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

				
8
Table 4. Formulation for one liter of HP media.
Component Amount
Special peptone 12 g
Yeast extract 5 g
Beef extract 5 g
NaCl 5 g
A calf serum with Fe 70 ml
Polymixin B 3500 units
Amphotericin B 7.5 mg
Vancomycin 10 mg
Trimethoprim 5 mg
Cefsulodin 5 mg
Urea 600 mg
Phenol red 100 mg
1 N HCl 0.8 ml
Agar 15 g
Distilled water 1 liter
a aseptically added post-autoclaving and tempering to 50oC.
b 1 N HCl added drop-wise to cause color change from red to yellowish.
Selectivity of modified HP media
The local well water used in these experiments contained about 400 native
heterotrophic
bacteria per 100 ml, some of which were identified as Flavobactrum, Serratia,
Citrobacter,
Pasteurella, Ochrobactrum, and Rahnella, using the API 20 E identification
system (Table 5).
In addition to the native flora, seven additional strains of bacteria and
an H. pylori cocktail were
added at levels of about 10,000 cfu/ 100 mls each. Dilutions of the adulterated
well water were
then plated onto BHI agar w/ 7% calf serum (positive control) and HP agar.
The plates were
incubated under microaerophillic conditions for up to 7 days and monitored
for colony
development. The positive control media without the selective agents became
overgrown with
bacterial colonies within 24 hours of incubation. The HP agar plates however
contained only
colonies of H. pylori over the seven day incubation. Colonies were presumptively
recognizable
within 72 + 8 hours (c.a. 14% less) because of the pH indicator and resultant
red halo indicating
urease production. In addition to shorter incubation periods, interference
from background
bacteria and/or molds was not problematic because of the increased levels
of antibiotics.
					
				
					
9
TABLE 5. Growth (+) and inhibition (none) of water related bacterial strains
on
conventional media with and without Helicobacter pylori selective supplements,
and on
newly formulated HP agar media.
Bacterial strains Solid media
Supplementary without with bHP formula
asupplement supplement
Acinetobacter
Aeromonas
Bacillus
E. coli
Enterobacter cloacea
Enterococcus faecalis
Helicobacter
Pseudomonas aeruginosa
+
+
+
+
+
+
+
+
+
none
none
+
none
none
+
none
none
none
none
none
none
none
+
none
Flora indigenous to well water
Flavobactrum
Serratia
Citrobacter
Pasteurella
Ochrobactrum
Rahnella
+
+
+
+
+
+
+
none
none
+
+
none
none
none
none
none
none
none
DISCUSSION
Five conventional media formulations showed that all were comparably nutritious
in
culturing H. pylori as well as the native and added background organisms
(Table 5). However,
developing an acceptable selective media for H. pylori in water presented
the classical
microbiological problem of finding a media that is nutritionally rich enough
to resuscitate and
grow this fastidious organism while managing to inhibit the growth of all
the other organisms
found in water samples. Helicobacter is a relatively slow-growing bacteria
usually requiring
about 4 days to develop discernible colonies. This genus can easily be overgrown
on solid media
by robust strains that grow readily within 24 hours, and thereby conceal
the presence of the
pathogen. The antibiotic resistance spectrum of H. pylori is well defined
but the concentrations
in media vary widely depending on the matrix in which the research is done.
For example,
trimethoprim and polymyxin B were used at 5 mg and 3,500 IU, respectively,
to isolate H. pylori
in a water matrix (Penner, 2000) but were increased to 40 mg and 62,000 IU
for selectivity in
cattle stomach (Stevenson, 2000). In this study, we focused on antibiotic
levels established by
commercial vendors (Oxoid, Becton Dickinson) and adjusted these components
in order to
achieve acceptable results.