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

				
1
DEVELOPMENT AND APPLICATION OF A PLATING MEDIA FOR DETECTION
OF HELICOBACTER PYLORI IN WATER
Alan J. Degnan*, Jon H. Standridge
Environmental Health Division
Wisconsin State Laboratory of Hygiene
2601 Agriculture Dr.
P.O. Box 7996
Madison, WI 53707-7996
ABSTRACT
In the U.S. alone, about 5,000,000 people are diagnosed annually with ulcers,
1,000,000
are hospitalized, 40,000 undergo surgery, and 6,500 die from ulcer-related
complications (Poms,
2001: Levin, 1998). Once thought to be a result of stress and/or diet, ulcers
are now almost
exclusively attributed to infection with the bacteria Helicobacter pylori.
Laboratory diagnosis of
H. pylori has become a standard procedure in the management of dyspeptic
patients. Although
transmission of the organism through the fecal/oral route is the assumed
infection route, the
possible mechanisms of human infection such as food, person to person contact,
water or fomites
are not clearly understood. There are a few reports in the literature suggesting
transmission of
H. pylori to humans via groundwater (Hegarty, 1999; Hulten, 1995 & 1998).
Methods of
detection used in those studies were relatively complex and costly (polymerase
chain reaction;
immunomagnetic separation) and unfortunately, didn't determine if the detected
organisms were
in fact viable or infectious. The work reported here focused on the development
of a
microbiological plating media that selects viable H. pylori organisms from
samples containing
mixed microbial populations, which could then be used for routine screening
of ground and/or
surface water for the presence of H. pylori. Efforts have resulted in a media
formulation that
allows the growth of Helicobacter while subsequently excluding common waterborne
microbial
background contaminants such as gram positive cocci and bacilli, enterobacteriaceae,
gram
negative bacilli, fungi, and pseudomonads. The laboratory-tested plating
media was used to
survey a cross section of Wisconsin groundwaters to further evaluate the
efficacy of the media
for recovering H. pylori from water samples and to begin a data base of H.
pylori occurrence.
INTRODUCTION
A scientific breakthrough occurred in 1982 when J. R. Warren and B. Marshall
isolated a
bacterium and showed that it caused gastritis and stomach ulcers that affect
millions of humans
worldwide (Marshall, 1984). Today that claim has been proven to the extent
that the National
Institute of Health recommends treatment with antibiotics for all patients
with peptic ulcers,
which are almost exclusively attributed to infection with the bacteria Helicobacter
pylori
(Graham, 1991). The scope of gastric illnesses around the world is vast.
In the U.S. alone,
estimates as high as 50% of adult Americans carry the pathogen, most asymptomatically,
and in
less-developed countries human carriers represent up to 90% of the populations
(Munangi,
1997).
The source of human infection is not yet known and until recently, the natural
reservoir
for H. pylori was thought to be the human gastrointestinal tract (Axon, 1996).
However,
isolation of Helicobacter from non-human sources such as livestock (Vaira,
1992), domestic cats
				
      
      
				
				
2
(Otto, 1994), and vegetables (Hopkins, 1993) prompted researchers to look
at environmental
sources as vectors for human infection. Previous efforts suggest the presence
of H. pylori in
ground water, surface water, and other drinking water (Mazari-Hiriart, 2001;
Yingzhi, 2002;
Hegarty, 1999; Hulten, 1998; Hulten, 1995; Shahamat, 1993), therefore implying
a waterborne
route of transmission to humans. The complex methods used in these studies
(polymerase chain
reaction; immunomagnetic separation; autoradiography; enzyme immunoassay)
are expensive to
run, subject to numerous interferences and do not differentiate between viable
and non-viable
organisms. A low cost and effective test to isolate viable H. pylori, similar
to selective media
used for Salmonella or E. coli, from ground and surface water would enable
the drinking water
industry to routinely screen samples. This study focused on efforts towards
development of a
plating media that selects viable H. pylori from real world water samples
containing mixed
microbial populations.
MATERIALS & METHODS
Media development
Bacterial strain management
Five clinical infection (no environmental isolates are available in U.S.
or European type
culture collections) Helicobacter pylori strains were obtained from the American
Type Culture
Collection (ATCC; Manassas, VA) or the Wisconsin State Laboratory of Hygiene
culture
collection (WSLH; Madison, WI; Table 1) and cultured on Brain Heart Infusion
agar (BHI;
Becton Dickinson, Sparks, MD) supplemented with 7% calf serum. The plates
were incubated
under a microaerophillic gas mixture (5% C02; 10% H2; 85% N2; Praxair, Inc.,
Danbury, CT) at
37oC. Frozen stock cultures were prepared by picking isolated colonies from
agar plates and
homogenizing in sodium phosphate buffer to about 107 colony forming units
(cfu)/ml using a
McFarland nephelometer. Each of the five isolates was then frozen in BHI
broth containing 10%
glycerol. Sufficient quantities were prepared to complete the entire study
in order to avoid
multiple passing of strains, which can sometimes lead to phenotypic variability.
The non-
Helicobacter bacteria used to create the complex spiked water samples (Table
1) were obtained
from the WSLH culture collection, grown on BHI agar slants at 35oC and then
stored at 4oC for
up to 3 weeks before re-culturing. The bacteria are listed in table 1.
TABLE 1. Bacterial strains and sources used in preparing water samples containing
known levels of contaminants.
Helicobacter pylori aATCC 43504
Helicobacter pylori ATCC 700392
Helicobacter pylori ATCC 49503
Helicobacter pylori bWSLH 95-10882
Helicobacter pylori WSLH 409013
Acinetobacter WSLH
Aeromonas WSLH
E. coli WSLH
Pseudomonas aeruginosa WSLH
Enterobacter cloacea WSLH
Enterococcus faecalis WSLH
Bacillus WSLH
a American Type Culture Collection.
b Wisconsin State Laboratory of Hygiene culture collection.
				
