Visual display of the Development and application of a plating media for detection of Helicobacter pylori in water

				
5
The requested sample group consisted of wells where a specific request to
test for H.
pylori was received from the well owner. These requests most likely resulted
from the fact that a
resident in the home served by the well had suffered from an H. pylori infection.
For this group,
0.5 liter was concentrated to 5 ml via centrifugation. Four mls of the final
concentration was
spread onto HP agar plates (0.5 ml per plate) and incubated as described.
For the requested
sample group, two additional methods were employed in an attempt to recover
H. pylori. In
addition to the culture another 1 ml aliquot was assayed using immunomagnetic
separation
(IMS). Briefly, the 1 ml aliquot is mixed with microscopic magnetic beads
(Dynabeads M-450,
Dynal ASA, Oslo, Norway) coated with antibodies specific to H. pylori. The
quantity of beads
and duration of contact mixing time was determined by manufacturer's recommendations
to
provide the optimal exposure of capture beads to target organism. During
this process the
bacteria immunologically attach to the magnetic particles. A magnetic tube
rack is then used to
retrieve the beads and attached H. pylori. The retrieved beads are then plated
onto the HP agar.
A third procedure involving direct fluorescent antibody staining of a filtered
sample was also
employed as a non culture method control on some samples. For the fluorescent
antibody (FA)
staining, 0.5 liter of sample water was passed through a 0.4 micron pore
size filter to capture H.
pylori. The filter was treated with fluorescent stain (IgG Fluor, Chemicon
International,
Temecula, CA) attached to H. pylori-specific antibodies (Biodesign International,
Saco, ME) that
attach to the target cell if present. Cells reflecting green under fluorescent
light microscope and
helical, horse-shoe, or folded shape were considered H. pylori (note: non-viable
as well as viable
cells will fluoresce). IMS was used when samples contained visibly high particulate
matter and
FA staining was used to assay clear water.
RESULTS & DISCUSSION
Media evaluation
Growth of pure cultures on conventional media.
Table 2 lists the five conventional media formulations evaluated. Each of
these media
was evaluated for its ability to recover H. pylori from among a population
of 7 spiked strains of
bacteria and various indigenous strains contained in a sample of well water
(Table 5). As
expected, media without the antibiotic supplement allowed the growth of all
organisms tested.
The rapid growth of the non H. pylori bacteria covered the plates precluding
any chance of
detecting the slow growing H. pylori organisms. The addition of selective
supplements provided
some measure of selective pressure, however, some of the organisms (Acinetobacter,
E. coli,
Flavobactrum, Pasteurella, Ochrobactrum) were not inhibited and unacceptable
levels of
overgrowth still occurred. The selectivity profiles were identical among
the five conventional
media formulations, although it was noted that H. pylori colonies formed
most rapidly (84 hours)
on HPSPA media.
Since the selectivity of the five conventional media proved inadequate to
isolate H. pylori
from the complex flora water samples it was determined that an enhanced selective
media would
be required. To develop the formula for this new media, components were individually
evaluated
to determine their contribution to the selective and nutritive properties
necessary to isolate H.
pylori from a mixed population of microbial contaminants. Some nutritive
components (yeast
extract, beef extract, special peptone [Oxoid], NaCl) were incorporated at
conventional
concentrations without further evaluation. In order to develop a media with
enhanced selectivity
for Helicobacter, a list of selective, nutritional, and differential components
were evaluated
(Table 3). The properties of an improved formulation would include a broader
inhibition