four family members were infected with the same H. pylori strain with a genotype of vacA s1a/m2.


CONCLUSIONS
This research lays the foundation for developing a routine and direct noninvasive method to detect the presence of H. pylori in fecal specimens. It is especially convenient for diagnosing children and infants, as samples can be obtained from soiled diapers. Culturing H. pylori from fecal samples in certain cases is important for antibiotic resistant studies prior to treating infected patients and for strain genotyping in epidemiological studies to determine transmission.
Liang
Shuwen
S
Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, USA.
Redlinger
Thomas
T
D000328:Adult; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods*; D001944:Breath Tests; D002374:Catalase / Q000378:metabolism; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D005838:Genotype; D005840:Gentian Violet; D016480:Helicobacter pylori / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D006801:Humans; D010105:Oxygenases / Q000378:metabolism; D010619:Phenazines; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D014508:Urea / Q000032:analysis
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; C035863:Gram's stain; D010619:Phenazines; C074689:VacA protein, Helicobacter pylori; D014508:Urea; D002374:Catalase; D010105:Oxygenases; D005840:Gentian Violet