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Journal of Clinical Microbiology, May 2000, p. 1984-1987, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Growth Supplements for Helicobacter
pylori
Xiuping
Jiang and
Michael P.
Doyle*
Center for Food Safety and Quality
Enhancement and Department of Food Science and Technology,
University of Georgia, Griffin, Georgia 30223-1797
Received 26 July 1999/Returned for modification 9 December
1999/Accepted 2 February 2000
 |
ABSTRACT |
The growth response of Helicobacter pylori in broth was
determined in the presence of ferrous sulfate, sodium pyruvate, and mucin (porcine stomach). The addition of either ferrous sulfate and
sodium pyruvate or mucin to brain heart infusion broth with 7% horse
serum (BHI-HS) enhanced the growth of H. pylori. The best
growth of strain NB2-1, which was the slowest growing of 10 H. pylori strains evaluated, occurred in the presence of 0.05% ferrous sulfate and 0.05% sodium pyruvate. The addition of 0.3% mucin
to BHI-HS reduced the lag time of H. pylori by 48 h
and enhanced the growth. On the basis of the results for 10 H. pylori strains, the combination of ferrous sulfate (0.025%),
sodium pyruvate (0.025%), and mucin (0.15%) in BHI-HS counteracted
the inhibitory effects of the antibiotics used in culture media for
selective growth of H. pylori. Results suggest that these
supplements may be useful for enhancement of the growth of H. pylori in enrichment media.
| |
TEXT |
Helicobacter pylori
induces chronic gastric inflammation and gastric ulcer and may also be
a contributory factor in human gastric carcinoma (10, 17).
Besides its natural habitat in the human stomach, other sources of
H. pylori and its mode of transmission are unknown
(4). Identification of the reservoirs and routes of
infection of H. pylori has been impeded by the difficulty of
isolating the pathogen from foods and environmental sources. This is
largely attributed to the fastidious nature of H. pylori and
its relatively low growth rate, thereby limiting its growth in a
competitive environment.
H. pylori requires for growth a nutrient-rich medium, serum,
and a microaerobic environment. Under the best conditions, on a
nonselective agar-containing medium, 2 to 3 days is generally required
for the formation of small colonies, whereas the implementation of
growth in a liquid medium can be more challenging (12). Some supplements such as whole blood, serum, yeast extract, lysed human erythrocytes, IsoVitaleX, hemin, cyclodextrin, and cholesterol have
been determined to enhance H. pylori growth in culture broth (6, 9, 18). However, growth of H. pylori in
liquid media is influenced by the initial inoculum concentration and
also strain variation (12, 19). Generally, the lower the
initial inoculum of H. pylori, the longer the lag time. To
determine the occurrence of H. pylori in environmental and
food sources, a selective enrichment medium is needed that will enable
growth of a small population of helicobacters to detectable levels in
an environment that may contain large populations of competitive
microorganisms. The purpose of the study described here was to identify
supplements to culture media that would enhance the growth of H. pylori under culture conditions with antibiotics that suppress the
growth of competitive microorganisms.
Bacterial strains and culture conditions.
A total of 10 H. pylori strains were used for this study. Five H. pylori strains, strains NB2-1, 1324P-1, 26695, G2-1, and WV 99, were obtained from Douglas Berg (Washington University, St. Louis,
Mo.), and five fresh clinical isolates of H. pylori, strains
D5131, D5135, D5136, D5178, and D5251, were obtained from Ben Gold
(Emory University and Centers for Disease Control and Prevention,
Atlanta, Ga.). Bacteria were grown on plates of brain heart infusion
(BHI) agar supplemented with 7% horse serum (HS). Cultures were
incubated at 37°C for 3 days in a GasPak jar (BBL Microbiology
Systems, Cockeysville, Md.) whose atmosphere was evacuated three times
and replaced with a microaerobic gas mixture composed of 5% oxygen,
10% carbon dioxide, and 85% nitrogen.
Growth studies.
Cultures were prepared by the procedures
described previously by Jiang and Doyle (8). Briefly,
3-day-old cultures were harvested from BHI agar-HS plates. A washed
suspension of ca. 5 × 108 H. pylori cells
per ml was prepared in fresh BHI broth-HS and was inoculated into fresh
BHI broth-HS to provide a final inoculum of approximately 5 × 103 CFU per ml.
