Mol. Cells, Vol. 5, No. 5, pp. 508-513
Intrafamilial Transmission of Helicobacter pylori Detected by Random
Amplified Polymorphic DNA Fingerprinting
Myung-Je Cho l*, Woo-Kon Lee I, Young-Seok Jeonl, Kyung-Hee Kim., Seung-Hee Kim I,
Seung-Chul Baikl, Kwang-Ho Rhee l, Yoon-Ok Kim 2, Hee-Shang Yoon 2 and Nam-Soo Kim3t
IDepartment of Microbiology and 2Department of Pediatrics, College of Medicine, Gyeongsang
National University, Chinju 660-280. Korea; 3Plant Molecular Biology and Biotechnology Research
Center, Gyeongsang National University, Chinju 660-701, Korea
(Received on August 14, 1995)
Random amplified polymorphic DNA (RAPD) fingerprinting was adapted for the epidemiological study of Helicobacter pylori. Of one hundred 10-mer primers screened, one primer (*I: 243;
5' -GGGTGAACCG-3') producing apparent polymorphic profiles for discriminating Helicobacter
pylori isolates was selected. The genomic DNAs of lOS isolates of Helicobacter pylori were
subjected to polymerase chain reaction for RAPD fingerprinting, and 4 amplified fragments
(900 bp, SOO bp, 700 bp, and 600 bp) were found to be useful in determining types of RAPD
fingerprinting. The lOS isolates of Helicobacter pylori were grouped into 11 types, as determined
by band profiles of 4 amplified fragments. Type I was found most frequently. The RAPD
type of Helicobacter pylori was not disease-specific since the types were diverse in the isolates
from patients. Helicobacter pylori isolates from members of three families showed identical
RAPD types, strongly suggesting intrafamilial transmission of Helicobacter pylori.
Helicobacter pylori is a causative agent of type-B gastritis and also is strongly associated with the development of peptic ulcer as well as gastric adenocarcinoma
(Hazell et al., 1986; Lee et al., 1991; Nomura et al.,
1991; Parsonnet et al., 1991; Rhee et al., 1988, The
Eurogast Study Group, 1993).
Most Korean people become carriers of Helicobacter
pylori from early childhood (Baik et al., 1990; Rhee
et al., 1990). To prevent Helicobacter pylori infection,
its mode of transmission should be known. The mode
of transmission of Helicobacter pylori is suspected as
fecal-to-oral or oral-to-oral (Lee, 1994; Park et al., 1992;
Thomas et al., 1992). If there were a reliable typing
system of Helicobacter pylori. it would contribute to
elucidating the mode of transmission of Helicobacter
pylori.
Biotyping systems based on detection of phenotypic
characteristics such as cytotoxin production (Figura
et al., 1989), hemagglutinating activity (Huang et al.,
1988), motility (Eaton et al., 1992), and enzyme production (Megraud et al., 1985) did not provide a useful
tool for Helicobacter pylori isolates. Immunoblot analysis (Burnie et al .. 1988) and protein fingerprinting (Megraud et al., 1985) have been reported to group Helicobacter pylori isolates, but the usefulness of these methods is limited or remains to be further evaluated.
Restriction endonuclease analysis (Majewski and
Goodwin, 1988; Simor et al., 1990) of Helicobacter pylori
DNA may be too sensitive for sub typing because all
isolates from different patients have individually unique genomic patterns. And use of a single gene probe
like the ribosomal RNA gene (Tee et al., 1992) or a
cloned Helicobacter pylori chromosomal DNA fragment
(Li et al., 1993) in Southern analysis has revealed too
many patterns.
The analysis of the restriction patterns of polymerase chain reaction (PCR) amplified specific gene fragment has been reported to be useful as an epidemiological tool for Helicobacter pylori isolates (Foxall et
al., 1992; Fusimoto et aI., 1994). However, this method
requires target DNA sequence information.
