relapse) results from a recrudescence of infection attributable either to incomplete eradication by therapy or to reinfection. Therefore, precise methods of strain ...
JOURNAL OF CLINICAL MICROBIOLOGY, May 1994, p. 1203-1210
Vol. 32, No. 5
0095-1137/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Comparison of PCR-Based Restriction Length Polymorphism Analysis of Urease Genes with rRNA Gene Profiling for Monitoring Helicobacter pylori Infections in Patients on Triple Therapy R. J. OWEN,`* J. BICKLEY,1 A. HURTADO,1 A. FRASER,2 AND R. E. POUNDER2 National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT,' and Royal Free Hospital School of Medicine, London NW3 2QG,2 United Kingdom Received 29 October 1993/Returned for modification 9 December 1993/Accepted 7 February 1994
Multiple isolates of Helicobacter pylori from antral biopsies of nine patients were examined by DNA fingerprinting. Analysis of rRNA gene patterns and HaeIII restriction fragment length polymorphism of PCR-amplified urease genes were compared and used to study colonization before and after failed triple therapy. H. pylori isolates from a single biopsy shared the same HaeIHl DNA fingerprint regardless of the isolation method (plate or broth). DNA pattern types of paired strains of H. pylori were distinct between patients and were not grossly affected by treatment except for one patient with an altered strain type. H. pylori infections were generally associated with several subpopulations of strains, evident from the subtypic variation before and after treatment, detectable by both DNA fingerprinting methods. The urease gene patterns also provided evidence that some cultures of H. pylori probably contained a mixture of genomic subtypes. The study suggests that triple therapy has the elfect either of inducing minor genomic variations or of changing the proportions of different subtypes of H. pyloni. It was concluded that urease gene profiling provides a simple yet reliable method of establishing whether treatment failures are attributable to incomplete eradication of H. pylori.
strains, both before and after treatment (10, 29). However, such molecular profiling methods are time-consuming and technically complex for nonspecialized laboratories, so their potential applications are restricted. The advent of PCR technology in clinical microbiology has resulted in the development of new DNA fingerprinting techniques, and the analysis of restriction fragment length polymorphisms (RFLPs), in particular urease gene sequences (6, 9, 37), has considerable potential for fingerprinting isolates of H. pylon. In the present study, we examined the usefulness of PCRRFLP analysis of urease A and B genes in comparison with rRNA gene profiling as a basis for a simplified yet discriminatory approach to fingerprinting strains of H. pylon from patients before and after failed triple therapy.
Seroepidemiological surveys show that gastric colonization with Helicobacterpylor (11) has a prevalence of at least 40% in human populations throughout the world (18, 40). Most infected persons are asymptomatic, yet there are significant associations with type B gastritis and duodenal ulceration and, to a lesser degree, gastric ulcer disease and gastric cancer (8, 12). Drug therapies to decrease acid secretion are highly effective for ulcer healing, but their withdrawal can be expected to be followed by recurrence of the duodenal ulceration within 2 years (3), although the chances are reduced if the associated H. pylon infection is eradicated by use of antibiotics (13, 36). Therapeutic intervention increasingly involves a regimen with one or more antibiotics (e.g., amoxicillin and metronidazole) in combination with either tripotassium dicitrato bismuthate or a gastric acid antisecretory drug (4, 5). To determine the effectiveness of different treatments, it is essential to know whether treatment failure (particularly ulcer relapse) results from a recrudescence of infection attributable either to incomplete eradication by therapy or to reinfection. Therefore, precise methods of strain characterization are necessary to monitor H. pyloni infections. Strains of H. pylon differ in some conventional phenotyping tests such as serology (19) and biotyging (27), but none offers sufficient discrimination for strain typing. In contrast, analyses of genomic DNA reveal considerable diversity between isolates of H. pylon, with genomic DNA restriction endonuclease digest patterns (16, 17, 21, 24, 30, 38) and rRNA gene patterns (ribopatterns) (10, 25, 26, 29, 31, 35) providing a high degree of discrimination of
MATERIALS AND METHODS Bacterial strains and culture media. The 47 clinical isolates from nine patients (A to I; see Table 1) were obtained from gastric tissue collected at the Royal Free Hospital during endoscopy of patients presenting with symptoms of dyspepsia, histologically confirmed gastritis, and duodenal ulcer. At least two antral biopsies were taken from each patient before treatment was initiated (culture numbers suffixed by a and b). Patients were treated with tripotassium dicitrato bismuthate [Brocades (Great Britain) Ltd., Weybridge, England] (one De-Nol tablet four times a day) for 2 weeks together with tetracycline (500 mg four times a day) and metronidazole (400 mg three times a day) for the first week. A follow-up endoscopy was performed 6 weeks after the completion of the triple therapy (culture numbers suffixed by c and d). This study was approved by the Ethical Practices Committee of Hampstead Health Authority.