Prior to inoculation of H. pylori, appropriate volumes of
filter-sterilized (pore size, 0.2 µm; Nalgene sterile syringe filter) ferrous sulfate and sodium pyruvate (FP; Sigma Chemical Co., St. Louis,
Mo.) solutions (5%) were added aseptically to 100 ml of sterile BHI
broth-HS to provide final concentrations of each component in FP of
0.025, 0.05, 0.075, and 0.10%. Porcine stomach mucin (Sigma Chemical
Co.) was added to BHI broth at final concentrations of 0.15, 0.30, and
0.45% prior to heating in an autoclave. H. pylori-selective
supplement (Dent) containing vancomycin (10 mg/liter), trimethroprim
lactate (5 mg/liter), cefsulodin (5 mg/liter), and amphotericin B (5 mg/liter), purchased from Oxoid (Oxoid, Hampshire, England), and filter
sterilized (pore size, 0.2 µm; Nalgene sterile syringe filter)
polymyxin B solution (2,500 U/liter) were added to 100-ml volumes of
BHI broth-HS for studies with antibiotics. A combination of ferrous
sulfate (0.025%), sodium pyruvate (0.025%), and mucin (0.15%) was
used with the antibiotics.
H. pylori was inoculated as described above, and cultures
were incubated at 37°C in a GasPak jar under microaerobic conditions with agitation at 150 rpm.
Enumeration of H. pylori.
At appropriate times,
cultures were serially diluted (1:10) in 0.01 M phosphate-buffered
saline (pH 7.2), and H. pylori was enumerated in duplicate
by surface plating onto BHI agar-HS. The plates were then incubated at
37°C for 3 to 7 days in GasPak jars under microaerobic conditions.
Generation times were derived from the exponential growth rates and
were determined in duplicate.
Results.
H. pylori strain NB2-1, the slowest
growing of 10 H. pylori strains evaluated, did not grow
within 4 days in BHI broth-HS at 37°C with agitation under
microaerobic conditions. However, the bacteria grew well in BHI
broth-HS supplemented with ferrous sulfate and sodium pyruvate and held
in the same GasPak jar under the same environmental conditions (Fig.
1). Growth was visible at 24 h in
the broths supplemented with 0.05, 0.075, and 0.1% FP, with generation
times of 3.2 to 4.3 h during 72 to 96 h of growth. Growth was
delayed about 24 h in BHI broth-HS supplemented with 0.025% FP,
but thereafter, the generation time was 3.7 h.
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FIG. 1.
Growth of H. pylori strain NB2-1 at 37°C
with agitation under microaerobic conditions in BHI broth-HS with
different concentrations of ferrous sulfate and sodium pyruvate (FP).
Symbols:  , BHI broth-HS;  , BHI broth-HS with 0.025% FP;  , BHI
broth-HS with 0.05% FP;  , BHI broth-HS with 0.075% FP; ---, BHI
broth-HS with 0.1% FP. The arrow indicates that H. pylori
was not detected at the minimum level of sensitivity (<10 CFU/ml).
|
|
Growth of H. pylori strain NB2-1 was also enhanced by
supplementing BHI broth-HS with mucin (Fig.
2). Porcine gastric mucin at
concentrations of 0.15, 0.30, and 0.45% reduced the lag time by 24 to
48 h compared with that for growth in unsupplemented BHI broth-HS.
The generation times for H. pylori in broths supplemented with mucin at 0.15, 0.30, and 0.45% were 4.3, 3.8, and 4.0 h, respectively. The addition of mucin at either 0.3 or 0.45% had a
similar effect on enhancing the growth of strain NB2-1.
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FIG. 2.
Growth of H. pylori strain NB2-1 at 37°C
with agitation under microaerobic conditions in BHI broth-HS with
different concentrations of mucin. Symbols: , BHI broth-HS; , BHI
broth-HS with 0.15% mucin; , BHI broth-HS with 0.3% mucin; ,
BHI broth-HS with 0.45% mucin. The arrow indicates that H. pylori was not detected at the minimum level of sensitivity (<10
CFU/ml).
|
|
The five antibiotics used in this study had an inhibitory effect by
delaying the growth of all strains except strain D5131 (Fig.
3). The addition of 0.15% mucin and
0.025% FP to BHI broth-HS with antibiotics reduced the lag time by
24 h for strains D5135 and D5178, by 48 h for strains 26695, G2-1, D5131, and D5136, and by at least 72 h for strains NB2-1,
1324P-1, WV 99, and D5251. Except for strains 1324P-1 and D5251, there
was little difference in the growth rates of helicobacters in broth
supplemented with mucin and FP or with mucin, FP, and antibiotics. With
the low inoculum level (ca. 1 × 102 CFU/ml), the
growth rates of H. pylori in the presence and absence of
mucin, FP, and/or antibiotics were similar to that observed with a
higher inoculum level (ca. 5 × 103 CFU/ml; data not
shown). Overall, the counts for all H. pylori strains
evaluated were substantially greater during the first 24 h of
incubation when grown in BHI broth-HS supplemented with FP and mucin
than in BHI broth-HS with no supplements.