William et al. (1990) and Welsh & McClelland
(1990) demonstrated that bacteria could be distinguished based on the banding patterns of their DNAs
by RAPD fingerprinting. Akopyanz et al. (1992) showed that the DNA diversity of Helicobacter pylori isolates can be detected by RAPD fingerprinting. Taylor
et al. (1995) reported consistency among the REA,
PCR-RFLP, and/or RAPD fingerprinting in genotyping Helicobacter pylori isolates.
In this study, we used RAPD fingerprinting as a
tool to show evidence of intrafami1ial transmission
of Helicobacter pylori.
* To whom correspondence should be addressed.
t Present address. Department of Agronomy, Kangwon National University. Chunchon 200-701. Korea.
The abbreviations used are: PCR polymerase chain reation;
RAPD, random amplified polymorphic DNA.
© 1995 The Korean Society for Molecular Biology
Myung-Je C ho et at.
Vol. 5 (1995)
Table I. Helicobacter pylori isolated from normal healthy per-
sons
without gastric symptoms and patients with gastric
Table 2. PCR-amplified patterns of Helicobacter pylon genome using primer :1* 243
disorders
Patterns
N o. of isolates
Sources
Healthy
Patients
Patients
Patients
Patients
Patients
Tota l
509
persons
with acute gastritis
with chro nic gastritis
with ulcers
with atrophic gastritis
with gastric ca ncer
type
type
type
type
type
type
type
type
type
type
type
25
9
58
9
4
3
108
Materials and Methods
Isolation and identification of Helicobacter pylori
For this study 108 isolates of Helicobacter pylori were
obtained from patients with gastric disorders at Gyeongsa ng University Hospital a nd normal healthy persons
without gastric symptoms (Table I ). Gastric biopsy
specimens were inoculated on Mueller-Hinton agar
containing 10% bovine serum, va ncomycin (10 J.lglm l),
nalidixic acid (25 J.lglml), a nd amphotericin B ( I J.lg
I ml). Plates were incubated at 37 "c for 4-5 days in
10% CO 2. All isolates were identified as H. pylori on
the basis of a small tra nslucent colony, gra m-negative
spiral, and urease production (Rhee et a/., 1988). Growth from a single colony was suspended in I ml of
I % peptone water with 30% glycerol and stored frozen
in liquid nitrogen.
Culture
Frozen isolates were revived on th e Mueller-Hinto n
aga r pl ates. One loop of ove rnight-grown bacteria was
inoculated into 30 ml of the mixed medium of Mueller-Hinton a nd brucell a broth (\ : I) enriched with I
giL of dimethyl-beta-cyclodextrin (Nihon Shokuhin
Ka ko Co. LTD, Japan). The medium was placed in
a n anaerobic jar. Th e atm osphere of the a naerobic
jar was cha nged to a microaerophilic co ndition (5%
O 2, 10% CO 2• and 85% N 2) and was gently shaken
(200 rpm) ove rnight at 37 °C. The optical density of
the medium was between I a nd 1.4 00 at 600 nm.
Preparation
~f
chromosomal DNA
Bacteri a l cell s were harvested by centrifugatio n for
10 min a t 3,000 rpm (Bec kman model TJ-6, U.S.A.),
a nd then washed with TE buffer (pH 8.0). The bacteri al pellet was resuspended in 0.6 ml of lysozyme solution (0.5% lysozyme in 25 mM Tris-HCl, pH 8.0, 50
mM glucose. 10 mM EDTA) a nd incubated fo r 30
min at 37 °C. Th en a one-tenth volume of 10% SDS,
10 mg/ml proteinase K. and I mglml RNase A were
added to th e solution and incubated for 2 h at 37
°C. Th e so lution was extracted sequentia lly with eq ual
volumes of phenol/chlorofornl/isoamyl alcohol (25 : 24
: I) and chloroform. To the extracted mixture were
I
II
III
IV
V
VI
VII
VIII
IX
Amplified band (bp)
900
900,
900,
900,
900,
900,
900,
900,
800
700
,
800,
800,
,
700,
600
,
,
700,
700,
700,
700,
700,
600
600
600
800,
X
XI
800,
600
600
added a o ne-tenth volume of 3 M sodium acetate (PH
5.2) and 2.5 volume of ethanol. Precipitated DNA was
spooled out with a glass bar, washed in 70% ethanol,
dissolved with 200 J.1.l of TE buffer, and stored at -20
"c. Approximately, 20-30 J.lg of DNA was obtained
fro m 30 ml of broth culture. The frozen DNA solutions were simulta neously thawed and diluted into 5
ngl I of concentration j ust before RAPD fingerprintmg.