* Corresponding author. Mailing address: National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: 081 200 4400. Fax: 081 200 7874.
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1204
OWEN ET AL.
The biopsy specimens for culture were placed directly in a selective enrichment medium (20) that consisted of brucella broth (Difco Laboratories, East Molesey, United Kingdom) supplemented with 10% fetal calf serum, 1% (vol/vol) IsoVitaleX (BBL Microbiology Systems, Becton Dickinson, Cowley, United Kingdom), polymyxin B (1,000 U/ml), vancomycin (10 ,ug/ml), and amphotericin B (2 jig/ml). Specimens were transported to the microbiology laboratory within 5 h. Each biopsy specimen was then placed in 5 ml of selective enrichment medium and incubated at 37°C on a gyratory platform in a variable-atmosphere incubator (Don Whitley Scientific Ltd., Shipley, United Kingdom) under microaerobic conditions (5% 02, 5% C02, 2% H2, and 88% N2). After 48 h, a sample from each flask was subcultured onto Oxoid brain heart infusion agar (Oxoid, Basingstoke, United Kingdom) supplemented with 5% horse blood and 1% IsoVitaleX. Biopsies were also cultured directly on Campylobacter selective medium according to the Skirrow formula: blood agar base with vancomycin (10 ,ug/ml), polymyxin B (2.5 U/ml), and trimethoprim (5 pug/ml). Positive growth was identified by Gram staining and rapid (within 10 min) hydrolysis of urea (32). Multicolony sweep cultures were preserved at - 196°C on glass beads in Oxoid nutrient broth no. 2 containing 10% (vol/vol) glycerol. H. pylori NCTC 11637 (type strain, originating from B. Marshall and S. Goodwin, Perth, Australia) was included for reference purposes. Isolation of genomic DNA. Bacterial genomic DNA was extracted and rapidly purified either by the cetyltrimethylammonium bromide method according to the DNA miniprep protocol of Wilson (42) for PCR-RFLP analysis or by the guanidium isothiocyanate reagent method (34) for RNA gene analysis. The pellet of DNA was redissolved in 60 RI of TE (10 mM Tris-HCl and 1 mM EDTA [pH 8.0]). The concentration and purity of the DNA samples were determined by absorbance readings at 230, 260, and 280 nm. Restriction digestion, vacublotting, and RNA gene hybridization. All DNA samples (5 ,ug) were digested for 4 h at 37°C with HaeIII (2 to 3 U/,ig of DNA) in the buffer recommended by the manufacturer (Boehringer Mannheim, Poole, United Kingdom). The digested DNA was electrophoresed at 30 V for 18 h in a horizontal 0.7% (wt/vol) agarose (Gibco-BRL Ltd., Paisley, United Kingdom) gel (20 cm long) in a buffer containing 89 mM Tris-HCl, 89 mM boric acid, and 2 mM disodium EDTA (pH 8.3). After electrophoresis, the gels were stained with ethidium bromide and photographed. The DNA fragments were transferred to nylon membranes (Hybond-N; Amersham International) by vacuum-assisted transfer blotting (VacuGene XL) (Pharmacia-LKB, Milton Keynes, United Kingdom), and the membranes were then prehybridized and hybridized by previously described procedures (31). The biotinylated copy cDNA probe used was prepared from a mixture of 16S and 23S rRNAs from H. pyloni NCTC 11637, using Moloney mouse leukemia virus reverse transcriptase (GibcoBRL). Biotinylation was achieved by the incorporation of biotin-16-dUTP according to previously described methods (33). The membranes were washed after hybridization for 18 h at 42°C, and the hybridized probe was detected colorimetrically with the BluGENE (Gibco-BRL) nonradioactive detection kit as recommended by the manufacturer. The pattern of bands containing rRNA gene sequences was designated the
ribopattern. Synthetic oligonucleotides. The oligonucleotide primers described by Foxall et al. (9) were synthesized commercially (Pharmacia-LKB). The forward primer had the sequence 5'-AGGAGAATGAGATGA-3' (bp 308 to 322 [15]), and the reverse primer had the sequence 5'-ACT'TFTATTGGCTG