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FIG. 3.
Growth of 10 H. pylori strains at 37°C with
agitation under microaerobic conditions. Symbols: , BHI broth-HS;
, BHI broth-HS with antibiotics (vancomycin [10 mg/liter],
trimethroprim lactate [5 mg/liter], cefsulodin [5 mg/liter],
amphotericin B [5 mg/liter], and polymyxin B [2,500 U/liter]); ,
BHI broth-HS with 0.025% FP and 0.15% mucin; , BHI broth-HS with
0.025% FP, 0.15% mucin, and antibiotics. The arrow indicates that
H. pylori was not detected at the minimum level of
sensitivity (<10 CFU/ml).
|
|
Discussion.
Microaerophilic bacteria such as campylobacters
are more sensitive than aerobic bacteria to toxic forms of oxygen
derivatives such as hydrogen peroxide, singlet oxygen, and superoxide
ions (7). A combination of ferrous sulfate, sodium
metabisulfite, and sodium pyruvate (FBP) has successfully been used to
enhance the growth and aerotolerance of Campylobacter spp.
by neutralizing the toxic effect of oxygen (3). Even under
optimal atmospheric conditions, the growth of Campylobacter
jejuni was enhanced with iron supplementation (0.025%
FeSO4) (15). Many selective media for isolation
of campylobacters contain some or all of these compounds at
concentrations in the range of 0.025 to 0.05% (2, 3). However, sodium metabisulfite is inhibitory to the growth of H. pylori (5). Therefore, in this study we chose FP as a
medium supplement for H. pylori growth. Among the
concentrations of FP studied, the best growth of H. pylori
occurred in 0.05% FP. Theoretically, both ferrous iron and pyruvate
undergo oxidative reactions under physiological conditions in the
living cells. Hence, the function of FP for H. pylori is
likely the same as that of FBP for Campylobacter spp.
Mucin, a high-molecular-weight glycoprotein, is the principal
constituent of mucus, in which H. pylori occurs naturally in vivo. Studies have revealed that H. pylori binds to human
gastric and salivary mucin in vitro (13, 16). The sulfated
carbohydrates of mucin are responsible for this interaction. Namavar et
al. (13) suggested that H. pylori uses its
temporary attachment to mucin as a means of "tracking" toward the
epithelium in vivo. Our observations by microscopic examination
revealed that H. pylori cells often form clumps during the
early stages of incubation in a liquid growth medium. Cellini et al.
(1) also observed that the coccoid forms of H. pylori tended to aggregate in clusters. By attaching to mucin in
the medium, H. pylori may form cell clumps that protect the
cells from the harmful effects of the oxygen dissolved in the
surrounding liquid. The increased viscosity provided by mucin in
supplemented broth may also create a more favorable microaerobic
environment for H. pylori growth initiation in a liquid environment. Slomiany et al. (14) reported that
H. pylori can enzymatically degrade mucin, suggesting that
mucin may serve as a nutrient for growth of H. pylori.
Antibiotics have been widely used as selective agents in enrichment and
plating media for bacterial isolation. Most strains of H. pylori are inherently resistant to vancomycin, cefsulodin, trimethroprim lactate, polymyxins, and amphotericin B (11). However, the level of resistance to different antibiotics varies among
strains of H. pylori, as well as among some injured
bacterial cells that occur in the environment. Our studies have
revealed that both FP and mucin can counteract the inhibitory effect of a combination of five antibiotics used to select for H. pylori. In this study, we tested five fresh clinical isolates of
H. pylori, i.e., strains D5131, D5135, D5136, D5178, and
D5251, in addition to five laboratory-adapted strains. Results revealed
that FP and mucin have the same effect on enhancing the growth of both
laboratory-adapted strains and clinical isolates recently obtained from
infected patients.
In conclusion, we have determined that both FP and mucin
can greatly reduce the growth lag time and enhance
the growth of H. pylori in a liquid culture medium. The
influence of these supplements in counteracting the inhibitory effects
of antibiotics on H. pylori may be beneficial for the
formulation of enrichment media for the isolation of H. pylori directly from the natural environment and foods.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the U.S. Department of
Agriculture as part of the Alliance for Food Protection.
We thank Douglas Berg, Washington University, and Benjamin Gold,
Centers for Disease Control and Prevention and Emory University, for
providing isolates of H. pylori.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for Food
Safety and Quality Enhancement, University of Georgia, 1109 Experiment St., Griffin, GA 30223-1797. Phone: (770) 228-7284. Fax: (770) 229-3216. E-mail: MDOYLE{at}CFSQE.GRIFFIN.PEACHNET.EDU.
| |
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Journal of Clinical Microbiology, May 2000, p. 1984-1987, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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