Visualization of Helicobacter species within the murine cecal mucosa using specific fluorescence in situ hybridization.
BACKGROUND
Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species.


METHODS
Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections.


RESULTS
Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts.


CONCLUSIONS
We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.
Chan
Vivian
V
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
Crocetti
Gregory
G
Grehan
Martin
M
Zhang
Li
L
Danon
Stephen
S
Lee
Adrian
A
Mitchell
Hazel
H
D000818:Animals; D002432:Cecum / Q000382:microbiology* / Q000473:pathology; D016998:Helicobacter / Q000235:genetics / Q000302:isolation & purification*; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology; D044923:Helicobacter hepaticus / Q000235:genetics / Q000302:isolation & purification; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016753:Interleukin-10 / Q000172:deficiency / Q000235:genetics; D007413:Intestinal Mucosa / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D016753:Interleukin-10