J. CLIN. MICROBIOL.
GT-3' (bp 2718 to 2703 [15]). After dilution in TE to a concentration of 10 ,uM, aliquots were stored at - 30°C. PCR amplification. A 1-plA sample of the template DNA solution was added to 100 pL of reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, 0.05 mM each deoxynucleotide (Pharmacia Biosystems, Milton Keynes, United Kingdom), and 0.2 ,uM each oligonucleotide primer. Thermus aquaticus (Taq) polymerase (2.5 U) (Boehringer Mannheim) was added, and the reaction mixture was overlaid with 60 pu1 of mineral oil to prevent evaporation. PCR was performed with an OmniGene automatic thermal cycler (Hybaid Ltd., Teddington, Middlesex, United Kingdom). The amplification cycle consisted of an initial denaturation of target DNA at 95°C for 5 min followed by denaturation at 94°C for 1 min, primer annealing at 40°C for 1 min, and extension at 72°C for 2 min. The final cycle included extension for 5 min at 72°C to ensure full extension of the product. Samples were amplified through 30 consecutive cycles. The completed reaction products were analyzed by electrophoresis of a 10-pA aliquot through 1.0% (wt/vol) agarose gels at 85 V for 1.5 h, and the bands were visualized by staining with ethidium bromide and excitation under UV light on a transilluminator. Restriction endonuclease digestion of PCR product. A 90-pA volume of the product was transferred to a fresh tube, and DNA was precipitated by adding 2 volumes (approximately 180 plI) of ethanol (95%, vol/vol). After gentle mixing for 10 min, the samples were centrifuged at 14,000 x g for 5 min. The pellet was dried under a vacuum for 10 min and redissolved in 5 pA of TE. The concentrated DNA was added to 11 plA of sterilized distilled water, and 1 pA of spermidine, 2 pA of HaeIII buffer, and 1 pA of HaeIII (Northumbria Biologicals Ltd., Cramlington, United Kingdom) were added consecutively, giving a final volume of 20 pA. This mixture was incubated at 37°C for 4 h according to the manufacturer's instructions. The digestion was stopped with 10 plA of stop mix solution. The digested DNA (15 plA) was analyzed by submarine gel electrophoresis at 24 V for 18 h using 3% (wt/vol) NuSieve 3:1 agarose (FMC Bioproducts, Flowgen Instruments Ltd., Sittingbourne, United Kingdom) and stained and photographed as described above for genomic HaeIII digests. Fragment size estimation. DNA fragment sizes in the Southern blot hybridization patterns were calculated from migration distances by an automated gel reader and analysis system (IBI, New Haven, Conn.). Biotinylated lambda phage (Gibco-BRL Ltd.) digested with HindIll was used to provide the size markers. Three lanes (outside left and right and center) containing the markers were included on each gel. DNA fragment sizes in the PCR-RFLP patterns were estimated from migration distances by the MolMatch automated gel reader and analysis system (UVP Ltd., Cambridge, United Kingdom). A 123-bp ladder (Gibco-BRL Ltd.) was used to provide size markers. Computation of strain similarities. To compare DNA patterns (ribopatterns and urease gene PCR-RFLP profiles) from different gels, the fragments were coded according to size to minimize errors when determining similarities by computerassisted methods of analysis. The Southern blot hybridization patterns were screened for bands within 21 different size ranges up to 10 kb, and positive (presence) and negative (absence) results were recorded by established procedures (25). Bands of faint intensity were excluded. Double bands falling within a given range were scored as a single band. Computed similarities among strains were estimated by means of the Dice coefficient (negative matches excluded), and clustering of strains was based on the unweighted pair group
VOL. 32, 1994
H. PYLORI GENE PROFILING AND TRIPLE THERAPY
method to facilitate the generation and plotting of a dendrogram (39). All computations were performed with the DNAGE program, a modification of previously described pattern analysis software (7). A similar analytical procedure was applied to the urease gene RFLP profiles.