      
      
				
				
3
Preparation of conventional media
Conventional dehydrated media preparations were chosen for selection of H.
pylori from
mixed microbial populations based on the published clinical literature. The
five types of media
chosen for evaluation were BHI + 7% calf serum (Osaki, 1998), Brucella Agar
(Poms, 2001),
Campylobacter Agar Kit Skirrow (Corry, 1995), Columbia Blood Agar Base (Baronn,
1994;
Becton Dickinson, Sparks, MD), and HPSPA (Jiang, 2000; Stevenson, 2000).
Table 2. compares
ingredients of these media to demonstrate the common and unique features
of each. The
inclusion of either Helicobacter pylori selective supplement (Oxoid Limited,
Hampshire,
England) or Campylobacter selective supplement S (Becton Dickinson) provided
antibiotics to
prevent background while permitting H. pylori growth. Positive control consisted
of the five
conventional media without the selective supplements while negative controls
consisted of
uninoculated plates. All media were prepared according to manufacturer's
or authors'
instructions.
TABLE 2. Formulations of conventional media used to culture and select Helicobacter
pylori.
Brain Heart Infusion Brucella Agar Columbia Agar aHPSPA Campylobacter Agar
10 g proteose peptone 10 g peptamin 10 g pantone 15 g spec. peptone 15 g
proteose peptone
NA beef heart inf. 10 g tryptone 10 g bitone 2 g porcine mucin 2.5. ml liver
digest
NA calf brains inf. 2 g yeast extract 3 g beef heart dig. 5 g yeast extract
5 g yeast extract
2 g dextrose 1 g dextrose 1 g dextrose 5 g beef extract
5 g NaCl 5 g NaCl 5 g NaCl 5g NaCl 5g NaCl
15 g agar 15 g agar 15 g agar 15 g agar 12 g agar
2.5 g disodium phos. 0.1 g Na bisulfate 0.5 g ferrous sulfate
70 ml calf serum w/fe 70 ml calf serum w/fe
0.6 g urea
0.5 g Na pyruvate
bselective supplement selective supplement selective supplement selective
supplement cselective supplement
a Formula published by Jiang and Doyle. J. Clin. Microbiol. 2000.
b Vancomycin (10 mg/L), Cefsulodin (5 mg/L), Trimethoprim (5 mg/L), and Amphotericin
B (5 mg/L).
c Vancomycin (10 mg/L), Trimethoprim (5 mg/L), and Polymyxin B (2500 IU/L)
Initial conventional test media screen
Evaluation of the efficacy of the conventional media formulations used in
clinical microbiology
laboratories was carried out as follows. Each strain listed in Table 1 was
separately cultured on
each of the five media listed in Table 2 in order to evaluate the growth
and inhibition spectra of
individual formulations. First, pure colonies of each strain were picked
from solid growth media
(BHI agar plates) and homogenized in sodium phosphate buffer (4%). Serial
dilutions of each
pure homogenate were immediately spread (0.1ml/plate) onto each type of solid
media with and
without selective supplement added. All plates were incubated under microaerophillic
conditions at 37oC for up to seven days and examined to determine the presence
or absence of
colonies on the media compared to the positive control of a non-selective
media.
Selectivity of media formulations for H. pylori from samples spiked with
a cocktail of non
H. pylori bacteria.
Well water samples containing native flora (Table 5; identified using API
20 E
identification system; Biomerieux Vitek, Inc., Hazelwood, MO) were further
spiked with the
seven strains of background bacteria described above, and the H. pylori,
to represent a highly
contaminated water sample (10,000 cells per strain per 100 mls). The spiked
water sample was
then serially diluted and 0.1 ml of each dilution was spread onto duplicate
plates of each of the
					
				
				
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
				
      
      
				
				