Source of arbitrary primers
Arbitrary oligonucleotides for RAPD fingerprinting
were selected from the UBC RAPD Primer Synthesis
P roject Oligonucleotide Set 100/3 which was purchased from the Nucleic Acid-Protein Service Unit, University of British Columbia, Canada. Selected oligonuleotides were synthesized with an Automated Synthesizer (Applied Biosystems Model 380B, U.S.A.) and
purified with a n oligonucleotide purification cartridge
(Applied Biosystems, U.S.A.).
RAPD fingerprinting
PCR amplification for RAPD fingerprinting was
carried out as described previously (William et al.,
1990). The reaction mixture was 25 J.1.l containing 50
mM KCl, 10 mM T ris-HC\, pH 9.0, 0.1 % Triton X100, 2 mM MgCh, 50 J.1.M each of dATP, dCTP,
dGTP, dTTP, 1 J.1.M of primer, 10 ng of genomic
DNA, a nd 0.75 U of AmpliTaq DNA polymerase
(Perkin Elmer Cetus, U.SA.). Amplification was perfo rmed by a n ASTEC Program Tern Control System
PC7OO. The cycle program was 39 cycles of 94 "c for
I min, 35 "c for I min, and 72 "c for 2 min and
followed by I cycle of 94 "c for 1 min, 35 °C for 1
min, a nd 72 °c for 10 min. After amplification reaction, 10 J.1.l of product was subj ected to electrophoresis in a mixed gel of 2% NuSieve GTG agarose
(FMC BioProducts, U.SA.) and 1% agarose (lBI Inc.,
U.SA) at 50 V for 2 h in a MUPID chamber (Advance
Corp., Japan). The gel was stained with ethidium bromide, a nd photographed under a UV transilluminator.
Intrafa milial Transmission of Helicobacter pylori
510
M 1
2 3
4
5
6
7 8
Mol. Cells
RAPD Patterns
9 10 11 12 13 1415
1/
III
IV
V
VI
VI/ VIII
IX
X XI
bp
A
900800700600M
1
2
3
4
5
6
7
8
9 10 11 12 13 1415
kb
12,0
U
B
Figure 3. Patterns of the amplified DNA of Helicobacter pylori
isolates in RAPD fingerprinting using primer # 243. Patterns
are based on the amplification of 900 bp. 800 bp, 700 bp.
and 600 bp DNA fragments generated from 108 isolates of
1,0
0,5
Helicobacter pylon'.
Figure I. RAPD fingerprinting of Helicobacter pylori isolates.
The serial numbers refer to Helicobacter pylori isolates. M,
I kb DNA ladder marker; A, primer #24 1; B, primer #243.
M 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
A
M
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
B
Figure 2. RAPD fingerprinting of Helicobacter pylori isolates.
The numbers refer to Helicobacter pylori isolates. M. I kb
DNA ladder marker; A, primer # 272; B. primer # 299.