TABLE 1. Bacterial strains examined and molecular typing results Isolate reference ratient
A
RESULTS Culturing from biopsies. H. pylon was identified in at least one pre- and one posttreatment antral biopsy specimen from each of the nine patients. A total of 47 isolates were obtained, comprising 31 pretreatment isolates (designated a and b) and 16 posttreatment isolates (designated c and d). There were twice as many pretreatment isolates because each original biopsy sample was divided into two for broth and plate cultures. Seventeen (55%) of the pretreatment cultures of H. pylori were isolated by direct culture on Campylobacter selective medium. For most (6 of 9) patients, four pretreatment and two posttreatment isolates were obtained (Table 1). Ribosomal DNA gene patterns. Genomic DNA from all of the isolates, except for A651/92 (patient H), was cut by HaeIII, which was the restriction endonuclease that in previous studies (16, 20) gave the most clearly resolved digest patterns and hybridization patterns for analysis. In Fig. 1A, the ribopatterns for patient A (lanes 1 to 6) showed significant differences between pre- and posttreatment isolates. Three patients (B to D) exhibited minor variations between strains within multiple isolate sets. Figure 1B shows the ribopatterns for a further five patients (E to I), of whom two had just one type of antral H. pylori before and after triple therapy. All the isolates were designated by a ribotype (r-type) on the basis of their HaeIII ribopatterns, which were coded according to the fragment loci defined above, and similarities between patterns were determined by numerical methods. The dendrogram (Fig. 2) illustrates the similarities between the HaeIII ribopatterns for the pre- and posttreatment sets obtained from all patients. On the basis of previous analyses (29), isolates linked at the 82% similarity level were defined as the same ribotype, whereas subtypes (single band differences) were isolates that grouped at the 98% similarity level. With these criteria, each of the patients had a genotypically unique set of H. pylori isolates that were clearly different from each other and from NCTC 11637 (reference strain). There were fewer similarities of H. pylori strains among patients than within patient sets at the 60% or lower similarity level. Overall, there were 11 different ribotypes among the multiple isolates of H. pylori. Three patients had isolates with apparently identical overall ribopatterns from all their antral biopsy specimens (patients B, F, and I), although close examination of patient B isolates a, b, and a* revealed the presence of a possible 1.68-kbp doublet whereas other isolates from that individual had a single band at that position. A further five patients (C, D, E, G, and H) had two different strain subtypes which in most cases emerged after treatment, except in patient C, who had two subtypes of pretreatment isolates. Two isolates from patient A grouped at about 50% similarity, but other isolates in the set showed clear evidence of