4
five conventional media, supplemented with either Helicobacter pylori or
Campylobacter
selective supplement (Table 2). Positive controls were the same as described
above. All plates
were incubated in microaerophillic atmosphere at 37oC for up to 7 days to
provide the optimal
environment for culturing Helicobacter.
Formulation of enhanced selectivity (HP) media
HP media preparation requires a sequential addition of components. The mixture
of
special peptone, beef extract, yeast extract, NaCl, phenol red, agar, and
water was autoclaved for
20 minutes at 121oC and then tempered to 50oC. Then calf serum with iron,
antibiotics
(vancomycin trimethoprim, cefsulodin, Amphotericin B, and Polymixin B), and
urea were
aseptically added with constant stirring. Finally 0.8 ml of 1N HCl was added
drop-wise to the
media as color changed from red to yellow/orange (final pH 5.7 @ 45oC, pH
6.0 @ 22oC). The
media was then poured into petri plates.
Table 3. Media components evaluated over a range of concentrations to develop
the final HP
formulation.
Component Range tested per liter a optimum level
Porcine mucin 0 - 4 g O
Ferrous sulfate 0 - 500 mg O
Na pyruvate 0 - 500 mg O
Polymixin B 0 - 4000 units 3500 units
Amphotericin B 0 - 7.5 mg 7.5 mg
Vancomycin 0 - 10 mg 10 mg
Trimethoprim 0 - 5 mg 5 mg
Cefsulodin 0 - 5 mg 5 mg
Urea 0 - 1 mg 600 mg
Phenol red 0 - 200 mg 100 mg
1 N HCl 0 - 2 ml 0.8 ml
a optimum level for recovering H. pylori while inhibiting background flora.
Well sample testing
About three hundred fifty private drinking water wells across Wisconsin were
screened
using HP agar, immunomagnetic separation (IMS), and/or fluorescent antibody
staining (FA) for
the presence of H. pylori. The samples were grouped into two types, designated
random or
requested.
Random samples were collected by taking aliquots from samples sent to the
WSLH
Water Bacteriology Department for routine coliform testing. For this group,
fifty to one hundred
ml aliquots were centrifuged at 2500 x g for 15 minutes and then all but
the bottom 5 mls of
supernatant was carefully siphoned away. 0.5 ml aliquots of the resulting
5 mls (or dilutions
thereof) were then spread plated onto HP agar plates and incubated in a microaerophilic
environment at 37oC for up to 7 days. Colonies appearing after 3 days with
reddish "halos"
(urease +), 1-2 mm in diameter, catalase and urease positive were considered
positive.
Subsequent microscopic examination showing helical, horseshoe, and/or coccoid
morphology
was used for verification. About 275 wells were represented in the grouping
of random samples.
					
				

					
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