Results
Primer selection
One hundred IO-mer primers from a UBC RAPD
Primer Synthesis Project Oligonucleotide Set 100/ 3
Table 3. RAPD patterns of Helicobacter pylori isolates
Patterns
type I
type II
type III
type IV
type V
type Vl
type VlI
type VlII
type IX
type X
type XI
Total
No. of isolates (%)
32 ( 29.6)
7 ( 6.S)
19 ( 17.6)
4 ( 3.7)
14 ( 13.0)
3 ( 2.8)
16 ( 14.8)
8 ( 7.4)
I ( 0.9)
3 ( 2.8)
I ( 0.9)
108 (100.0)
were evaluated using genomic DNAs of 15 Helicobacter
pylori isolates to select suitable primer(s) for RAPD
fingerprinting. Following PCR amplified samples were
analyzed by agarose gel electrophoresis. Of 100 primers 4 primers produced multiple a mplified DNA
ba nds in all IS Helicobacter py lori as shown in Figures
I and 2. Primer # 243 (5'-GGGTGAACCG-3' and 70
% G + C) was finally chosen as the primer for RAPD
fingerprinting because RAPD patterns obtained with
thi s primer were more reproducible a nd more apparent for discriminating Helicobacter pylori isolates than
those obtai ned with the others.
RAPD markers jor genomic typing
Genomic DNAs of 108 Helicobacter pylori isolates
were subjected to PCR using primer # 243 for RAPD
fingerprinting. For the analysis of the fingerprinting
profiles. 4 amplified DNA fragments (900 bp. 800 bp.
700 bp. a nd 600 bp) were chosen as marke rs for the
classification of genomic polymorph isms of Helicobactel' pylori isolates (Fig. 3). All of the 108 isolates had
Vol. 5 (1995)
Myung-Je Cho et al.
511
Table 4. The RAPD patterns of Helicobacter pylori isolates recovered from healthy persons without gastric symtoms and
patients with gastric disorders
Sources
Healthy persons
Acute gastritis
Chronic gastritis
U1cers
Atrophic gastritis
Cancer
Total
RAPD patterns
Total
25
9
58
9
4
3
108
5
4
19
2
II
III
IV
V
Vl
VlI
3
5
2
9
3
2
4
3
1
9
2
3
3
7
2
4
14
3
1
I
I
32
7
2
1
19
at least o ne o r more of 4 amplified DNA fragments.
RAPD types of Helicobacter pylori
Based on the band profiles of 4 ma rkers in RAPD
fingerprinting, 108 isolates of Helicobacter pylori were
classified into 11 types as shown in Figure 3. The
patterns of 4 DNA fragments are summarized in T able 2. Of 108 isolates: more than 70% were classified
into type I, type III, type V, and type VII and type
I was the most frequent (fable 3). There was diversity
in the isolates from patients, indicating RAPD types
are not disease-specific (fable 4).
RAPD types of isolates from family members
Three independent isolates of Helicobater pylori from
a member of each family were recovered to determine
their RAPD types. Th e RAPD types of 33 isolates
from 11 members of three families are shown in Figure 4. In family 1, 3 isolates of a father. 2 isolates
of a mother, 1 isolate of offspring one, a nd 3 isolates
of offspring two had identical RAPD type (type IV),
whereas 1 isolate of a mother and 2 isolates of offspring one had another RAPD type (type Ill). In
family 2, a father and offspring one were infected with
an identical type of strain (type VI). In family 3, all
isolates of a father a nd offspring two had an identica l
type (type IV), whereas those of offspring one a nd
three had another type (type I).
Discussion
An appropriate primer by which a number of apparent DNA bands could be formed in all clinical isolates at PCR is needed for RAPD fingerprinting to cl assify the genomic polymorphism of pathoge nic bacteria.