different strain types before and after treatment. RFLP analysis of ure genes. PCR with the two urease primers amplified a 2.41-kbp fragment from within the urease A and B genes of NCTC 11637 and of 24 isolates of H. pylon from six patient sets selected mainly on the basis of ribotype diversity. Restriction analysis of the PCR product showed multiple HaeIII sites within the fragments yielding three to six bands (Fig. 3), depending on the strain, with sizes between 140 bp and 1.67 kbp (Table 2). A 520-bp fragment was the most
1205
B
C
D
E
F
G
H
Study no. (Fig. 1)
Control
Molecular typeb
Biopsy'
r-type
1 2 3 4 5 6
A600/92 A601/92 A616/92 A617/92 A671/92
7 8 9 10 11 12
A608/92 A609/92 A624/92 A625/92 A673/92 A674/92
13
14 15 16 17 18
A610/92 A611/92 A626/92 A627/92 A675/92 A676/92
Ia lb Ia*
19 20 21 22 23 24
A614/92 A615/92 A630/92 A631/92 A677/92 A678/92
Ia lb Ia*
25 26 27 28 29
A672/92
Ia
lb Ia*
Ib* lIc Ild Ia
Ib*
Ia
Ib* IIc+ IId+
u-type
1 1 1 1 4 4
5 5 3a 3a
7 7 7 7 7 7
2a 2a 2b 2b
Sa
Sb
5a
Ib*
Sa Sa Sa
IIc
Ild
6 6 6 6
9a
Ib*
lIc
9a 9a 9a 9b
Id
9c
A657/92 A658/92 A679/92 A680/92
Ia* Ib* IIc Ild
6a 6b 6b 6b
30 31 32
A644/92 A645/92 A659/92 A683/92
Ia lb Ia* Ild
8 8 8 8
33 34 35 36 37 38
A648/92 A649/92 A663/92 A664/92 A684/92 A685/92
Ia' Ib+ Ia* Ib* IIc lId
2a 2a 2a 2a 2b 2b
4 4
39
Ia lb Ia* Ib* IIc IId+
3a NT 3a 3a 3b
3a
41 42 43
A650/92 A651/92 A665/92 A666/92 A686/92 A687/92
3b
2c
44 45 46 R
A667/92 A668/92 A688/92 NCTC 11637
Ia* Ib*
11 11 11 10
1
40
I
Laboratory no.
lId
7 7 7 7
3b 3b
2a 3a
a I, pretreatment, biopsies a and b; II, posttreatment, biopsies c and d. *, broth culture (others were plate cultures); +, possible mixed culture (see text). b r-type, mi gene type (ribotype); u-type, ureA -ureB PCR-RFLP type; NT, not typeable because DNA was not digested by HaeIII.
common and was present in 92% of strains (23 of 24). Seven unique fragment profile types and subtypes were identified among the 24 isolates and were designated urease types u-1 to u-7 (Table 2). The types were defined from results of a
1206
OWEN ET AL.
2 3 4 5 6 7 8
M 1
!a b
di
b a* b* c dila b a* b* c B A
M 25 26 27 28 29 30 31 32 R
1:
4:
f, :.
f:.. ....
:.
s. .n ... UsM " .: ::
f:!M :,:-.,.f .f -,
:n:"::, I.:.
s-x
.:.." t. P f: ..f .. ....::,. ..
MUMbA
Aiaw
la* b*
c
di la b a* d I.
E
-_a 2.3 _-2.0
kbp
b a* b* c
di
D
M 33 34 35 36 37 38 39 40 41 42 43 44 45 46 M
.........
........
.95
_--9.5 _-6.7
b* c _diLa
a*
C
B W.s
._iJ~
12 R M 13 14 15 16 17 18 19 20 21 22 23 24 M
9 10 1
A
la
J. CLIN. MICROBIOL.
...
a
......
b La* b*
F
G
c
d
!a
a* b*
c
-_ 6.7 -_ 4.3
_2.3 -_m2.0 kbp
d ;la* b* dl
H
FIG. 1. Ribopatterns (HaeIII digests) of DNA from strains of H. pylori. Lanes are numbered according to the strain numbers shown in Table 1. (A) isolates from patients A to D; (B) isolates from patients E to I. Lanes a and b, pretreatment plate isolates; lanes a* and b*, pretreatment broth isolates; lanes c and d, posttreatment plate isolates. Lanes M, size markers from HindIII digest of bacteriophage lambda; lanes R, DNA from reference strain NCTC 11637.