6
spectrum that includes E. coli, Acinetobacter, Flavobactrum, Pasteurella,
and Ochrobactrum, as
well as inhibition of molds.
The growth of H. pylori was not enhanced by the presence of the various concentrations
of ferrous sulfate, sodium pyruvate, or porcine mucin applied. Therefore
these components were
omitted from the final formulation. However, more rapid colony formation
on HPSPA media
was attributed to the presence of Special Peptone and calf serum with iron,
so these remained as
nutritional components. Increasing the levels of vancomycin or Cefsulodin
above 10 mg/L
resulted in retarding the growth of Helicobacter, as did increasing trimethoprim
above 5 mg/L.
Conversely, reducing below these levels allowed background contamination.
Therefore these
concentrations (as contained in the selective supplement) were considered
optimal. Increasing
the level of amphotericin B from 5 to 7.5 mg/L was adequate to broaden the
spectrum to inhibit
all apparent background bacteria. The addition of polymyxin B at 3500 International
Units (IU)
appreciably reduced the occurrence of mold contaminants while having little
deleterious effects
on H. pylori colony development.
The novel color indicator system to differentiate H. pylori among non-urease
producers
greatly enhanced the utility of the media (Figure 1). Colony growth and subsequent
urease
production reduced urea to ammonium and bicarbonate, thus neutralizing a
discreet area around
each colony. This area of neutralization was marked by a zone of red around
the colony as pH of
the media changed from about 6.0 to >7.5. Incorporating the color indicator
accelerated
presumptive identification of H. pylori colonies by at least 12 hours (from
84 down to 72 hours).
The final formulation of HP agar is listed in Table 4.
FIGURE 1. Helicobacter pylori on HP or Brain Heart Infusion agar plates.
					
				
				
7
HP agar Brain Heart Infusion agar
					
				
				
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.
					
				
				
10
Commercial supplements allowed some background strains (E. coli, Acinetobacter,
Flavobactrum, Pasteurella, and Ochrobactrum) to grow during the moist, warm
(37oC), and
relatively lengthy (up to 7 days) incubation. In spite of extraordinary care
in maintaining
sterility, molds also frequently developed and overwhelmed the plates. Thus
some of the
selective components were increased after individually evaluating each antibiotic.
Increasing
amphotericin from 5 to 7.5 mg/L and adding polymyxin B at 3,500 IU/L was
adequate to solve
the contamination problems, while having no apparent deleterious effects
on H. pylori growth.
Although Jiang and Doyle (2000) reported that porcine mucin, ferrous sulfate,
and
sodium pyruvate were important in improving recovery of H. pylori cells,
there was no
appreciable difference in the presence or absence of these components in
this study. The concept
of using the characteristic urease production appears in a clinical assay
(CLO rapid urease test;
Delta West Pty Ltd, Bentley, Australia) but has not been previously reported
in solid media
designed for culturing Helicobacter. The addition of this color system substantially
reduces the
time required (about 14%) to grow colonies to the point of presumptive visual
identification.
Even on HP media plated with heavy bacterial loads, the occasional development
of a
background colony was easily differentiated from H. pylori.
In summary, the HP formulation provides a media with superior selectivity
for H. pylori from
mixed microbial populations in water and reduces the time required to complete
the assay.
Well water survey
Random samples
About 275 samples representing private Wisconsin wells were randomly chosen
from
those sent to the Wisconsin State Laboratory of Hygiene for routine coliform
testing. The
samples were centrifuged and reduced volumes were plated onto HP media. Assays
were
accompanied with positive (reverse osmosis water spiked with H. pylori) and
negative (nonspiked
water) controls to monitor performance. H. pylori was not detected in any
of these
samples which were incubated for 7 days at 37oC. About 95% of the plates
were barren
following incubation, which demonstrates that HP agar is effective in inhibiting
growth of
background contamination, even though some of these samples contained heavy
flora.
Requested samples
About 75 wells were screened for the presence of H. pylori at the request
of owners
suffering from peptic ulcers or related symptoms. HP agar in addition to
immunomagnetic
separation and/or fluorescent antibody staining were used to screen these
samples. In addition to
Helicobacter screening, information regarding the history of the wells, well
location, consumers'
symptoms, and coliform/E. coli presence in the sample was recorded.
Although this subset of samples was relatively unsanitary (up to 104 heterotropic
bacteria/ml), with about 25% positive for coliforms and one containing E.
coli, none were
positive for Helicobacter. Demographics of these wells reveal that 95% were
neither tested nor
disinfected since being drilled. The locations of the wells were widely distributed
across the state
(Figure 2) and the quality of water ranged from clear and sterile to turbid
and unsafe. The results
from HP agar were confirmed by two supporting methodologies which are accepted
as reliable
and sensitive by previous research groups. Therefore, interpretation of these
data must conclude
that water, as far as Wisconsin, U.S.A. well water is concerned, is not a
likely vector for the
transmission of H. pylori to humans.
					
				
				
11
FIGURE 2. Wisconsin counties containing private wells sampled randomly (blue)
or requested by residents suffering gastrointestinal problems (black).
ACKNOWLEDGEMENTS
This is work supported by the Wisconsin Department Natural Resources Groundwater
Coordinating Council. The authors wish to acknowledge the valuable contributions
of Becky
Leidner, Media Specialist and Linda Peterson, Senior Microbiologist from
the Wisconsin State
Laboratory of Hygiene.
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