In this study, IO-mer primers from the UBC RAPD
Primer Synthesis Project Oligonucleotide Set 100/3
were arbitrarily chosen to select a primer for th e
RAPD fingerprinting of H. pylori genomic DNAs. This
set contains 100 kinds of IO-mer oligonucleotides of
which the G + C content is 50% or greater. except for
one primer. William et al. (1990) showed th at G + C
content in a n oligonucleotide IO-mer should be 40%
or greater to generate detectable levels of amplification
products. Akopyanz et af. (1992) observed that most
3
16
VlII
IX
X
4
3
1
8
3
XI
IO-mer primers with 6()01o or greater G+C content yielded an amplified DNA array, whereas most IO-mer
primers with 50% G+C content did not. The present
study showed that 4 primers with 60% or greater G + C
content produced amplified DNA fragments in all of
the 15 clinical isolates of H. pylori at PCR (Figs. 1
and 2). However, other primers with less than 60%
G + C content in th is primer set did not produce amplified DNA fragments in all of them, which was consistent with the observation of Akopyanz et af. (1992)
Although most techniques reported so far, including
REA, ribotyping, and PCR-based RFLP, succeeded
in differentiating the cl inical isolates, they showed too
many patterns to estab lish a practically applicable typing scheme. In this study, we used only 4 marker
DNA fragments to screen a large number of clinical
isolates with the naked eye without a ny soph isticated
devices. By this subtyping method, we found useful ness in tracing intrafamilial transmission of Helicobacter pylori.
Several epidemiological studies (Banatvala el al.,
1993; Cullen el al., 1993; Kuipers el al., 1993; Rhee
eI aI., 1990) have suggested that Helicobacter pylori infection is predominantly acquired at a young age. Children ordinari ly have close contact with their parents;
therefore parent strains have the potential of being
carried over to the chi ldren.
Familial clusters of antibody preva lence for HelicobaCler pylori suggested the possibility of intrafamilial
tra nsmission (Drum el al.. 1990; M itchell el al., 1992).
Simor el al. (1990) a nd Majewski and Goodwin (1988)
showed that different strai ns of Helicobacler pylori were
isolated a mo ng family members whereas Rauws el al.
( 1989) reported th at eight members of one fami ly were
confirmed to be infected with an identica l strain. In
contrast. Tee el al. (1992) showed family members
might acquire infection from other members of the
fami ly or different sources. However. they did not consider mixed infections by various types of Helicobacler
pylori because a single iso la te from an infected individual was used for typing. So the possibi lity of intrafamilial tra nsmi ssion of Helicobacler pylori had to be
clarified by using mUltiple isolates from members of
a fami ly. Three fami lies tested in thi s study consisted
of parent(s) and their children. RAPD types of all
Intrafamilial Transmission of Helicobacter pylori
512
~ Pf --, - Pm- ,- f1----, ,-
f2 --,
,-
Mo!. Cells
P1 -, ,--- f1---, ,-- f2 ----: r- Pf-, ,--- f1-,
r-
f2---,
r-
f3--,
kb
21.02.0-
0.90.5-
Family 1
Family 2
Family 3
Figure 4. RAPD fingerprintings of genomic DNAs of Helicobacter pylori recovered from family members. Pf, father; Pm,
mother; n, f2, 13, their offspring.
or part of isolates recovered from 4 of 7 children were
shown to be identical to those of their parents (Fig.
4). Mother's isolates in family 2 and family 3 were
not available for typing in this study. Therefore, the
souces of Helieobaeter pylori infection of 3 children,
who were infected with different isolates from their
fathers', could not be determined. Considering that
all isolates of the children in family I were identical
to their parents' strains, however, it was not ruled out
that these children might be infected with their mothers' strains. Anyway, these results suggested that
some children acquire Helieobaeter pylori infection
from their parents.
Mixed infection by Helieobaeter pylori strains might
be possible (Hirschi et aI., 1994; Prewett et al., 1992;
Taylor et al., 1995). It was reported that 20% of patients
were colonized with different Helieobaeter pylori strains.
In this study, mother and offspring one in family I
were shown to be infected with 2 RAPD types of Helieobaeter pylori, suggesting that they may carry more
than one strain.
Acknowledgments
This work was supported by the Genetic Engineering Research Program (1993), Ministry of Education,
Korea.
N. S. Kim was supported by the Brain Pool Program. Korea Research Foundation.
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