numerical analysis of ure gene RFLP profiles (data not shown), which was done by the method used for the analysis of ribopatterns. The U-type of each strain of H. pyloni is given in Table 1. For most strains, the sum of the sizes of the ureA-ureB gene RFLPs was similar to the size of the original 2.41-kbp PCR product after allowing for minor errors in size estimation. Some exceptions, which are given below, have been described in more detail in another study (14), in which single colonies were typed by PCR-RFLP pattern analysis and further evidence confirming that mixed genomic types were present was obtained. Strains A673/82 and A674/82 (patient B). The urease gene RFLPs (u-2b) for these two posttreatment isolates were 646 bp larger than the PCR product, which suggested that the profile was a composite of profile u-2a and another novel profile with
the 676-bp fragment replaced by a 640-bp fragment to give a profile containing 310-, 450-, 460-, 520-, and 640-bp fragments. Strains A648/82 and A649/82 (patient G). The urease gene profile (u-4) for these two pretreatment isolates contained 1,117 extra bases (Fig. 3), which suggested that the profile was a composite of two profiles, the first containing 310-, 460-, 500-, 520-, and 630-bp fragments and the second containing 310-, 500-, 520-, and 1,107-bp fragments. The occurrence of the low-intensity bands of 460 bp and high-intensity bands of 500 and 520 bp was attributed to the presence of different concentrations of PCR product resulting from an unequal distribution of strain types within the culture. However, a distinct feature of pure cultures was the uniform intensity of the different fragments in the PCR-RFLP profiles. Strain A687/92 (patient H). The urease gene RFLP (u-2c)
VOL. 32, 1994
H. PYLORI GENE PROFILING AND TRIPLE THERAPY
100
Patient A
Patient G
90
80
Percentage Similarity 60 70 50
1207
M R 21 22 23 24 33 34 37 38 M 40
30
~~~1~~~~
20
r-1
r-2a
r-2b
738 _
r-3a Patient H
r-3b Patient A
Patient
C
Patient
E
Patient
B
r-4
492
-
369
-
r-5a
bp
r-5b r-6a
r-6b
r-7
a --.-
Patient F
Patient D
L r-8
d ILa
D
r-9a r-9b
Patient
c
b
c
d
G
FIG. 3. HaeIII restriction digest patterns of the 2.41-kbp PCR product from amplified ureA and ureB. Multiple isolates of H. pylon were from patients D (strains 21 to 24, type u-7) and G (strains 33 and 34, type u-4; strains 37 and 38, type u-3b). Lane M, fragment size marker (123-bp ladder; Gibco-BRL); lane R, reference strain (NCTC
Lr-9c NCTC 11637
b
r-10
11637).
Lr1 1
FIG. 2. Dendrogram of the cluster analysis to show similarities based on the HaeIII ribopatterns and rRNA gene types (r-1 to r-11) of H. pylori isolates from nine patients (A to I) and the type strain NCTC 11637. Numbers on the horizontal axis indicate the percent similarities as determined by the Dice coefficient.
for this posttreatment isolate was 1,136 bp larger than the PCR product, which suggested that the profile was possibly a composite of profiles u-2a and u-3a, which were both present in the pretreatment cultures. DISCUSSION
The study demonstrated that the rRNA gene patterns of multiple isolates of H. pylon from antral biopsies of an infected patient before and after triple therapy were generally more similar to other multiple isolates from that individual than to any isolate from different individuals undergoing a similar treatment regimen. The nine patients examined were receiving triple therapy and were all infected by H. pylon with unique genotypes according to their ribopatterns. These observations were in concordance with the well-documented genomic diversity of H. pylon isolates from different individuals worldwide (1, 31, 41). The results specifically confirm and extend our data from previous comparisons of DNA profiles of pre- and posttreatment biopsies from other United Kingdom patients in showing that sequential and multiple isolates of H. pylon are generally the same (10, 23, 28, 30, 35). Compiled results of such studies based on some 47 individuals show that 51% of patients were apparently each infected by a single unique strain type of H. pylori whereas 40% were infected by multiple subtypes and the remainder (9%) had two different strains (22). Comparable values for the present study were 33, 56, and 11%, respectively, with the main difference being the increased proportion of
patients that we found were infected with subtypes of H. pylon (Table 3). In a recent study of H. pyloni in the United Kingdom (35), multiple isolates from the gastric antrum, body, and duodenum of a series of 15 patients with dyspepsia were compared prior to treatment. Ribopatterns showed that although the majority of those patients (87%) harbored a single strain throughout their stomach and duodenum, as many as 47% of the patients harbored multiple strain subtypes. Such a high level of diversity between strains was not evident in a HindIII ribotyping analysis of Australian isolates from different anatomical sites (41), but evidence of genomic subtype diversity based on total DNA restriction digest analysis was found in H. pylon from patients in the Netherlands (21). In the present study, only two patients (C and E) had multiple TABLE 2. HaeIII fragment length polymorphisms of the ureA -ureB 2.41-kbp PCR product from isolates of H. pylon Pattern
No. of
Fragment lengths (bp)
u-1 u-2a
1C 3 (2) 2 (1) 1 5 (2) 2(1) 2 (1) 2 (1) 4 (1) 4 (1)
520, 460, 450, 380, 310, 290 676, 520, 460, 450, 310 676, 640, 520, 460, 450, 310 1,120, 676, 520, 460, 450, 310 1,120, 520, 460, 310 1,120, 520, 460, 310, 140 1,107, 630, 520, 500, 460, 310 940, 580, 380, 234, 170, 140 1,670, 520, 234 790, 520, 450, 380, 234
u-2b u-2c u-3a u-3b u-4 u-5 u-6 u-7
Sum
(D)b
2,410 (0) 2,416 (+6) 3,056 (+646) 3,536 (+1,136) 2,410 (0) 2,550(+140) 3,527 (+1,117) 2,444 (+34) 2,424 (+14) 2,374 (-36)
a Numbers of patients are shown in parentheses. b D is the size difference (in base pairs) between the sum of the RFLPs and the original PCR product (2.41 kbp). c NCTC 11637, type strain of H. pylon.
1208
OWEN ET AL.
J. CLIN. MICROBIOL.
TABLE 3. Comparison of DNA typing results for H. pylon from different patients before and after treatment by the two molecular typing methods DNA typing result'
Patient
mi (r-type)
ure (u-type)
A
++
B
0 0
++ +
C D E F G H I
+ +
0 0
0
NT NT
+ +
++ +
0
NT
" Symbols: + +, new type after treatment; +, new or variation in subtypes after treatment; 0, no change in type or subtype after treatment; NT, not tested.
subtypes of H. pylon before treatment, yet after treatment four patients (D, E, G, and H) had new or different subtypes. In contrast, there was less diversity in strain types before and after treatment because we found evidence of two clearly distinct strains in only one individual (patient A). Other investigators in France (2), Australia (17), and the Netherlands (16), using either DNA digest analysis or ribotyping, have also reported the presence of several distinct strain types of H. pyloi that were simultaneously coexisting and infecting a single patient. However, in a recent study of H. pylori isolated from patients in Italy prior to treatment, a higher level of strain diversity was detected for most (85%) of the subjects colonized by strains with different types or subtypes (28). We suggest that the most likely explanation for the apparently lower degree of DNA variation within patient sets in the present study is that the sets were inherently more homogeneous or that different strain types or subtypes of H. pylon were present both before and after treatment but had not been detected because of sampling error. We chose to take sweep cultures from original isolation plates to reduce such errors. The alternative approach of picking single colonies was not adopted because of the risk of increased sampling error unless at least 10 colonies from each culture were picked and analyzed. Because of the complexity of the molecular techniques, that approach was considered impractical in the present study. The fact that 24 colonies from a single biopsy culture all had the same molecular fingerprint when analyzed by DNA digest analysis (5a) supported our contention that for most analyses, sweep cultures were acceptable provided a confirmatory PCR test was used when there was evidence of ribotype variation between different cultures from the same individual. It appears unlikely from this triple-therapy study and a previous study on omeprazole (10) that treatment specifically induces genomic changes in H. pylon which result in stably modified genotypes, although treatment may be a factor in promoting minor (subtype) changes. Metronidazole in treatment also may result in shifting the relative sizes of subpopulations of each constituent genotype if they possess different susceptibilities (23). The observed H. pylon strain differences alternatively could be interpreted as reinfection by a different strain type or subtype either from another site in the stomach or from an external source. However, the probability of that occurring within the 6-week follow-up period is likely to be low. Our study is the first systematic description of defined molecular types and subtypes within H. pyloni based on multiple isolates taken before and after triple therapy, and the
analytical methods used allowed a precise comparison of the infecting organisms. In general, the DNA ribopatterns of most strains were essentially stable despite therapy, and we conclude that failure of therapy was probably due to incomplete eradication of infecting H. pylori, which in most (67%) of the patients was originally a mixed population of types or subtypes. Several recent studies demonstrated diversity among H. pylori isolates on the basis of PCR-RFLP analysis of urease genes and indicated that such profiles provide alternative reproducible and convenient strain markers for clinical identification (1, 9, 37). Romero-Lopez et al. (37) established that HaeIII-based ureA-ureB PCR-RFLP profiles were almost as discriminatory as HaeIII ribopatterns, so strains of H. pylori exhibiting ribotype and subtype variation in the present study were selected for further investigation by that technique. A comparison of the two molecular profiling methods showed excellent concordance between pre- and posttreatment profile results (Table 3) for four of the six patient sets compared (A, C, G, and H) despite the facts that the analytical procedures differed significantly and that variation was measured at unlinked and functionally distinct genetic loci. Detailed comparison of our data also shows that ureA-ureB PCR-RFLP profiles provided better discrimination than ribopatterns for isolates in some sequential isolate sets (B and G), whereas the ribopatterns were more discriminatory for strains from patient D. One evident advantage of ureA-ureB PCR-RFLP profiling is that it can be applied to strains for which total genomic DNA is not digested by HaeIII, a fact now documented for about 25% of strains of H. pylorn (31). A second important advantage of using ureA-ureB PCR-RFLP profiling is that it provides a direct demonstration that cultures could be mixed if profiles contain major fragments that exceed the total size of the ureA-ureB PCR product. It was possible in the present study to identify three cultures that were confirmed elsewhere to be a mixture of different genomic subtypes (14). Such a precise test has important implications for checking the purity of biopsy cultures because it is much simpler and quicker to perform than detailed analyses of individual colonies, although such a PCR technique could be applied to single colonies if necessary. For patient A, both DNA profiling methods were in agreement that pre- and posttreatment sets of H. pylori isolates had different genotypes. The agreement between the techniques therefore adds further support to the view that the observed genomic differences are genuine and not artifacts of the methods (21). We conclude that DNA fingerprinting by ribotyping and urease gene profiling provides discriminatory and complementary data for identifying strains of H. pylori while monitoring therapy. Furthermore, by its application to H. pylori isolates from infected persons, it was possible to define subtypic variation in more detail, even though the H. pyloni genome was overall highly stable after failed eradication treatment. In the present series of patients, failed therapy appeared by both molecular typing methods almost certainly to be recrudescence of infection due to incomplete eradication. It should be emphasized that these molecular techniques must be viewed essentially as providing profiles for specific strain identification and are less suitable for general typing of H. pylori. Ribotyping (HaeIII) was shown to be too discriminatory for that purpose (31), but the applicability of ureA-ureB PCR-RFLP profiles for typing needs to be further evaluated because matching profiles for strains originating from different individuals have been described (9, 37), and some similarities were evident in the present study (Table 2, types u-2a and u-3a). The increased use in the future of such techniques for monitoring strain colonization should enable clinical microbiologists to provide gastro-
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H. PYLORI GENE PROFILING AND TRIPLE THERAPY
enterologists with valuable information for developing effective therapies for H. pylori infection and particularly for resolving treatment failures.
18.
ACKNOWLEDGMENTS A.H. is the recipient of a training grant under the COMETT program of the Commission of the European Community. Karen Griffin is thanked for assistance with typing.
19. 20.
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